NEUROPSYCHOPHARMACOLOGY 
The Fifth Generation 
 of Progress 
 
An Official Publication of the American College of 
Neuropsychopharmacology 

 
5th Edition 
2002

Lippincott Williams & Wilkins

Philadelphia

530 Walnut Street, Philadelphia, PA 19106 USA LWW.com

0-7817-2837-1

Copyright © 2002 The American College of Neuropsychopharmacology.

All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form or by any means,
including photocopying, or utilized by any information storage and retrieval system without written permission from the copyright
owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals
as part of their official duties as U.S. Government employees are not covered by the above-mentioned copyright.

The publishers have made every effort to trace the copyright holders for borrowed materials. If they have inadvertently overlooked
any, they will be pleased to make the necessary arrangements at the first opportunity.

Printed in the USA

Library of Congress Cataloging-in-Publication Data

Neuropsychopharmacology: the fifth generation of progress: an official publication of the American College of
Neuropsychopharmacology / editors, Kenneth L. Davis… [et al.].

p. ; cm.

Includes bibliographical references and index.

ISBN 0-7817-2837-1

1. Neuropsychopharmacology. 2. Neurobehavioral disorders. I. Davis, Kenneth L., 1947-II. American College of

Neuropsychopharmacology.

[DNLM: 1. Psychopharmacology—methods. 2. Diagnostic Imaging—methods. 3. Drug Evaluation. 4. Neurotransmitters—physiology.

QV 77 N4968 2002]

RM315 .N4825 2002

616.8′0461—dc21

2001038463

Care has been taken to confirm the accuracy of the information presented and to describe generally accepted practices. However,
the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the
information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of
the contents of the publication. Application of this information in a particular situation remains the professional responsibility of the
practitioner.

The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in
accordance with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in
government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to
check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is
particularly important when the recommended agent is a new or infrequently employed drug.

Some drugs and medical devices presented in this publication have Food and Drug Administration (FDA) clearance for limited use in
restricted research settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device
planned for use in their clinical practice.

10 9 8 7 6 5 4 3 2 1
Editors

Kenneth L. Davis MD

Esther and Joseph Klingenstein Professor of Psychiatry and Chairman

Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

Dennis Charney MD

Chief
Mood and Anxiety Disorder Research Program, National Institute of Mental Health Bethesda, Maryland

Joseph T. Coyle MD

Eben S. Draper Professor of Psychiatry and of Neuroscience; Chairman

Department of Psychiatry, Harvard Medical School, Boston, Massachusetts

Charles Nemeroff MD, PhD

Reunette W. Harris Professor and Chairman

Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia

SECTION EDITORS

Samuel H. Barondes MD

Jeanne and Sanford Robertson, MD, Director

Center for Neurobiology and Psychiatry, Department of Psychiatry, University of California, San Francisco, San Francisco, California

Dennis Charney MD

Chief
Mood and Anxiety Disorder Research Program, National Institute of Mental Health, Bethesda, Maryland

Joseph T. Coyle MD

Eben S. Draper Professor of Psychiatry and of Neuroscience; Chairman

Department of Psychiatry, Harvard Medical School, Boston, Massachusetts

Kenneth L. Davis MD

Esther and Joseph Klingenstein Professor of Psychiatry and Chairman

Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

Robert Desimone PhD

Scientific Director
National Institute of Mental Health, Director, Intramural Research Program, National Institute of Mental Health, Bethesda,
Maryland

Ronald Duman PhD

Departments of Psychiatry and Pharmacology, Laboratory of Molecular Psychiatry, Yale University School of Medicine, New Haven,
Connecticut

Marian W. Fischman PhD

Professor of Behavioral Biology

Department of Psychiatry, College of Physicians and Surgeons of Columbia University and, The New York Psychiatric Institute, New
York, New York

Eric Hollander MD

Professor of Psychiatry; Director of Clinical Psychopharmacology; Director of the Compulsive; Impulsive and Anxiety Disorders

Program Clinical Director

Seaver Autism Research Center, Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

David Kupfer MD, PhD

Thomas Detre Professor and Chair

Department of Psychiatry, University of Pittsburgh Medical Center Western Psychiatric Institute and Clinic, Pittsburgh,
Pennsylvania

Herbert Meltzer MD
Bixler Professor of Psychiatry and Pharmacology
Vanderbilt University School of Medicine, Nashville, Tennessee

Charles Nemeroff MD, PhD

Reunette W. Harris Professor and Chairman

Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia

Eric Nestler MD, PhD

Lou and Ellen McGinley Distinguished Professor and Chairman Department of Psychiatry
The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Charles P. O'Brien MD, PhD

Chief of Psychiatry; Philadelphia VA Medical Center Kenneth Appel Professor and, Vice Chair of Psychiatry
University of Pennsylvania, Philadelphia, Pennsylvania

Carol Tamminga MD

Professor of Psychiatry and Pharmacology

University of Maryland School of Medicine; Deputy Director, Maryland Psychiatric Research Center, Baltimore, Maryland

Daniel Weinberger MD

Chief
Clinical Brain Disorders Branch, National Institutes of Health, National Institute of Mental Health, Bethesda, Maryland

Secondary Editors

Charley Mitchell

Acquisitions Editor

Ray Reter

Developmental Editor

Patrick Carr

Production Editor

Tim Reynolds

Manufacturing Manager

Mark Lerner

Cover Designer

Compositor: Maryland Composition

Printer: Courier Westford

CONTRIBUTORS

Henry David Abraham M.D.

Lecturer
Department of Psychiatry, Harvard Medical School, Boston, Massachusetts

Paul D. Acton Ph.D

Research Assistant Professor

Radiology, University of Pennsylvania, Philadelphia, Pennsylvania

George K. Aghajanian M.D

Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut

Huda Akil Ph.D.

Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan

Meenakshi Alreja Ph.D.

Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut

James C. Anthony Ph.D

Professor
Johns Hopkins University, School of Hygeine and Public Health, Baltimore, Maryland

Paul S. Appelbaum M.D

Professor and Chair

Department of Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts

Jean-Michel Arrang Ph.D.

Research Director
Unité de Neurobiologie et Pharmacologie Moléculaire, INSERM, Paris, France

Gary Aston-Jones Ph.D.

Department of Psychiatry, University of Pennsylvania, VAMC, Philadelphia, Philadelphia

C. Bruce Baker M.D., J.D.

Assistant Professor
Department of Psychiatry, Yale University School of Medicine, Connecticut Mental Health Center, New Haven, Connecticut

Vaishali P. Bakshi Ph.D

Department of Psychiatry, University of Wisconsin at Madison, Madison, Wisconsin

Peter A. Bandettini Ph.D

Director
Functional Neuroimaging Facility, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland

Robert F. Barnes M.D

University of Washington School of Medicine, Department of Psychiatry and Behavioral Sciences, Seattle, Washington

Samuel H. Barondes M.D

Jeanne and Sanford Robertson M.D. Director

Center for Neurobiology and Psychiatry, Department of Psychiatry, University of California, San Francisco, San Francisco, California

Vincenzo S. Basile B.Sc., B.Ed.

Research Scientist
Psychiatric Neurogenetics, Centre for Addiction and Mental Health, Clarke Division, Toronto, Ontario, Canada

Russell Bauer Ph.D.

University of Florida, Health Science Center, Department of Clinical Psychology, Gainesville, Florida

Kevin L. Behar

Neal L. Benowitz M.D

Department of Psychiatry, University of California, San Francisco, San Francisco, California

Karen F. Berman M.D

Chief
Unit on Integrative Neuroimaging, Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of Health,
Intramural Research Program, Bethesda, Maryland

Wade Berrettini M.D., Ph.D.

University of Pennsylvania School of Medicine, Center for Neurobiology and Behavior, Philadelphia, Pennsylvania

Edward H. Bertram III M.D.

Associate Professor
Department of Neurology, University of Virginia, Charlottesville, Virginia

Joseph Biederman M.D.

Chief
Joint Program in Pediatric Psychopharmacology, Massachusetts General and McLean Hospitals; Professor, Department of Psychiatry,
Massachusssets General Hospital, Boston, Massachusetts

Randy D. Blakely M.D., Ph.D

Department fo Pharmacology, Vanderbilt University, Nashville, Tennessee

Daryl E. Bohning Ph.D

Professor
Department of Radiology, Medical University of South Carolina, Charleston, South Carolina

Robert J. Boland M.D

Miriam Hospital, Department of Psychiatry, Providence, Rhode Island

David L. Braff M.D

Department of Psychiatry, University of California, San Diego, La Jolla, California

James Robert Brasic M.D., M.P.H.

Postdoctoral Fellow
Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore,
Maryland
Monique Breteler M.D., Ph.D.
Department of Epidemiology and Biostatistics, Erasmus Medical Center, Rotterdam, Netherlands

Michael Brownstein M.D.

National Institutes of Health, National Institute of Mental Health, Bethesda, Maryland

Alastair Buchan FRCPC

Department of Clinical Neuroscience, Foothills Hospital, Calgary, Alberta, Canada

Christian Büchel M.D.

Department of Neurology, Hamburg University, Hamburg, Germany

Robert E. Burke M.D

Professor of Neurology and Pathology

Departments of Neurology and Pathology, Columbia University, New York, New York

David J. Burn F.R.C.P., M.D., M.A., M.B.B.S

Consultant and Service Lecturer in Neurology

Department of Neurology, Regional Neurosciences Centre, Newcastle upon Tyne, United Kingdom

Alistair Burns M.D.

Department of Old Age Psychiatry, Withington Hospital, West Disbury, Manchester, United Kingdom

Joseph D. Buxbaum Ph.D

Assistant Professor of Psychiatry

Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

Daniel Buysee M.D.

Associate Professor of Psychiatry

University of Pittsburgh Health System, WPIC, Pittsburgh, Pennsylvania

William A. Carlezon Jr. Ph.D

Assistant Professor of Psychiatry

Harvard Medical School, McLean Hospital, Belmont, Massachusetts

James V. Cassella Ph.D

Neurogen Corporation, Branford, Connecticut

V. S. Caviness Jr. CMA

MGH-East, Charlestown, Massachusetts

Britton Chance M.D.

Department of Biophysics and Physical Biochemistry, University of Pennsylvania, Johnson Research Foundation, Philadelphia,
Pennsylvania

Dennis Charney M.D.

Chief
Mood and Anxiety Disorder Research Program, National Institute of Mental health, Bethesda, Maryland

A. R. Childress
University of Pennsylvania School of Medicine, Department of Psychiatry, Addiction Treatment, Philadelphia, Pennsylvania

Emil F. Coccaro M.D

The University of Chicago, The Pritzker School of Medicine, Department of Psychiatry, Chicago, Illinois

C. Keith Conners M.D.

Behavioral Neurology Department, Durham, North Carolina

Jeremy D. Coplan M.D

Columbia University, New York Psychiatric Institute, New York, New York

Nancy H. Covell Ph.D

Project Director
Research Division, Connecticut Department of Mental Health and Addiction Services, Hartford, Connecticut, and, Research
Associate, Department of Psychology, University of Connecticut, Storrs, Connecticut

Joseph T. Coyle M.D

Eben S. Draper Professor of Psychiatry and Neuroscience; Chairman

Department of Psychiatry, Harvard Medical School, Boston, Massachusetts

Merit E. Cudkowicz

Anders M. Dale M.D

NMR Center, Charlestown, Massachusetts

Richard Davidson M.D.

Department of Psychology, University of Wisconsin, Madison, Wisconsin

Michael Davidson
Sheba Medical Center, Tel Aviv, Israel

Linda Davies M.Sc.

Health Economics, Reader, Health Economics, School of Psychiatry and Behavioural Sciences, Univesity of Manchester, Manchester,
United Kingdom

Kenneth L. Davis M.D

Esther and Joseph Klingenstein Professor of Psychiatry and, Chairman

Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

Michael Davis Ph.D.

Department of Psychiatry, Emory University School of Medicine, Atlanta, Georgia

Mahlon R. DeLong M.D

Emory University, Department of Neurology, Atlanta, Georgia

Robert Desimone Ph.D.

Scientific Director
National Institute of Mental Health; Director, Intramural Research Program/National Institute of Mental Health, Bethesda,
Maryland

Errol B. DeSouza Ph.D

Research and Development, Neurocrine Bioscience, Inc., San Diego, California

William L. Dewey
Virginia Commonwealth University, Department of Pharmacology and Toxicology, Richmond, Virginian

Vincenzo M. Di Marzo Ph.D.

Primo Richercatore, Istituto Chimica Biomoleculare, Consiglio Nazionale Ricerche, Pozzuoli (NA), Italy

David F. Dinges Ph.D

Professor of Psychology in Psychiatry

Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania

Cynthia M. Dorsey Ph.D

Assistant Professor
Department of Psychiatry, Harvard Medical School, Boston, Massachusetts, and, Director, Sleep Research Laboratory, Behavioral
Psychopharmacology Research Laboratory, McLean Hospital, Belmont, Massachusetts

Wayne C. Drevets M.D

Chief
Section on Mood and Anxiety Disorders Imaging, Molecular Imaging Branch, National Institute of Mental Health, Bethesda, Maryland

Karen Duff Ph.D.

Nathan Kline Institute, Dementia Research Program, Orangeburg, New York

Ronald S. Duman Ph.D

Professor of Psychiatry and Pharmacology, and, Director

Abraham Ribicoff Research Facilities, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven,
Connecticut

Gary E. Duncan Ph.D

Research Associate Professor

Department of Psychiatry, University of North Carolina, Chapel Hill, North Carolina

Jane L. Eisen M.D

Assistant Professor
Department of Psychiatry and Human Behavior, Brown Medical School, Providence, Rhode Island
Mary-Anne Enoch M.D.

Visiting Associate
Laboratory of Neurogenetics, NIAAA, National Institutes of Health, Bethesda, Maryland

Charles M. Epstein M.D

Emory University, Department of Neurology, Atlanta, Georgia

Susan M. Essock Ph.D

Mount Sinai School of Medicine, Department of Psychiatry, New York, New York

Alan Evans M.D.

Department of Neurology, McGill University, Montreal Neurological Institute, Montreal, Quebec, Canada

Dwight L. Evans M.D

Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania

StephenV. Faraone Ph.D.

Associate Professor
Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts

Marian W. Fischman Ph.D

Professor of Behavioral Biology

Department of Psychiatry, College of Physicians and Surgeons of Columbia University and, The New York Psychiatric Institute, New
York, New York

Joanna S. Fowler Ph.D

Senior Chemist
Chemistry Department, Brookhaven National Laboratory, Upton, New York

Teresa R. Franklin Ph.D

Substance Abuse Fellow

Department of Psychiatry, University of Pennsylvania, and, Philadelphia Veterans Affairs Medical Center, Philadelphia,
Pennsylvania

Alan Frazer Ph.D.

Chair and Professor

Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas

Robert Freedman M.D.

Professor
Department of Psychiatry and Pharmacology, University of Colorado, Denver, Colorado

Nelson B. Freimer M.D

University of California, San Francisco, Department of Psychiatry, San Francisco, California

Linda K. Frisman Ph.D

Director of Research
Research Division, Connecticut Department of Mental Health and Addiction Services, Hartford, Connecticut, and, Research
Professor, Department of Psychology, University of Connecticut, Storrs, Connecticut

Karl Friston
Wellcome, Department of Cognitive Neurology, London, United Kingdom

Paul J. Fudala Ph.D

Research Associate Professor of Pharmacology in Psychiatry

Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania, and, Clinical Toxicologist, Department of
Behavioral Health Service, Veterans Affairs Medical Center, Philadelphia, Pennsylvania

Masahiro Fujita M.D., Ph.D.

Assistant Professor
Department of Psychiatry, Yale University School of Medicine, West Haven, Connecticut

Fred H. Gage Ph.D

Professor
Laboratory of Genetics, The Salk Institute, La Jolla, California

Stephen J. Garlow M.D., Ph.D

Assistant Professor
Department of Psychiatry and Behavioral Science, Emory University, Atlanta, Georgia

L. Garrido
Massachusetts General Hospital-East, Charlestown, Massachusetts

Nori Geary Ph.D.

Research Professor
Department of Psychiatry, Weill Medical College of Cornell University, New York, New York

Mark S. George M.D

Departments of Psychiatry, Radiology and Neurology, Medical University of South Carolina, Charleston, South Carolina

Mark A. Geyer Ph.D

Professor
Department of Psychiatry, University of California, San Diego, La Jolla, California

Patricia S. Godman-Rakic Ph.D.

Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut

Donald C. Goff M.D

Director
Schizophrenia Program, Massachusetts General Hospital, Boston, Massachusetts, and, Associate Professor, Department of Psychiatry,
Harvard Medical School, Boston, Massachusetts

Terry E. Goldberg Ph.D

National Institutes of Mental Health, Bethesda, Maryland

David Goldman M.D.

Chief
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland

Patricia S. Goldman-Rakic Ph.D.

Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut

Jack M. Gorman M.D

Columbia University, Department of Psychiatry, New York, New York

Anthony A. Grace M.D

University of Pittsburgh, Department of Neuroscience and Psychiagtry, Pittsburgh, Pennsylvania

Jean-Michel Gracies M.D., Ph.D.

Assistant Professor
Department of Neurology, The Mount Sinai Medical Center, New York, New York

Steven H. Graham M.D., Ph.D

Associate Professor and Vice-Chair

Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania, and, Associate Director for Research, Department of
Geriatric Research Education and Clinical Center, Veterans Affairs Pittsburgh Health System, Pittsburgh, Pennsylvania

Michael F. Green Ph.D

Professor
Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, Los Angeles, California

Paul E. Greenberg M.A., M.S

Analysis Group, Inc., Cambridge, Massachusetts

David J. Greenblatt M.D

Dept of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts

Dimitri E. Grigoriadis Ph.D

Senior Director
Department of Pharmacology and Lead Discovery, Neurocrine Biosciences, Inc., San Diego, California

Amy Grogg PharM.D.

Associate Director
CNS Outcomes Research, Department of Outcomes Research, Janssen Pharmacetica, Titusville, New Jersey
Jerold S. Harmatz B.A

Assistant Professor of Medicine

Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Masachusetts

Vahram Haroutunian Ph.D.

Associate Professor
Department of Psychiatry, Mount Sinai School of Medicine and, Bronx Veterans Affairs Medical Center, New York, New York

James C. Harris M.D

Director
Developmental Neuropsychiatry, Professor of Psychiatry and Behavioral Sciences and Pediatrics, Johns Hopkins University School of
Medicine, Baltimore, Maryland

Philip D. Harvey Ph.D

Professor of Psychiatry
Mount Sinai School of Medicine, New York, New York

Robert W. Hickey M.D

Assistant Professor
Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania

Makoto Higuchi Ph.D.

University of Pennsylvania, School of Medicine, Department of Pathology and Laboratory Medicine, Philadelphia, Pennsylvania

Steven A. Hillyard M.D

University of California, San Diego, Department of Neuroscience, La Jolla, California

Robert M. A. Hirschfeld M.D.

Titus H. Harris Chair; Professor and Chair

Department of Psychiatry and Behavioral Science, University of Texas Medical Branch, Galveston, Texas

J. Allan Hobson M.D.

Professor of Psychaitry
Massachusetts Mental Health Center, Boston, Massachusetts

Eric Hollander M.D.

Professor of Psychiatry; Director of Clinical Psychopharmacology; Director of the Compulsive, Impulsive and Anxiety Disorders

Program; Clinical Director Seaver Autism Research Center

Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

Fahmeed Hyder
Department of Diagnostic Radiology, Yale University, School of Medicine, New Haven, Connecticut

Steven E. Hyman M.D

National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland

Robert B. Innis M.D

Professsor
Department of Psychiatry and Pharmacology, Yale University School of Medicine, West Haven, Connecticut

Bruce G. Jenkins Ph.D

Director
Neurochemical Imaging, MGH-NMR Center/Radiology, Massachusetts General Hospital, Charlestown, Massachusetts

J. David Jentsch Ph.D.

Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania

David S. Johnson M.D., Ph.D

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, Langley Porter Psychiatric Institute, University
of California, San Francisco, San Francisco, California

Reese T. Jones M.D

Langely Porter Psychiatric Institute, University of California, San Francisco, San Francisco, California

Paramjit Joshi M.D.

Chair
Department of Psychiatry, Children's National Medical Center, Washington, DC
Ned H. Kalin M.D

Professor and Chair

Department of Psychiatry, University of Wisconsin Medical School, Madison, Wisconsin

Peter W. Kalivas Ph.D

Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina

John Kane M.D.

Hillside Hospital, Glen Oaks, New York

Shatij Kapur M.D., Ph.D.

Department of Psychiatry, Clark Institute; University of Toronto, Ontario, Canada

Walter Kaye M.D.

Western Psychiatric Institute & Clinic, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Paul E. Keck Jr. M.D.

c/o Husseini K. Manji, M.D Department of Psychiatry, Wayne State University School of Medicine, Detroit, Michigan

John H. Kehne Ph.D

Executive Director
Department of Behavioral Biology, Neurogen Corporation, Branford, Connecticut

Martin B. Keller M.D

Professor and Chairman

Brown University School of Medicine, Butler Hospital, Providence, Rhode Island

David Kennedy M.D.

Morphometric Analysis Laboratory, Neuroscience Center, Charlestown, Massachusetts

James L. Kennedy M.D., FRCP(C)

Head
Neurogenetics Section, Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada

Justine M. Kent M.D

Columbia University, Department of Psychiatry, New York, New York

Matcheri S. Keshavan M.D

Professor of Psychiatry
Department of Psychiatry, Western Psychiatric Institute and Clinic, Pittsburgh, Pennsylvania

Ronald C. Kessler Ph.D

Harvard Medical School, Boston, Massachusetts

Clint Kilts M.D.

Emory University School of Medicine, Department of Psychiatry, Atlanta, Georgia

Joel E. Kleinman M.D., Ph.D

Clinical Brain Disorders Branch, National Institutes of Mental Health Neuroscience Center, Washington, DC

Violetta Klimek Ph.D.

Assistant Professor
Department of Psychiatry, University of Mississippi Medical Center, Jackson, Mississippi

Jacqueline D. Kloss Ph.D

Assistant Professor of Psychology

Department of Psychology, Sociology, Anthropology, Drexel University, and, Adjunct Assistant Professor of Psychology, Department
of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania

George F. Koob Ph.D

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California

Kathy L. Kopnisky
National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland

Thomas R. Kosten M.D

Professor of Psychiatry
Department of Psychiatry, Yale University School of Medicine, and, Chief, Department of Psychiatry, Veterans Affairs Connecticut
Healthcare System, West Haven, Connecticut

Chris M. Kozma Ph.D

Senior Research Director

Strategic Outcomes Services of Care Science, Inc., Philadelphia, Pennsylvania

Mark S. Kramer M.D., Ph.D

Senior Director
Department of Clinical Neuroscience, Merck & Co., West Point, Pennsylvania, and, University of Pennsylvania, Philadelphia,
Pennsylvania

Mary Jeanne Kreek M.D.

The Rockefeller University, Laboratory of the Biology of Addictive Diseases, New York, New York

Suchitra Krishnan-Sarin Ph.D.

Yale University School of Medicine, Department of Psychiatry, New Haven, Connecticut

John H. Krystal M.D

Psychiatry Service
Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut

David Kupfer M.D.

Thomas Detre Professor and Chair

Department of Psychiatry, University of Pittsburgh Medical Center, Western Psychiatric Institute and Clinic, Pittsburgh,
Pennsylvania

Marta Kutas Ph.D.

Professor of Cognitive Science and Neurosciences

Department of Cognitive Science, University of California, San Diego, La Jolla, California

Ariel J. Lang Ph.D

Assistant Professor in Residence

Department of Psychiatry, University of California, San Diego, San Diego, California, and, Psychologist, Veterans Affairs San Diego
Health System, San Diego, California

Marc Laruelle M.D.

New York Psychiatric Institute, Department of Psychiatry, New York, New York

Paul Leber M.D.

Director
Neuro-Pharm Group, LLC, Potomac, Maryland

James F. Leckman M.D

Neison Harris Professor of Child Psychiatry and Pediatrics

Child Study Center, Yale University School of Medicine, New Haven, Connecticut

Virginia M.-Y. Lee Ph.D.

The John H. Ware 3rd Professor in Alzheimer's Research and Co-Director

Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania

Robert H. Lenox M.D

Vice President
Head CNS Group, Aventis Pharmaceuticals, Bridgewater, New Jersey

Jane Leserman Ph.D.

Research Associate Professor

Department of Psychiatry, University of North Carolina School of Medicine, Chapel Hill, North Carolina

Michael L. Leski Ph.D

Instructor
Department of Psychiatry, McLean Hospital, Belmont, Massahusetts

David A. Lewis M.D

Professor
Department of Psychiatry and Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania
Alfred J. Lewy M.D
Oregon Health Sciences University, Sleep & Mood Disorders Laboratory, Portland, Oregon

Jeffrey A. Lieberman M.D

Department of Psychiatry, University of North Carolina Chapel Hill, Chapel Hill, North Carolina

J. Listerud
University of Pennsylvania School of Medicine, Department of Psychiatry, Addiction Treatment, Philadelphia, Pennsylvania

Shauna N. MacMillan B.Sc.

Research Assistant
Department of Psychiatry and Behavioral Neurosciences, Wayne University School of Medicine, Detroit, Michigan

Pierre Magistretti M.D., Ph.D.

Inst de Physiologie, Univ Lausanne, Lausanne, Switzerland

Ramin Mahmoud M.D.

Director
Outcome Research, Janssen Research Foundation, Titusville, New Jersey

Nikos Makris M.D., Ph.D.

Instructor
Department of Neurology, Harvard Medical School, Boston, Massachusetts

Robert C. Malenka M.D., Ph.D

Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, Palo Alto, California

Andrea Manca M.Sc.

Research Fellow
Centre for Health Economics, University of York, York, United Kingdom

Husseini K. Manji M.D

Department of Psychiatry, Wayne University School of Medicine, Detroit, Michigan

John D. Mann M.D

Department of Neurology, University of North Carolina, Chapel Hill, School of Medicine, Chapel Hill, North Carolina

J. John Mann M.D.

Chief of Neuroscience
Department of Psychiatry, Columbia University, New York, New York

Russell L. Margolis M.D

Associate Professor and Attending Psychiatrist

Department of Psychiatry, Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland

Athina Markou
The Scripps Research Institute, Department of Neuropharmacology, La Jolla, California

Billy R. Martin Ph.D

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia

Mario Masellis B.Sc., M.Sc.

Research Scientist
Department of Psychiatric Neurogenetics, Clarke Division, Centre for Addiction and Mental Health, Toronto, Ontario, Canada

Karen I. Mason Ph.D

Neuropsychology Fellow
Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, School of Medicine, Los Angeles,
California

Graeme F. Mason

Helen S. Mayberg M.D

Baycrest Center, Rotman Research Institute, Toronto, Ontario, Canada

W. Vaughn McCall M.D.

Professor
Department of Psychiatry and Behavioral Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Una D. McCann M.D
Mt. Auburn Hospital, Department of Psychiatry and Behavioral Sciences, Lexington, Massachusetts

Robert W. McCarley M.D

Harvard Medical School, Department of Psychiatry, Brockton, Massachusetts

William M. McDonald M.D

Department of Psychiatry, Wesley Woods Health Center, Atlanta, Georgia

Christopher J. McDougle M.D

Raymond E. Houk Professor of Psychiatry; Professor of Pediatrics and Neurobiology

Department of Psychiatry, Indiana University School of Medicine, Indianapolis, Indiana

L. Alison McInnes M.D., M.S.

Assistant Adjunct Professor

Department of Psychiatry and Neurogenetics, University of California, San Francisco, San Francisco, California

Ian G. McKeith M.D

Department of Old Age Psychiatry, Wolfson Research Centre, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom

Thomas McLellan Ph.D.

Philadelphia Veterans Affairs Medical Center, Philadelphia, Pennsylvania

Gavan P. McNally

Research Fellow
Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan

James H. Meador-Woodruff M.D.

Senior Associate Research Scientist

Mental Health Research Institute, and, Associate Professor, Department of Psychiatry, University of Michigan, Ann Arbor, Michigan

Brian Meissner PharM.D.

Janssen Outcomes Fellow

Department of Pharmacy, University of South Carolina, Columbia, South Carolina

Herbert Meltzer M.D.

Bixler Professor of Psychiatry and Pharmacology

Vanderbilt University School of Medicine, Nashville, Tennessee

Wallace B. Mendelson M.D

Professor
Departments of Psychiatry, Medicine, and Clinical Pharmacology, University of Chicago, Chicago, Illinois

Kathleen Merikangas Ph.D.

Professor of Epidemiology
Public Health and Psychiatry, Yale University School of Medicine, New Haven, Connecticut

Emmanuel Mignot M.D., Ph.D.

Associate Professor
Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California

Mark A. Mintun M.D

Washington University School of Medicine, Department of Radiology, St. Louis, Missouri

Seiya Miyamoto M.D., Ph.D.

Visiting Research Fellow

Department of Psychiatry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, and, Department of
Neuropsychiatry, St. Marianna University School of Medicine, Kawasaki, Japan

Bita Moghaddam
Yale University School of Medicine, Department of Psychiatry, West Haven, Connecticut

Richard C. Mohs Ph.D

Research Service, Veterans Affairs Medical Center, Bronx, New York

Paul Morley M.D.

Receptors and Ion Channels Group, Institute of Biological Science, National Research Council of Canada, Ottawa, Ontario, Canada
John H. Morrison Ph.D
Mount Sinai Medical Center, New York, New York

Pierandrea Muglia M.D.

Postdoctoral Fellow
Neurogenetics Section, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, Toronto, Ontario,
Canada

Emanuela Mundo M.D.

Assistant Professor
Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada

Charles Nemeroff M.D., Ph.D.

Reunette W. Harris Professor and Chairman

Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia

Eric J. Nestler M.D., Ph.D

Lou and Ellen McGinley Distinguished Professor and Chairman

Department of Psychiatry, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Rachael Neve M.D.

McLean Hospital, Belmont, Massachusetts

Seiji Nishino M.D., Ph.D.

Senior Research Scientist

Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California

Ralph A. Nixon M.D., Ph.D

Nathan Kline Institute, Dementia Research Program, Orangeburg, New York

Eric Nofzinger M.D.

University of Pittsburgh Health System, Department of Psychiatry, Pittsburgh, Pennsylvania

Charles P. O'Brien M.D., Ph.D.

Chief of Psychiatry
Department of Behavioral Health, Philadelphia Veterans Affairs Medical Center, and, Kenneth Appel Professor and Vice Chair,
Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania

John T. O'Brien D.M.

Department of Old Age Psychiatry, Wolfson Research Centre, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom

Stephanie S. O'Malley Ph.D.

Yale University School of Medicine, Department of Psychiatry, New Haven, Connecticut

C. Warren Olanow M.D., F.R.C.P.L.

Professor and Chairman

Department of Neurology, Mount Sinai Medical Center, New York, New York

Berend Olivier Ph.D.

Department of Psychopharmacology, Utrecht University, Utrecht, Netherlands, and, Department of Psychiatry, Yale University
School of Medicine, Hawthorne, New York

Richard W. Olsen Ph.D

Professor of Neuroscience, Pharmacology, and Anesthesiology

Department of Molecular and Medical Pharmacology, University of California Los Angeles School of Medicine, Los Angeles,
California

Gregory A. Ordway Ph.D

Division of Neurobiology and Behavior Research

Department of Psychiatry, University of Mississippi Medical Center, Jackson, Mississippi

Michael J. Owen Ph.D

University of Wales College of Medicine, Department of Psychological Medicine, Cardiff, United Kingdom

Vural Özdemir M.D., Ph.D.

Senior Scientist
Department of Pharmacogenetics, R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey
Edward F. Pace-Schott M.S., M.A., L.M.H.C.

Instructor
Department of Psychiatry, Harvard Medical School, Boston, Massachusetts

Stefano Pallanti M.D.

Mount Sinai School of Medicine, Department of Psychiatry, New York, New York

G. M. Papadimitriou
Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts

S. Parvathy Ph.D.

Postdoctoral Fellow
Department of Psychiatry, Mount Sinai School of Medicine, New York, New York

David L. Pauls Ph.D

Child Study Center, Yale University School of Medicine, New Haven, Connecticut

Robert H. Perry M.B., Ch.B.

Department of Neuropathology, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom

Elaine Perry M.D.

MRC Neurochemical Pathology Unit, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom

John Petitto M.D.

Department of Psychiatry, University of Florida, College of Medicine, Gainesville, Florida

Ognen A. C. Petroff

Michael Phelps M.D.

Department of Molecular and Medical Pharmacology, Center Health Services, University of California, Los Angeles, School of
Medicine, Los Angeles, California

Marina Picciotto M.D.

Department of Psychiatry, Yale University School of Medicne, New Haven, Connecticut

Debra A. Pinals M.D

Director; Fellowship in Law and Psychiatry

Department of Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts

Daniel S. Pine M.D

Associate Professor of Clinical Psychiatry

Department of Psychiatry, Division of Child and Adolescent Psychiatry, Columbia University / New York State Psychiatric Institute,
New York, New York

Joseph Piven M.D.

Department of Psychiatry, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina

Mark Potenza M.D., Ph.D.

Director
Problem Gambling Clinic and, Assistant Professor, Department of Psychiatry, Yale University School of Medicine and, Connecticut
Mental Health Center, New Haven, Connecticut

William Z. Potter M.D., Ph.D

K. Ranga Rama Krishnan M.D.

Department of Psychiatry, Duke University Medical Center, Durham, North Carolina

Bruce R. Ransom M.D., Ph.D

Warren Magnuson Professor and Chair

Department of Neurology, University of Washington, Seattle, Washington

Murray A. Raskind M.D

Psychiatry Service, Seattle Veterans Affairs Medical Center, Seattle, Washington

Steven A. Rasmussen M.D

Brown University, Butler Hospital, Providence, Rhode Island

Scott L. Rausch M.D

Associate Professor
Massachusetts General Hospital, Charlestown, Massachusetts
C. E. Reeder Ph.D.

Professor
College of Pharmacy, University of South Carolina, Columbia, South Carolina

Allan Reiss M.D.

Professor of Psychiatry
Stanford University School of Medicine, Stanford, California

Charles Reynolds M.D.

Professor of Psychiatry and Neuroscience

University of Pittsburgh Health System, Pittsburgh, Pennsylvania

George A. Ricaurte M.D., Ph.D

Department of Neurology, Johns Hopkins Bayview Medical Center, Baltimore, Maryland

George S. Robertson Ph.D

Director of Pharmacology
Merck Frosst Canada, Inc, Kirkland, Quebec, Canada

Robert G. Robinson M.D

Paul W. Penningroth Professor and Head

Department of Psychiatry, The University of Iowa College of Medicine, Iowa City, Iowa

Catherine A. Roca M.D

Staff Psychiatrist
Behavioral Endocrinology Branch, National Institute of Mental Health, Bethesda, Maryland

B. R. Rosen
CMA, MGH-East, Charlestown, Massachusetts

David R. Rosenberg M.D

Director
Obsessive Compulsive Disorders Program, Wayne University School of Medicine, Detroit, Michigan

Robert Rosenheck M.D.

Veterans Affairs Medical Center, West Haven, Connecticut

Christopher A. Ross M.D., Ph.D

Professor and Attending Psychiatrist

Department of Psychiatry and Neuroscience, Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland

Douglas Rothman M.D.

Yale University School of Medicine, Department of Radiology, Magnetic Resonance Center, New Haven, Connecticut

David R. Rubinow M.D

National Institutes of Health, Bethesda, Maryland

Nadia M. Rupniak Ph.D

Department of Behavior Pharmacology, Merk Sharp & Dohme, Harlow, Essex, United Kingdom

A. John Rush M.D.

University of Texas Southwestern Medical Center, Department of Psychiatry, Dallas, Texas

David S. Russell M.D., Ph.D

Assistant Professor
Department of Neurology and Psychiatry, Yale University School of Medicine, New Haven, Connecticut

Neal D. Ryan M.D

Professor of Psychiatry
Joaquim Puig-Antich Professor in Child and Adolescent Psychiatry, Department of Child Psychiatry, University of Pittsburgh School
of Medicine, Pittsburgh, Pennsylvania

Elaine Sanders-Bush Ph.D.

Professor
Department of Pharmacology and Psychiatry, Vanderbilt University School of Medicine, Nashville, Tennessee

William R. Schafer Ph.D

Assistant Professor
Section of Neurobiology, University of California, San Diego, La Jolla, California

Helen E. Scharfman Ph.D

Director
Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, West Haverstraw, New York, and, Associate
Professor, Departments of Pharmacology and Neurology, Columbia University, New York, New York

Alan F. Schatzberg M.D

Department of Psychiatry, Atlanta, Georgia

Peter J. Schmidt M.D

Chief
Unit on Reproductive Endocrinology, Behavioral Endocrinology Branch, National Institute of Mental Health, Bethesda, Maryland

Marc Alan Schuckit M.D.

Professor of Psychiatry
Department of Psychiatry, San Diego Veterans Affairs Medical Center and, University of California, San Diego Medical School, San
Diego, California

Ann C. Schulte Ph.D

Associate Professor
Department of Psychology, North Carolina State University, Raleigh, North California

Robert Schwarcz Ph.D.

Director
Neuroscience Program, Maryland Psychiatric Research Center, Baltimore, Maryland

J. C. Schwartz M.D.
Unite de Neurobiologie et Pharmacol., Centre Paul Broca de I'inserm, Paris, France

Richard I. Shader M.D

Newton Centre, Massachusetts

Yvette Sheline M.D.

Department of Psychiatry, Washington University of St. Louis, St. Louis, Missouri

Jun Shen

Lisa M. Shin Ph.D

Assistant Professor
Department of Psychology, Tufts University, Medford, Massachusetts

Toni S. Shippenberg Ph.D

National Institutes of Health, National Institute on Drug Abuse, Integrative Neurosciences, Bethesda, Maryland

Robert G. Shulman

Nicola Sibson

Larry J. Siever M.D

Bronx Veterans Affairs Medical Center, Psychiatry Service, Bronx, New York

Daphne Simeon M.D.

Assistant Professor
Department of Psychiatry, Mt. Sinai School of Medicine, New York, New York

Daniel L. Small Ph.D

Receptors and Ion Channels Group

Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada

Gary W. Small M.D

Director
Center on Aging, Parlow-Solomon Professor on Aging, Professor, Department of Psychiatry and Biobehavioral Sciences, University of
California, Los Angeles, Neuropsychiatric Institute, Los Angeles, California

Gerard P. Smith M.D

Professor of Psychiatry (Behavioral Neuroscience)

Department of Psychiatry, Weill Medical College of Cornell University, New York, New York
A. Gregory Sorensen M.D.

Assistant Radiologist
Department of Radiology / NMR Center, Massachusetts General Hospital, and, Assistant Professor in Radiology, Department of
Radiology, Harvard Medical School, Boston, Massachusetts

Dan J. Stein M.B.

Director
MRC Research Unit on Anxiety Disorders, Department of Psychiatry, University of Stellenbosch, Cape Town, South Africa

Murray B. Stein M.D

Anxiety & Traumatic Stress Disorders, Department of Psychiatry, University of California, San Diego, La Jolla, California

William S. Stone Ph.D

Assistant Professor
Harvard University, Department of Psychiatry, Massachusetts Mental Health Center, Boston, Massachusetts

James S. Sutcliffe Ph.D

Assistant Professor
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee

Neal R. Swerdlow M.D

Professor
Department of Psychiatry, University of California, San Diego, School of Medicine, La Jolla, California

Martin P. Szuba M.D

Assistant Professor
Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania

Boris Tabakoff Ph.D.

Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado

John F. Tallman Ph.D

Scientific Director
Neurogen Corporation, Branford, Connecticut

Carol A. Tamminga M.D

Professor of Psychiatry and Pharmacology

University of Maryland School of Medicine, and, Deputy Director, Maryland Psychiatric Research Center, Baltimore, Maryland

Laurence H. Tecott M.D., Ph.D

Department of Psychiatry, University of California, San Francisco, San Francisco, California

Kenneth E. Towbin M.D

The George Washington University, School of Medicine, Department of Psychiatry, Washington, DC

John Q. Trojanowski M.D., Ph.D

University of Pennsylvania School of Medicine, Department of Pathology and Laboratory Medicine, Philadelphia, Pennsylvania

Ming T. Tsuang M.D., Ph.D

Stanley Cobb Professor of Psychiatry; Director

Harvard Institute of Psychiatry Genetics, Head, Department of Psychiatry, Harvard Medical School, Massachusetts Mental Health
Center, Boston, Massachusetts

David S. Tuch B.A

(Physics); Graduate Research Assistant

Massachusetts General Hospital, NMR Center, Charlestown, Massachusetts

Henriette van Praag M.D.

Research Associate
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California

Gabriel A. Vargas M.D., Ph.D

Research Fellow
Department of Psychiatry, University of California at San Francisco, San Francisco, California

Veronica J. Vieland Ph.D

Professor
Department of Biostatistics and Psychiatry, University of Iowa, Iowa City, Iowa

Nora D. Volkow M.D

Professor
Department of Psychiatry, SUNY, Stony Brook, Stony Brook, NY, and, Medical Department, Brookhaven National Laboratory, Upton,
New York

Joseph Volpicelli M.D.

Department of Psychiatry, University of Pennsylvania, Veterans Affairs Medical Center, Philadelphia, Pennsylvania

Lisa L. von Moltke M.D.

Tufts University School of Medicine, Department of Pharmacology and Experimental Therapeutics, Boston, Massachusetts

Mark Von Zastrow M.D.

University of California, San Francisco, Department of Psychiatry, San Francisco, California

B. Timothy Walsh M.D.

Ruane Professor of Pediatric Psychopharmacology

College of Physicians and Surgeons, Columbia University; Director, Eating Disorders Research Unit, New York State Psychiatric
Institute, New York, New York

Thomas H. Wassink M.D

Assistant Professor
Department of Psychiatry, University of Iowa College of Medicine, Iowa City, Iowa

V. J. Wedeen
MGH-East, NMR Center, Charlestown, Massachusetts

Daniel R. Weinberger M.D

Chief
Clinical Brain Disorders Branch, National Institutes of Health, National Institute of Mental Health, Bethesda, Maryland

Myrna M. Weissman Ph.D

Professor of Psychiatry and Epidemiology
Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York

Thomas Wichmann M.D.

Assistant Professor
Department of Neurology, Emory University, Atlanta, Georgia

Michael Williams Ph.D., DSc

Divisional Vice President
Neurological and Urological Diseases Research, Pharmaceuticals Products Division, Abbott Laboratories, Abbott Park, Illinois

Margaret G. Woerner Ph.D

Associate Director
Psychiatry Research, Hillside Hospital, North Shore-Long Island Jewish Health System, Glen Oaks, New York, Associate Professor,
Department of Psychiatry, Albert Einstein College of Medicine, Bronx, New York

Dean F. Wong M.D., Ph.D

Radiology Vice-Chair for Research Administration and Training; Professor of Radiology, Psychiatry, and Environmental Health

Sciences
Johns Hopkins Medical Institutions, Baltimore, Maryland

Scott W. Woods M.D

Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut

George Woody M.D.

Veterans Affairs Medical Center, Substance Abuse Treatment Unit, Philadelphia, Pennsylvania

Audrey J. Worth
CMA, MGH-East, Charlestown, Massachusetts

Ona Wu M.S.

Research Assistant
Department of Radiology, MGH NMR Center, Charlestown, Massachusetts
Larry J. Young Ph.D

Associate Professor
Department of Psychiatry and Behavioral Sciences, Emory University, Atlanta, Georgia

Michael J. Zigmond Ph.

Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania

Joseph Zohar

Associate Professor
Department of Psychiatry, Sheba Medical Center, Tel Hashomer, Israel
PREFACE
Neuropsychopharmacology: The Fifth Generation of Progress appears at an important moment in the history of psychopharmacology.
We have recently ended the decade of the brain, a decade that witnessed enormous progress in understanding fundamental
physiology of the central nervous system. The fruits of these basic science discoveries have already resulted in important progress in
the treatment of mental illness. The importance of these fundamental discoveries has recently been acknowledged by the awarding
of the Nobel Prize in Psychology or Medicine to three members of the College, Arvid Carlsson, Paul Greengard and Eric Kandel for
their discoveries on neuronal signaling. This edition in the Generation of Progress series details advances in both the basic science
and clinical application of recent research in psychopharmacology. It also demonstrates the prospects for even greater advances in
the future.

Charles P. O'Brien M.D., Ph.D.

ACNP President, 2001

Joseph T. Coyle M.D.

ACNP President, 2002

 Contents
EDITORS

PREFACE

Contents

Section I Neurotransmitters and Signal Transduction

Neurotransmitters and Signal Transduction - Introduction

1 Acetylcholine

2 Serotonin

3 Opioid Peptides and Their Receptors: Overview and Function in Pain Modulation

4 Norepinephrine

5 Regulation of G Protein-Coupled Receptors by Phosphorylation and Endocytosis

6 The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction

7 Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders

8 Neurogenesis in Adult Brain

9 Dopamine

10 Astrocytes

11 Synaptic Plasticity

12 Gaba

13 Substance P and Related Tachykinins

14 Histamine

15 Purinergic Neurotransmission

16 Neurotrophic Factors and Intracellular Signal Transduction Pathways

17 Regulation of Gene Expression

Section II Emerging Methods in Molecular Biology and Genetics

Emerging Methods in Molecular Biology and Genetics - Introduction

18 Using Human Genomics to Advance Neuropsychopharmacology

19 Gene Targeting and Transgenic Technologies

20 Gene Delivery into the Brain Using Viral Vectors

21 Neuropsychopharmacology of Worms and Flies

 22 Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors

23 Applying Functional Genomics to Neuropsychopharmacology

Section III Emerging Imaging Technologies and Their Application to Psychiatric Research

Emerging Imaging Technologies and Their Application to Psychiatric Research - Introduction

24 Automated 3D Analysis of Large Brain Mri Databases

25 In Vivo Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and

Functional Neuroenergetics

26 The Spatial, Temporal, and Interpretive Limits of Functional MRI

27 Diffusion Tensor Imaging

28 Activation Paradigms in Affective and Cognitive Neuroscience: Probing the Neuronal Circuitry Underlying Mood and

Anxiety Disorders

29 Interactions Among Neuronal Systems Assessed with Functional Neuroimaging

30 Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation

31 In Vivo Molecular Imaging: Ligand Development and Research Applications

32 Event-Related Potentials and Magnetic Fields in the Human Brain

Section IV Drug Discovery and Evaluation

Drug Discovery and Evaluation - Introduction

33 The Role of Preclinical Models in the Development of Psychotropic Drugs

34 Proof of Concept: Functional Models for Drug Development in Humans

35 Ethical Aspects of Neuropsychiatric Research with Human Subjects

36 Regulatory Issues

37 Pharmacogenomics and Personalized Therapeutics in Psychiatry

38 Pharmacokinetics, Pharmacodynamics, and Drug Disposition

39 The Role of Pharmaceuticals in Mental Health Care Outcomes

40 Issues in Clinical Trial Designs

Section V Disorders of Development

Disorders of Development - Introduction

41 The Molecular and Cellular Genetics of Autism

42 Current and Emerging Therapeutics of Autistic Disorder and Related Pervasive Developmental Disorders

43 Pathophysiology of Attention-Deficit/Hyperactivity Disorder

 44 Learning Disorders

45 Psychosis in Childhood and Its Management

46 Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain

Section VI Schizophrenia and related disorders

Schizophrenia and related disorders - Introduction

47 Schizophrenia: Course Over the Lifetime

48 Neurocognitive Functioning in Patients with Schizophrenia: An Overview

49 Molecular and Population Genetics of Schizophrenia

50 Animal Models Relevant to Schizophrenia Disorders

51 Endophenotypes in Studies of the Genetics of Schizophrenia

52 Neurochemistry of Schizophrenia: Glutamatergic Abnormalities

53 Neural Circuitry Approaches to Understanding the Pathophysiology of Schizophrenia

54 Functional Neuroimaging in Schizophrenia

55 Structural Magnetic Resonance Imaging Studies in Schizophrenia

56 Therapeutics of Schizophrenia

57 The Economics of the Treatment of Schizophrenia

58 Mechanism of Action of Atypical Antipsychotic Drugs

59 Neurochemical and Neuropharmacological Imaging in Schizophrenia

Section VII Anxiety and stress disorder

Anxiety and stress disorder - Introduction

60 Anxiety and Stress Disorders: Course Over the Lifetime

61 Genetic and other Vulnerability Factors for Anxiety and Stress Disorders

62 Animal Models and Endophenotypes of Anxiety and Stress Disorders

63 Neurobiological Basis of Anxiety Disorders

64 Neural Circuitry of Anxiety and Stress Disorders

65 Structural and Functional Imaging of Anxiety and Stress Disorders

66 Current and Emerging Therapeutics of Anxiety and Stress Disorders

67 The Economic Burden of Anxiety and Stress Disorders

68 Mechanism of Action of Anxiolytics

Section VIII Affective disorders

Affective disorders - Introduction

69 The Course of Depression

70 Risk Factors for Major Depression and Bipolar Disorder

71 Bipolar Disorders: Review of Molecular Genetic Linkage Studies

72 Molecular and Cellular Mechanisms in Depression

73 Neurocircuitry of Mood Disorders

74 Structural and Functional Imaging of Affective Disorders

75 Current and Emerging Therapeutics for Depression

76 Electroconvulsive Therapy: Sixty Years of Progress and a Comparison with Transcranial Magnetic Stimulation and

Vagal Nerve Stimulation

77 Current and Emerging Treatments for Acute Mania and Long-Term Prophylaxis for Bipolar Disorder

78 Cost-Effectiveness of the Newer Generation of Antidepressants

79 Mechanism of Action of Antidepressants and Mood Stabilizers

80 Hormonal and Gender Influences on mood Regulation

81 Depression and the Medically Ill

Section IX Alzheimer and other dementias

Alzheimer and other dementias - Introduction

82 Alzheimer Disease: From Earliest Symptoms to End Stage

83 Molecular Genetics of Alzheimer Disease

84 Transgenic Mouse Models of Alzheimer Disease

85 Cell and Molecular Neuropathology of Alzheimer Disease

86 Structural and Functional Brain Imaging of Alzheimer Disease

87 Current and Experimental Therapeutics of Alzheimer Disease

88 Alzheimer Disease: Treatment of Noncognitive Behavioral Abnormalities

89 Cost-Effectiveness of Therapeutics for Alzheimer Disease

90 Neuropsychiatric Manifestations of Hiv-1 Infection and Aids

91 Dementia with Lewy Bodies

92 Molecular Pathophysiology of Stroke

93 Current and Experimental Treatment of Stroke

 94 Tau Protein and Tauopathy

Section X Substance Use Disorders

Substance Use Disorders - Introduction

95 Neurocircuitry of Addiction

96 Molecular and Cellular Biology of Addiction

97 Recent Advances in Animal Models of Drug Addiction

98 Vulnerability Factors for Alcoholism

99 Molecular and Cellular Genetics of Alcohol Addiction

100 Ethanol Abuse, Dependence, and Withdrawal: Neurobiology and Clinical Implications

101 Alcoholism Pharmacotherapy

102 Pathophysiology and Treatment of Cocaine Dependence

103 Application of Imaging Technologies in the Investigation of Drug Addiction

104 Molecular and Cellular Neurobiology and Pathophysiology of Opiate Addiction

105 Current and Experimental Therapeutics for the Treatment of Opioid Addiction

106 Marijuana

107 Therapeutics for Nicotine Addiction

108 Psychedelic Drugs

109 Epidemiology of Drug Dependence

110 Neuroimaging of Cocaine Craving States: Cessation, Stimulant Administration, and Drug Cue Paradigms

Section XI Impulsive and Compulsive Disorders

Impulsive and Compulsive Disorders - Introduction

111 The Course and Clinical Features of Obsessive-Compulsive Disorder

112 The Pathophysiology and Genetics of Obsessive-Compulsive Disorder

113 Imaging and Neurocircuitry of OCD

114 Current and Experimental Therapeutics of Obsessive-Compulsive Disorder

115 The Behavioral Neuroscience of Eating

116 Psychopharmacology of Eating Disorders

117 Tourette Syndrome and Related Tic Disorders

118 Animal Models of Aggression

 119 Pathophysiology and Treatment of Aggression

120 Pathologic Gambling and Impulse Control Disorders

121 Compulsive and Impulsive Aspects of Self-Injurious Behavior

Section XII Movement Disorders and Proxysmal Disorders

Movement Disorders and Proxysmal Disorders - Introduction

122 Neurocircuitry of Parkinson’s Disease

123 Pathophysiology of Parkinson's Disease

124 Current and Experimental Therapeutics of Parkinson's Disease

125 Huntington Disease

126 Clinical Course and Cellular Pathology of Tardive Dyskinesia

127 Temporal Lobe Epilepsy: Renewed Emphasis on Extrahippocampal Areas

Section XIII Chronobiology and Sleep Disorders

Chronobiology and Sleep Disorders - Introduction

128 Basic Mechanisms of Sleep: New Evidence on the Neuroanatomy and Neuromodulation of the NREM-REM Cycle

129 Circadian Phase Sleep and Mood Disorders

130 Sleep Loss and Sleepiness: Physiological and Neurobehavioral Effects

131 Pathophysiological and Pharmacologic Aspects of the Sleep Disorder Narcolepsy

132 Basic Mechanisms of Sedative/Hypnotics

133 Current and Experimental Therapeutics of Insomnia

134 Sleep Disturbances Associated with Neuropsychiatric Disease

Color Plates

INDEX
 P.1

Section I
Neurotransmitters and Signal Transduction

Eric J. Nestler

Ronald S. Duman

Neurotransmitters and Signal Transduction - Introduction

Neuropsychopharmacology continues to be organized primarily according to the neurotransmitters that are utilized by various
populations of neurons for synaptic transmission. This is because the vast majority of psychotropic drugs presently used clinically to
treat neuropsychiatric disorders still have as their initial targets proteins that regulate the availability of a particular
neurotransmitter (e.g., presynaptic reuptake transporters, synthetic or degradative enzymes) or that serve as ligands for particular
neurotransmitter receptors. It is entirely appropriate then that this edition of Neuropsychopharmacology: A Generation of Progress
begins with a section devoted to the major neurotransmitter systems in the brain. Rather than provide a comprehensive review of
the now vast literature on neurotransmitter systems, the goal of this section is to highlight recent advances in the field.

Glutamate, as described in the chapter by Joseph Coyle, Michael Leski, and John Morrison, is the major excitatory neurotransmitter
in the brain. During the past decade, numerous subtypes of glutamate transporters and glutamate receptors have been identified
and characterized. Each of these represents a potentially exciting target for new pharmacotherapeutic agents. Richard Olsen
focuses on γ-aminobutyric acid (GABA), which serves as the major inhibitory neurotransmitter in the brain. GABA receptors and
GABA transporters are important targets for commonly used antianxiety, anticonvulsant, and antimanic medications. Agents with
improved specificity toward subtypes of these proteins may offer substantial benefit as future treatments.

The next several chapters focus on other small-molecule neurotransmitters, which are used by relatively small fractions of neurons
and generally serve to modulate the efficacy of glutamatergic and GABAergic synapses through diffuse projections throughout the
neuraxis. Such neurotransmitters include the catecholamines, norepinephrine, and dopamine. Norepinephrine, covered in a chapter
by Gary Aston-Jones, regulates mood, attention, and alertness and is a substrate for many commonly used antidepressants.
Dopamine, discussed in a chapter by Anthony Grace, plays a critical role in movement and reward. Accordingly, it is involved in
movement disorders such as Parkinson's disease and is a common target for most drugs of abuse. The catecholamines, along with
serotonin and histamine, are often referred to as monoamine neurotransmitters because they contain a single amine group.
Serotonin is critically involved in many brain functions and is the target for many commonly used antidepressants. George
Aghajanian and Elaine Sanders-Bush focus on new findings about serotonin, including the discovery and characterization of 14
distinct serotonin receptors and their physiologic functions. Histamine is discussed by Jean-Charles Schwartz and Jean-Michel Arrang.
Although it has been known for some time that histamine regulates alertness and sleep, new advances in histamine pharmacology
have been made possible by the cloning of three distinct histamine receptors. Finally, acetylcholine is often categorized along with
the monoamines because it too is concentrated in discrete regions of the brain, many of which project diffusely to other parts of the
brain. A major goal of neuropsychopharmacology research, as discussed by Marina Picciotto, Meenakashi Alreja, and J. David Jentsch,
continues to be the development of drugs that are selective for the many subtypes of cholinergic receptors expressed in the central
nervous system.

Many other types of molecules serve neurotransmitter functions. Michael Williams covers the so-called purinergic neurotransmitters,
which include adenosine and adenosine triphosphate. The last few years have seen the cloning and characterization of a vast
number of purinergic receptors,
 P.2

with very different transmitter selectivities and functional properties. It is believed that selective ligands at these various receptors
may serve as novel drugs in the treatment of Parkinson's disease, insomnia, anxiety, and pain, to name a few. Many types of
polypeptides serve as neurotransmitters; these molecules are often termed neuropeptides. Significant recent progress has been
made in understanding the physiologic role and pharmacology of certain neuropeptides, which are discussed in several chapters in
this section. Gavan McNally and Huda Akil cover the opioid peptides, including a newly discovered opioid-like peptide, termed
orphanin-FQ or nociceptin, that promotes nociception. Errol De Souza and Dimitri Grigoriadis review recent advances in the
understanding of corticotropin-releasing factor, including the identification of two main types of receptors for corticotropin-
releasing factor and other peptides (e.g., urocortin) that serve as endogenous ligands for the receptors. Nadia Rupniak and Mark
Kramer focus on substance P and related neurokinins. Long known to be involved in the regulation of pain perception, recent
evidence suggests that antagonists at certain neurokinin receptors may be effective antidepressants.

Despite the importance of neurotransmitter systems in neuropsychopharmacology, it must be emphasized that all the proteins that
account for neurotransmitter synthesis and degradation, reuptake, and receptors, and for neuropeptide transmitters themselves,
represent a small fraction of the perhaps hundreds of thousands of proteins expressed in the adult brain. A central promise of
neuropsychopharmacology as we enter a new century is to evaluate these vast arrays of other proteins as targets for entirely new
families of pharmacotherapeutic agents. Robert Malenka reviews what we know about synaptic plasticity, the processes by which
the efficacy of transmission at particular synapses is altered as a consequence of synaptic activity. Mark von Zastrow covers the
molecular and cellular mechanisms underlying receptor internalization, a process in which the numbers of many and perhaps most
types of neurotransmitter receptors on the plasma membrane are regulated by synaptic activity. David Russell and Ronald Duman
offer an overview of neurotrophic factors and their signaling pathways. Neurotrophic factors have long been recognized for their
role in neural growth and differentiation during development, and we now know they are also important for regulating the survival
and plasticity of adult neurons. Eric Nestler and Steven Hyman review the intracellular signaling pathways by which
neurotransmitters, acting on plasma membrane receptors, regulate gene expression. Such regulation represents a prominent
mechanism of long-term plasticity in the nervous system, including the actions of repeated exposure to psychotropic drugs (e.g.,
antidepressant action and drug addiction). Pierre Magistretti and Bruce Ransom discuss the role of glial cells in the central nervous
system—in particular, their control of the energy metabolism in the brain. Finally, Fred Gage and Henriette van Praag summarize
new knowledge of neurogenesis in the adult brain. The recent discovery that new neurons are born in certain regions of the brain
each day, and may be incorporated into the existing circuitry within those regions, raises new hope for the treatment of
neurodegenerative and other neuropsychiatric disorders. The subject matter of these last several chapters has not yet been
exploited pharmacologically, but it is believed that the next generation of progress will see new pharmacologic agents directed at
these nontraditional mechanisms.
 P.3

1
Acetylcholine
Marina R. Picciotto
Meenakshi Alreja
J. David Jentsch
Marina R. Picciotto and Meenakshi Alreja: Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut.
J. David Jentsch: Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania.
Acetylcholine (ACh) is critical for communication between neurons and muscle at the neuromuscular junction, is involved in direct neurotransmission in autonomic ganglia,
and has been implicated in cognitive processing, arousal, and attention in the brain (1 ). Cholinergic transmission can occur through muscarinic (G protein-coupled) or
nicotinic (ionotropic) receptors and is terminated by the action of cholinesterases. Seventeen different subunits of the nicotinic ACh receptor (nAChR) (2 ) and five
different subtypes of the muscarinic receptor (3 ) have been cloned to date, and a majority of those are known to be expressed in the brain. Although the anatomic
locations of cholinergic cell bodies and their projections have been known for some time (Fig. 1.1 ), recent studies using specific cholinotoxins, electrophysiology, or
molecular genetics have altered our view of the functional role of the cholinergic system in the brain. The anatomic, pharmacologic, and biochemical complexity of the
cholinergic system indicates an intricate involvement in nervous system function, and new advances in this field are discussed here.

FIGURE 1.1. Anatomy of major cholinergic pathways in the brain.

The principal source of cholinergic input to the cortex and
hippocampus is the basal forebrain complex, whereas the
pedunculopontine and laterodorsal tegmental areas innervate
brain stem and midbrain targets preferentially. Cholinergic
interneurons are found in the olfactory tubercle, striatum,
nucleus accumbens, and islands of Calleja. BFC, basal forebrain
complex; VTA, ventral tegmental area; IPN, interpeduncular
nucleus; PPT, pedunculopontine tegmental nucleus; LDT,
laterodorsal tegmental nucleus.

 KNOCKOUT OF MUSCARINIC- AND NICOTINIC-RECEPTOR SUBUNITS

 ROLE FOR CHOLINERGIC NEURONS IN AROUSAL AND SLEEP
 ROLE FOR CHOLINERGIC NEURONS IN MOTIVATION AND REWARD
 ROLE FOR CHOLINERGIC NEURONS IN COGNITIVE PROCESSES
 ROLE FOR CHOLINERGIC NEURONS IN STIMULUS PROCESSING
 CONCLUSIONS
 SUMMARY
 ACKNOWLEDGMENTS

KNOCKOUT OF MUSCARINIC- AND NICOTINIC-RECEPTOR SUBUNITS

Part of "1 - Acetylcholine "
A particularly useful tool in identifying the role of individual molecules in the physiologic and behavioral functions of the cholinergic system are transgenic animals
that lack specific subunits or subtypes of muscarinic receptors or nAChRs. These animals, termed “knockout” mice, can be generated by means of genetic
engineering techniques and have been extremely useful in determining the functional role of many proteins that have been identified through molecular cloning (see
refs. 4 and 5 for a review of this technology). Mice lacking the α3 (6 ), α4 (7 ), α5 (8 ), α7 (9 ), α9 (10 ), β2 (11 ), β3 (12 ), or β4 (13 ) subunit of the nAChR have
been reported. In addition, mice lacking the M1 (14 ), M2 (15 ), and M4 (16 ) subtypes of the muscarinic receptor have been generated. These mice have already
been used to demonstrate the role of particular receptor subtypes in the physiologic effects of ACh in muscle, the peripheral ganglia, and the central nervous system
(Table 1.1 ).

TABLE 1.1. KNOCKOUTS OF MUSCARINIC AND NICOTINIC

SUBUNITS

The function of ACh has been best studied at the neuromuscular junction, where signaling occurs through the muscle form of the nAChR. In the embryo, the nAChR at the
neuromuscular junction is a pentamer made up of two α, one β, one γ, and one δ subunit. After birth, the γ subunit is replaced by the ε subunit, so that the physiologic
properties of the receptor are altered. In mice in which the ε subunit has been knocked out, the neuromuscular junction nAChRs remain in the embryonic form; the
consequence is survival past birth with progressive muscle degeneration and lethality by 2 to 3 months of age (17 ). These experiments demonstrate that maturation of the
neuromuscular junction nAChR is necessary for muscle cell function and survival and imply that the kinetics of ACh neurotransmission are critical for the health of muscle fibers
in adulthood.
Cholinergic neurotransmission within the sympathetic ganglia occurs through several receptor subtypes. In the peripheral nervous system, the issue of which ACh-receptor
subtypes are involved in cholinergic neurotransmission has been addressed both by knocking out muscarinic and nicotinic subunits and by treating sympathetic neurons from
isolated chick sympathetic ganglia with antisense oligonucleotides (short stretches of DNA that can inhibit the translation of a particular protein of interest) to decrease the
expression of α3, α4, and α7 nAChR subunits. Antisense experiments have indicated that the α3 nAChR subunit plays a primary role in nicotinic transmission in sympathetic
ganglia and that the α7 subunit also contributes to the observed currents (18 ,19 ). These data are in agreement with electrophysiologic and immunoprecipitation studies of
nAChR subunits from ganglionic neurons (20 ). Although the results of studies using this powerful technique are compelling,
 P.4

some problems have been noted with antisense approaches, including issues of specificity, so that it is useful to complement these
studies with other techniques that can be used to manipulate levels of nAChR subunits.

In studies of knockout mice, disruptions of two nicotinic-receptor subunits expressed in sympathetic ganglia, α7 (9 ) and β2 (11 ), do
not grossly alter ganglionic function. In contrast, if the β2 and the β4 nAChR-subunit mutations are combined (13 ), or if the α3
nAChR subunit is knocked out (6 ), mutant mice die perinatally of severe autonomic failure. These experiments suggest that a
nicotinic cholinergic receptor composed of the α3/β4 or β2 subunit, or both, is responsible for mediating direct neurotransmission by
ACh between ganglionic neurons.

Muscarinic function has also been studied in the autonomic ganglia with knockout technology. Mutation of the M1 muscarinic
receptor is sufficient to abolish the M current, a muscarine-mediated potassium current, in the sympathetic ganglia, but M1
mutation does not significantly perturb ganglionic function (14 ). In contrast, mice lacking
 P.5

the M2 muscarinic-receptor subtype do not show carbachol-induced bradycardia, confirming that the effect of ACh on sympathetic
control of heart rate is mediated through the M2 receptor (15 ). In addition, knockout animals have been very useful in determining
which subtypes of muscarinic receptors are responsible for the effects of ACh on modulation of calcium channels in sympathetic
neurons (21 ). A slow, voltage-independent modulation is mediated by M1 receptors, whereas a fast, voltage-dependent modulation
is mediated through M2, and neither is affected in M4 knockout mice.

The function of ACh in the brain has also been examined in electrophysiologic experiments with mice lacking cholinergic-receptor
subtypes. For example, a rapidly desensitizing nicotinic current in the hippocampus is mediated through an α7-containing receptor
(9 ). Mice lacking the α7 subunit appear grossly normal in behavioral experiments (22 ), but future experiments should determine
whether these currents contribute to nicotine-induced improvements in cognitive function or to nicotine-induced seizure activity.
Antisense experiments have also demonstrated that the α5 nAChR subunit can alter the electrophysiologic properties of nAChRs
containing the α4 and β2 subunits in vivo (23 ). Mice lacking the β2 subunit have been used to characterize four classes of nAChR in
the brain by means of pharmacologic and electrophysiologic techniques (24 ) and to extend the existing pharmacologic
characterization of nicotinic-receptor subtypes (Fig. 1.2 ). Future experiments using mice lacking individual nAChR α subunits should
allow a finer definition of these receptor classes.

FIGURE 1.2. Nicotinic ligand binding in brain slices from wild-type and β2-subunit knockout mice. Mice lacking individual
subunits of the nicotinic acetylcholine receptor (nAChR) can be used to distinguish between subclasses of receptors. For
example, although β2-subunit knockout mice lack the highest-affinity subclass of nicotine binding sites, the frog toxin
epibatidine, shown here, still reveals β4 subunit-containing nAChRs in the medial habenula (remaining binding shown in panel
at top, far right). Binding of epibatidine in brain slices through thalamus (top) or striatum (bottom) is shown in wild-type
heterozygous (β2 +/-) and homozygous (β2 -/-) β2-subunit knockout mice.

A significant development in thinking about nicotinic-receptor function has been the idea that nicotine exerts many of its functions
in brain through the regulation of neurotransmitter release, at least partly through terminal and preterminal nAChRs (25 ,26 ).
Experiments on synaptosomes (nerve terminals) isolated from mice lacking the β2 subunit of the nAChR have shown that presynaptic
regulation of GABA release by nicotine is mediated through β2 subunit-containing receptors in most brain areas (27 ). This is also
likely to be the case for other neurotransmitters because the efflux of rubidium, a radioactive tracer that serves as a marker of
neurotransmitter vesicle fusion, is mediated through β2 subunit-containing receptors in most brain areas (28 ). More recently,
dopamine release from striatal synaptosomes has been shown to be disrupted in β2-subunit knockout mice, while ACh release in the
interpeduncular nucleus is preserved (29 ). This suggests that a distinct nAChR subtype, most likely containing the β4 subunit,
mediates nicotine-elicited ACh release in the interpeduncular nucleus.

Systems-level function and behavioral effects of ACh have also been examined in knockout mice. Muscarinic agonist-induced seizures
are dependent on the presence of the M1 receptor because M1 knockout mice are resistant to pilocarpine-induced seizure activity
(14 ). Interestingly, although the M1 receptor has been implicated in the modulation
 P.6

of potassium channels in the hippocampus in pharmacologic experiments, muscarinic modulation of potassium channels is unchanged
in the hippocampus of M1 knockout mice (30 ). In contrast, the pharmacologic effects of muscarinic agonists on movement,
temperature control, and antinociception appear to be mediated through the M2 receptor because these responses are absent in M2
knockout mice (15 ). M4 receptors are also involved in locomotion; these knockout animals exhibit increased basal locomotor activity
and a potentiated locomotor response to D1-selective dopaminergic agonists (16 ).

Like the M2 receptors, the α4/β2 subtype of nAChR is implicated in antinociceptive cholinergic pathways. Mice lacking either of
these subunits show decreased nicotine-induced analgesia (7 ). In behavioral experiments, the β2 nicotinic subunit mediates the
ability of nicotine to improve avoidance learning and may also be involved in the circuitry underlying this form of associative
learning in wild-type mice (11 ). In addition, this subunit appears to be necessary for the mouse to experience the reinforcing
properties of nicotine because animals without the β2 subunit will not self-administer nicotine (31 ). Extensions of these
experiments to mice lacking other subunits of the nicotinic receptor should allow identification of the receptor subtypes that are
activated by smoking in humans and result in tobacco addiction. An interesting effect of ACh on neuronal survival was demonstrated
in mice lacking the β2 nAChR subunit (32 ). Mice that lack this cholinergic-receptor subtype show progressive neuronal loss with age
in cortical and hippocampal brain areas, which appears to lead to age-related impairments in spatial learning. These experiments
demonstrate that the effects of ACh on cognition, antinociception, locomotion, and overall neuronal activity are differentially
mediated through the various subtypes of muscarinic and nicotinic receptors, and that the various roles of ACh may be separated
pharmacologically, suggesting new targets for rational drug design.

ROLE FOR CHOLINERGIC NEURONS IN AROUSAL AND SLEEP

Part of "1 - Acetylcholine "

Traditionally, the basal forebrain complex, the primary source of cholinergic innervation to the telencephalon (Fig. 1.1 ), was
thought to be involved in arousal or sleep regulation. Either lesions or electric stimulation of subregions of the basal forebrain can
facilitate sleep and synchronize the EEG, and cholinergic drugs regulate EEG synchrony (33 ). Moreover, a correlation between
cortical ACh release and the state of behavioral activation or sleep has been observed in rodents. Thus, it was hypothesized that
cholinergic input to the neocortex from the basal forebrain is critical for regulating arousal (see ref. 34 for review).

The pontomesencephalic tegmentum is also critical for the sleep–wake cycle. These neurons largely do not innervate the neocortex
but project to the diencephalon (thalamus) and the basal forebrain complex. Stimulation of tegmental brainstem cholinergic
neurons can evoke cortical ACh release and EEG desynchrony, and these effects are blocked by reversibly decreasing the activity of
the basal forebrain (35 ). Moreover, application of cholinergic agonists to the basal forebrain produces behavioral activation and EEG
desynchrony (33 ). Although the brainstem cholinergic projections to the thalamus undoubtedly also contribute to EEG regulation
(36 ), these findings suggest that cholinergic projections to the basal forebrain from the pontomesencephalic tegmentum regulate
behavioral arousal.

It was subsequently noted that cholinergic tegmental projections largely formed connections with noncholinergic neurons within the
basal forebrain (37 ). This finding is critical because it could explain why stimulation of the horizontal diagonal band, preoptic area,
and substantia innominata, but not of the septal nucleus and nucleus basalis, produces sleep in the cat (33 ). The ratio of cholinergic
to noncholinergic neurons in the horizontal diagonal band, preoptic area, and substantia innominata is significantly lower than in the
septum and nucleus basalis. This observation has led to the hypothesis that activation of primarily noncholinergic neurons is
responsible for producing sleep after basal forebrain stimulation (33 ). These noncholinergic neurons are believed to be GABAergic
and achieve their effects through inhibition of cholinergic basal forebrain neurons and neurons within the brainstem reticular
formation. In contrast, stimulation of the nucleus basalis or septal nucleus produces behavioral activation and cortical ACh release,
and this is consistent with the notion that basal forebrain cholinergic neurons are involved in behavioral arousal (activation),
whereas noncholinergic basal forebrain neurons are involved in regulating the sleep state. These two effects are related (sleep vs.
arousal), but the qualitative contributions of the GABA and cholinergic systems to sleep and arousal are opposed.

ROLE FOR CHOLINERGIC NEURONS IN MOTIVATION AND REWARD

Part of "1 - Acetylcholine "

Cholinergic neurons have also been implicated in motivation and reward. The strongest evidence for the hypothesis that nAChRs are
involved in motivation and reward is that nicotine is abused by humans and is reinforcing in animals (see ref. 38 for review). The
effects of nicotine on tests of reinforcement and behavioral sensitization are primarily mediated through the mesolimbic dopamine
system (39 ). Indeed, the ventral tegmental area (VTA) may be sufficient to mediate the reinforcing properties of nicotine, as local
injection of nicotine or nicotinic agonists into the VTA can result in increased locomotion (40 ) or conditioned place preference (41 ).
 P.7

Basal forebrain cholinergic neurons may also be involved in modulating cortical processing of stimuli with conditioned or
unconditioned rewarding properties because these neurons are more responsive to stimuli with a high incentive value. Novel stimuli
that typically elicit orienting responses and attention in animals increase cortical ACh release, but this effect is diminished with
repeated exposure if the stimulus has no contingent incentive valence. In contrast, if the stimulus is repeatedly paired with an
incentive stimulus (e.g., food or foot shock), the (now-conditioned) stimulus can evoke ACh release even after multiple exposures
(42 ). These sorts of changes are reflected in the firing of “reinforcement-related” neurons within the primate basal forebrain (43 ).
Pontomesencephalic cholinergic neurons are also involved in motivation and reward, although these effects are likely mediated, in
part, by projections to the dopamine neurons within the VTA (44 ,45 ).

Stimulation of the VTA by the pedunculopontine tegmental nucleus (PPT) enhances mesostriatal dopamine transmission (45 ,46 ).
While a significant proportion of the PPT neurons that project to the tegmental dopamine neurons are noncholinergic (44 ), the
cholinergic input per se appears to stimulate dopamine neurons (47 ). Thus, ascending projections from the PPT to the dopamine
cells may regulate the ability of mesostriatal dopamine neurons to affect incentive/motivational processes.

This innervation of dopamine cells by cholinergic neurons may explain the finding that lesions of the PPT can modulate the
rewarding qualities of addictive drugs. Lesions of the PPT reduce the self-administration of nicotine (48 ) and opiates (49 ).
Moreover, conditioned place preference for food, opiates (50 ), morphine (51 ), and amphetamine (52 ) is blocked or reduced by PPT
lesions, whereas cocaine-induced reward is unaffected (53 ). Although the mesolimbic dopamine pathway is known to be involved in
drug reward (see ref. 54 for review), it is not yet known whether the influence of the PPT on drug reinforcement is through
cholinergic projections. It is also not known whether the effect of PPT lesions on these processes is mediated through projections to
areas other than the dopamine cell groups within the VTA.

The PPT may have another, more critical, role in motivation and reward via afferent inputs from the striatum (55 ). Excitotoxic
lesions of the PPT (that equivalently destroy both cholinergic and noncholinergic neurons) disrupt responding for conditioned
reinforcement and augment stimulant-induced orofacial stereotypy, yet no difference is observed in stimulant-induced locomotion
or other measures of food consumption (42 ,56 ). These data may implicate the PPT (and its innervation from the striatum) in
response selection when discrimination is involved because the disruption of responding for conditioned reinforcement resulted from
decreased discrimination of response between a lever associated with reinforcement and an inactive lever (56 ). However, a recent
study found that although PPT lesions increased sucrose consumption, similar lesions did not affect discrimination or contrast effects
(57 ). Nevertheless, the hypothesis of Winn (58 ) is that lesions of the PPT affect responding for rewarding stimuli similarly to lesions
of the frontal cortex, so that the role of the PPT, like that of the basal forebrain, is expanded into higher-order cognitive processes.

ROLE FOR CHOLINERGIC NEURONS IN COGNITIVE PROCESSES

Part of "1 - Acetylcholine "

Lesion Studies
The hypothesis of cholinergic involvement in learning and memory processes arose from several findings. Both destruction of the
basal forebrain complex and the administration of cholinergic antagonists produce profound deficits in a variety of forms of
cognition, including learning and memory (59 ,60 ).

The original finding that lesions of the basal forebrain could produce deficits in a variety of cognitive tasks suggested a role for ACh
in cognitive function. Electrolytic, radiofrequency, or nonspecific excitotoxic lesions of cholinergic subnuclei within the basal
forebrain (particularly the medial septum/diagonal band) profoundly impair performance on a variety of tests of learning, memory,
and attention, particularly the Morris water maze and passive avoidance learning (see ref. 59 for review). These deficits appeared
to be reversed following regeneration of cholinergic projections across a bridging graft (61 ) or after grafting of ACh-producing cells
in the hippocampus (62 ). These findings have been interpreted as support for the hypothesis of cholinergic involvement in cognitive
functions; however (as with arousal and sleep), noncholinergic neurons within the basal forebrain may likewise be involved in these
effects, and more specific approaches must be employed to address these issues.

Novel approaches for selectively destroying cholinergic neurons depend on the differential sensitivity of basal forebrain neurons to
excitotoxins and new types of immunotoxins. Systematic studies have demonstrated that cholinergic and noncholinergic neurons
within the basal forebrain are differentially sensitive to excitotoxic amino acids such as quisqualate, α-amino-3-hydroxy-5-methyl-4-
isoxazole propionic acid (AMPA) (Fig. 1.3 ), kainate, and N-methyl-D-aspartate (NMDA) (59 ). Based on the results of these studies,
new methods for preferentially destroying cholinergic neurons have been described (63 ). Moreover, an IgG–saporin toxin has been
developed that takes advantage of the fact that basal forebrain cholinergic neurons are particularly enriched with low-affinity
receptors for nerve growth factor (64 ). The toxin selectively binds to the receptor for nerve growth factor and then kills the neuron
expressing the receptor. More excitingly, recent studies suggest that IgG–saporin can be used to destroy the cholinergic innervation
of
 P.8

terminal regions into which the toxin is injected (65 ). These methods have been applied to studies of learning and memory in an
attempt to qualify earlier findings.

FIGURE 1.3. Acetylcholinesterase staining of the nucleus basalis magnocellularis after infusion of saline solution or AMPA to
destroy cholinergic neurons preferentially. Low concentrations of the glutamatergic agonist AMPA selectively destroy
cholinergic neurons (measured by acetylcholinesterase staining) and spare γ-aminobutyric acid (GABA) neurons (left). In
contrast, control sections show robust acetylcholinesterase staining after infusion of saline solution (right). This process allows
more specific cholinergic lesions to be generated, so that the function of the neurons in behavioral processes can be clarified.
(Courtesy of Professor Barry J. Everitt, University of Cambridge.)

Based on either excitotoxic or saporin lesions of the basal forebrain, the hypothesis for cholinergic function has been revised
considerably. Essentially, selective damage to cholinergic neurons of the basal forebrain has failed to produce the retrograde or
anterograde amnesia or deficits in learning that have been reported to result from nonspecific lesions of the basal forebrain
(59 ,66 ). Previously, the medial septal/diagonal band nuclei and their projections to posterior cortical regions were thought to be
critical for spatial learning and contextual conditioning (59 ). By means of saporin lesions, however, cholinergic depletion within the
hippocampus or posterior parietal cortex has been shown to result in impairments in latent inhibition or unblocking (65 ), with
sparing of spatial learning (67 ) and spatial working memory (68 ). Moreover, selective excitotoxic lesions of the medial
septum/diagonal band produce enhancements in contextual conditioning but impairments in discrete cue (trace) conditioning (69 ).
Both sets of data may suggest that the attentional processing of discrete stimuli is disrupted following cholinergic depletion from
posterior cortical regions. It is possible, however, that the depletion of ACh from caudal or rostral cortical regions alone may be
insufficient to impair performance of some tasks, whereas combined depletions may have more than additive effects (70 ).

Other investigators have further argued that the cholinergic innervation of rostral (e.g., frontal) cortex from the nucleus basalis is
also involved in attentional functions, such as vigilance or sustained, divided attention (59 ,71 ). Direct pharmacologic manipulation
of basal forebrain neurons has been used to alter activated cholinergic efflux in the frontal cortex and performance of tasks related
to stimulus processing or detection (72 ). Selective excitotoxic lesions or pharmacologic manipulation of the nucleus basalis has also
been reported to impair performance in a five-choice serial reaction task that requires animals to detect and respond to brief visual
stimuli (73 ). Interestingly, the observation that appetitive pavlovian learning for a discrete cue is enhanced after nucleus basalis
lesions (74 ) suggests that attentional processing of discrete cues may not be affected by depletion of ACh from the rostral
neocortex except when divided attention is required. The findings of these latter studies are also bolstered by advances in the
measurement of ACh transmission in vivo, which allows investigators to quantify directly the extent of the lesions produced by the
toxins for the first time (75 ). Taken together, the available data seem to suggest that basal forebrain cholinergic neurons are
capable of regulating the cortical processing of sensory stimuli within a variety of domains, which may be explained by a role for
basal forebrain ACh in the regulation of cortical processing.

Tegmental cholinergic neurons have also been implicated in cognitive processes (58 ,76 ). Although some of the effects of PPT
lesions on learning and memory may be related to generalized anxiety (76 ), PPT lesions also produce a set of behavioral deficits
that are consistent with executive dysfunction and impairments in frontal lobe functioning (58 ). In particular, PPT lesions result in
deficits of behavioral inhibition and motor perseveration. Notably, working memory performance does not seem to be affected by
destruction of the PPT (77 ). The position of the PPT as a modulator of dopaminergic systems (which affect frontal cortex function),
in addition to the influence of the frontal cortex on the PPT (mediated through the striatum), suggests that this nucleus is in an
excellent position to affect the functions of the frontostriatal system. Further research that attempts to control for the extent and
selectivity of PPT lesions is necessary.

Muscarinic Mechanisms
Although lesions of cholinergic nuclei have implicated ACh in various behavioral processes, it is also of interest to determine
 P.9

which cholinergic-receptor subtypes mediate these responses to ACh. Systemic infusions of the muscarinic-receptor antagonists
atropine and scopolamine produce an amnesic syndrome in humans (78 ), monkeys (79 ), and rats (80 ). Several lines of evidence
suggest that multiple central nervous system structures, including the medial septum/diagonal band region, are critical in mediating
the effects of muscarinic drugs on mnemonic functions (80 ). Infusions of muscarinic-receptor antagonists into a variety of cortical
regions, including the hippocampus, prefrontal cortex, and amygdala, can impair the cognitive functions associated with these
respective regions (81 ). Similarly, the effects of systemic muscarinic antagonists are attenuated by intraseptal injections of
muscarinic agonists, and intraseptal applications of muscarinic antagonists mimic the amnesic effects of systemic treatment with
muscarinic antagonists in experimental animals (82 ). These results suggest that activation of muscarinic receptors by ACh at
multiple forebrain sites, including within the somatodendritic regions of the cholinergic neurons, may be involved in the behavioral
dysfunction produced by muscarinic cholinergic antagonists.

Figure 1.4 presents the results of an experiment aimed at determining the relationship between in vivo cortical cholinergic
transmission and the cognitive effects of muscarinic-receptor antagonists. Scopolamine was administered systemically to rats
performing a test of working memory, the spatial delayed alternation task, both alone and in combination with FG7142, an
anxiogenic β-carboline that acts as an inverse agonist of the benzodiazepine site of the GABAA receptor. Consistent with previous
findings, scopolamine produced dose-dependent performance impairments when administered 45 minutes before testing on the
delayed alternation task, suggesting that decrements in cholinergic stimulation of muscarinic receptors result in cognitive
dysfunction. FG7142 (20 mg/kg) significantly elevated prefrontal cortical ACh release in vivo (measured in parallel studies), and
FG7142 on its own impaired delayed alternation performance. Interestingly, the fact that coadministration of FG7142 and
scopolamine did not affect the slope of the dose–response curve for scopolamine suggests that these two drugs act on different
mechanisms to impair delayed alternation performance. The additivity of these effects indicates that supranormal ACh transmission
produced by FG7142 likely does not contribute to the working memory deficits produced by this drug; moreover, the data indicate
that the impairments produced by scopolamine are independent of the level of ongoing cortical cholinergic transmission. Thus, it is
possible that the cognitive effects of muscarinic antagonists may not be solely the consequence of changes in cortical cholinergic
transmission.

FIGURE 1.4. The cognitive effects of scopolamine administration are insensitive to phasic changes in cortical acetylcholine
(ACh) release. Scopolamine dose-dependently impairs performance on a test of spatial working memory, the delayed
alternation task, in control rats and rats treated with FG7142, an inverse agonist of the benzodiazepine site of the γ-
aminobutyric acid subtype A (GABAA) receptor (left). Although FG7142 increases prefrontal cortical ACh release in vivo (right)
and produces performance deficits on its own (left), it does not alter the slope of the dose– response curve for scopolamine.

The septohippocampal pathway was first believed to convey only cholinergic fibers to the hippocampus (83 ); the noncholinergic,
GABAergic component was discovered almost two decades later (84 ). Work focusing on the GABA limb of the septohippocampal
GABA pathway has suggested that the septohippocampal GABA and cholinergic pathways may both be critical for the effects of
septal efferents on cognitive functioning (85 ). In support of this hypothesis, agents that increase impulse flow in the
septohippocampal GABA pathway, including muscarinic agonists, augment
 P.10

learning and memory (86 ), whereas agents that impair learning and memory decrease impulse flow in the GABA pathway (87 ).
Interestingly, impulse flow in the septohippocampal GABA pathway is maintained by ACh released via the tonic firing activity of
septohippocampal cholinergic neurons. This release occurs via local axon collaterals of septohippocampal neurons, which then
synapse on septohippocampal GABA neurons within the medial septum/diagonal band (Fig. 1.5 ). Thus, interaction between the
septohippocampal GABA pathway and muscarinic mechanisms within the medial septum/diagonal band may be crucial for learning
and memory (86 ).

FIGURE 1.5. Schematic representation of the septohippocampal pathway. The medial/septum diagonal band region is
composed primarily of cholinergic and GABAergic neurons, and the activity of both neuronal populations is regulated by locally
released γ-aminobutyric acid (GABA). The cholinergic neurons and a subpopulation of GABA neurons, containing the calcium-
binding protein parvalbumin (parv), project to the hippocampus via the fimbria/fornix. Muscarinic agonists may not increase
hippocampal acetylcholine release directly, but rather activate septohippocampal GABA neurons via M3 (and possibly M5)
receptors. Similarly, muscarinic antagonists disrupt impulse flow in the septohippocampal GABA pathway.

Cholinergic neurons, the primary source of ACh input to the hippocampus, innervate both the hippocampal pyramidal neurons and
subpopulations of GABAergic interneurons (88 ). In contrast, septohippocampal GABA neurons are very selective in their innervation
pattern, do not innervate the pyramidal cells at all, but innervate almost every type of hippocampal interneuron (89 ).
Septohippocampal GABA neurons are able to produce a powerful disinhibitory effect on pyramidal cells via this connectivity and so
enhance their excitability (90 ). Loss of cholinergic neurons severely disables the septohippocampal pathway by reducing both the
direct excitatory cholinergic drive and the indirect disinhibitory GABA drive to the hippocampus via locally released ACh. A
restoration of cholinergic function within the medial septum/diagonal band, not just in the hippocampus, could therefore be critical
for the treatment of cognitive deficits associated with the septohippocampal pathway.

At a molecular level, the excitatory effects of ACh on hippocampal pyramidal cells were at first thought to be mediated via the M1
subtype of muscarinic receptor, partly as a result of closing of M-type potassium channels, so that specific M1-receptor agonists
were developed. However, M1-receptor agonists were found to be of limited use in improving cognition. This might not be surprising
because studies of knockout mice lacking M1 receptors show no change in muscarinic enhancement of potassium currents in the
hippocampus (30 ). The finding that non-M1 receptors (M3 and possibly M5) mediate the effects of ACh in the medial
septum/diagonal band may further explain the limited effectiveness of M1 agonists in improving learning and memory functions and
supports the need for M3-selective agonists (85 ).

Nicotinic Mechanisms
Nicotinic systems are also involved in several important aspects of cognitive function, including attention, learning, and memory
(60 ). Nicotinic ACh receptors are expressed throughout the brain, including areas involved in cognitive function, such as the
hippocampus and frontal cortex (91 ). Nicotinic agonists improve performance on a variety of memory tasks, particularly following
lesions or aging, whereas nicotinic antagonists such as mecamylamine impair working memory function (60 ). The nAChR subtypes
involved in cognitive function are under investigation, and different subtypes may be involved in the performance of different
cognitive tasks. As mentioned above, experiments on knockout mice have implicated nAChRs containing the β2 subunit in both
passive avoidance learning (11 ) and maintenance of spatial learning during aging (32 ). Although the cellular basis for the effects of
nicotine are likely to be diverse, one site of action for nicotine, excitation of hippocampal GABAergic interneurons through both α7
and non-α7 subtypes of the nAChR, has been demonstrated by several groups (see ref. 92 for review). Further, although theta
rhythm in the hippocampus, a mechanism that appears to facilitate the induction of synaptic plasticity, is abolished by atropine
(93 ), it is converted to burst-mode activity by nicotinic antagonists (94 ).

Another major contributor to the cholinergic hypothesis of cognitive functioning was the discovery in the early 1980s that cholinergic
neurons in the basal forebrain degenerate in Alzheimer’s disease (95 ). Since then, loss/atrophy of cholinergic neurons has been
reported not only in Alzheimer’s disease but also in Parkinson’s disease, Lewy body dementia, progressive supranuclear palsy, and
several other disorders (96 ), although not all studies have reported losses in cholinergic neurons (97 ). In those that have reported
losses, the greatest reduction in numbers, of the order of 50% to 65%, has been observed in cholinergic neurons of the nucleus
basalis and the medial septal/diagonal band regions of patients pathologically verified as having Alzheimer’s disease (96 ). Loss of
high-affinity nAChRs has also been seen in the brains of patients with Alzheimer’s disease (98 ), and
 P.11

nicotinic agonists have been proposed as potential therapeutic agents to treat the disease (60 ).

ROLE FOR CHOLINERGIC NEURONS IN STIMULUS PROCESSING

Part of "1 - Acetylcholine "

Several lines of evidence suggest that cholinergic neurotransmission through nAChRs can affect stimulus processing. In support of
this notion, nicotine has been reported to alleviate some sensory gating deficits in schizophrenic patients (99 ), and animal studies
also suggest that nicotine may act to facilitate sensory inhibition, such as prepulse inhibition of startle in mice (100 ) and rats (101 ).
In another animal model of sensory processing, latent inhibition, nicotine can either enhance or disrupt sensory habituation,
depending on the preexposure parameters (102 ). Lesions of the nucleus accumbens or the pedunculopontine nucleus have been
shown to block prepulse inhibition (103 ), whereas lesions of the hippocampus, septum, medial raphe, and nucleus accumbens
disrupt latent inhibition (104 ), observations suggesting that nicotine may act in one or more of these brain areas to affect sensory
processing. Another brain area that is likely to mediate the effect of nicotine on sensory gating in schizophrenia is the hippocampus.
Postmortem studies have shown a reduced number of α-bungarotoxin-sensitive nAChRs (α7-containing nAChRs) in the hippocampus in
schizophrenic patients (105 ). Further, pharmacologic (106 ) and genetic (107 ) studies have suggested a role for the α7 nAChR in
prepulse inhibition in rodents.

A series of physiologic studies also supports the concept that ACh, acting on muscarinic receptors within the cerebral cortex,
promotes cortical responses to exogenous stimuli. ACh can produce a biphasic effect on membrane polarization in cortical neurons:
a rapid hyperpolarization followed by a prolonged depolarization (108 ). The inhibitory component was mediated through ACh-
induced activation of GABAergic interneurons that inhibited the pyramidal cells in a feed-forward manner. In contrast, the long-
lasting depolarization was mediated through direct effects of ACh on the cortical neuron. Subsequent studies suggested that this
effect is mediated by blockade of Im, a voltage-sensitive rectifying K+ channel (14 ). In addition, ACh reduced spike frequency
adaptation by blocking the after-hyperpolarization effect.

The net physiologic effect of these changes in cortical cell physiology may be to render pyramidal cells more responsive to afferent
input. Because the membrane is more depolarized, neurons are more likely to fire in response to a given excitatory stimulus; also,
the response to that stimulus may be prolonged because the after-hyperpolarization has been blocked. Thus, it seems plausible that
muscarinic cholinergic effects on cortical pyramidal cells may indeed promote stimulus access to the cortical circuit. Inasmuch as
attentional processing may represent the ability of stimuli to be processed actively within the neocortex, these physiologic actions
of ACh may be consistent with the reported behavioral effects of cholinergic lesions.

CONCLUSIONS
Part of "1 - Acetylcholine "

Recent studies using new physiologic techniques, cholinergic-selective toxins, and molecular genetic techniques have refined our
ideas about the role of ACh in the brain. In particular, it is clear that cholinergic and GABAergic pathways are intimately connected
in the hippocampus and basal forebrain complex and may combine to exert their effects on cognition, attention, and arousal.
Further, the subtypes of cholinergic receptors that mediate these effects of ACh are beginning to be elucidated with the use of
knockout mice that lack specific receptor subunits. These techniques have contributed to a minirevolution in our views of how ACh
contributes to cognitive processes. Research in this area is moving very quickly, and it is likely that these ideas will continue to be
refined as the new techniques are applied to previously described behavioral paradigms. Improvements in existing techniques—for
example, through the development of inducible and site-specific mutations in cholinergic-receptor subtypes—will also contribute to
further refinements in our view of cholinergic functions in the brain.

SUMMARY
Part of "1 - Acetylcholine "

Acetylcholine is critical for communication between neurons and muscle at the neuromuscular junction, is involved in direct
neurotransmission in autonomic ganglia, and has been implicated in cognitive processing, arousal, and attention in the brain. The
results of recent studies in which specific cholinotoxins, electrophysiology, or molecular genetic techniques were used have altered
our view of the functional role of the cholinergic system in the brain. Mice that lack specific subunits or subtypes of muscarinic or
nAChRs have recently been generated and used to demonstrate the role of particular receptor subtypes in physiologic effects of ACh
in muscle, peripheral ganglia, and the central nervous system. Roles for cholinergic neurons have been found in arousal and sleep,
motivation and reward, cognitive processes, and stimulus processing. The evaluation of these functions by means of novel
cholinotoxins and new electrophysiologic techniques have refined our ideas about the role of ACh in the brain. The evidence that
cholinergic and GABAergic pathways are intimately connected in the hippocampus and basal forebrain complex and may combine to
affect cognition, attention, and arousal is reviewed. In addition, the subtypes of cholinergic receptors that mediate these effects of
ACh are discussed based on studies of knockout mice that lack specific receptor subunits. Improvements in existing techniques—for
example, through the development of inducible and site-specific mutations in
 P.12
cholinergic-receptor subtypes—will contribute to further refinements in our view of cholinergic functions in the brain.

ACKNOWLEDGMENTS
Part of "1 - Acetylcholine "
M.R.P. is supported by grants DA10455, DA00167, and DA07290 from the National Institutes of Health and the Christiane Brooks Johnson Foundation.
M.A. is supported by grant DA09797 from the National Institutes of Health. We thank Professor B. J. Everitt for the use of his unpublished figure.

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2
Serotonin

George K. Aghajanian

Elaine Sanders-Bush

George K. Aghajanian: Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, Connecticut.

Elaine Sanders-Bush: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee.

Serotonin, or 5-hydroxytryptamine (5-HT), has been implicated in almost every conceivable physiologic or behavioral function—
affect, aggression, appetite, cognition, emesis, endocrine function, gastrointestinal function, motor function, neurotrophism,
perception, sensory function, sex, sleep, and vascular function (1 ). Moreover, most drugs that are currently used for the treatment
of psychiatric disorders (e.g., depression, mania, schizophrenia, autism, obsessive-compulsive disorder, anxiety disorders) are
thought to act, at least partially, through serotoninergic mechanisms (see elsewhere, this volume). How is it possible for 5-HT to be
involved in so many different processes? One answer lies in the anatomy of the serotoninergic system, in which 5-HT cell bodies
clustered in the brainstem raphe nuclei are positioned through their vast projections to influence all regions of the neuraxis.
Another answer lies in the molecular diversity and differential cellular distribution of the many 5-HT receptor subtypes that are
expressed in brain and other tissues.

During the past decade, molecular cloning techniques have confirmed that putative 5-HT receptor subtypes, predicted from
radioligand binding and functional studies (e.g., 5-HT1, 5-HT2, 5-HT3, 5-HT4), represent separate and distinct gene products. This
knowledge has revolutionized contemporary research on the serotoninergic system. Through the use of in situ messenger RNA (mRNA)
hybridization and immunocytochemical maps, studies of previously recognized 5-HT receptors could be directed more precisely
toward neurons and model cell lines that express these specific 5-HT receptor subtypes. Moreover, by the use of cloning techniques,
investigations could be initiated to determine the functional role of previously unrecognized 5-HT receptors (e.g., 5-HT5, 5-HT6, 5-
HT7). Concurrently, much progress has been made in delineating the signal transduction pathways of the various 5-HT-receptor
subtypes. The focus of this review is on the molecular and cellular aspects of individual 5-HT receptor subtypes and their
transduction mechanism, in addition to interactions between different receptor subtypes within a single neuron or region. The
implications of this work in understanding the global functions of the 5-HT system are discussed.

 5-HT RECEPTOR SUBTYPES: MOLECULAR AND CELLULAR ASPECTS

 INTRACELLULAR SIGNAL TRANSDUCTION PATHWAYS
 BEHAVIORAL CORRELATES
 OVERVIEW AND CONCLUSIONS
 ACKNOWLEDGMENTS

5-HT RECEPTOR SUBTYPES: MOLECULAR AND CELLULAR ASPECTS

Part of "2 - Serotonin "

Molecular Biology
In the first half of the last decade, the cloning of the major known families of 5-HT receptors was accomplished. More recently,
attention has turned to issues of transcriptional and post-transcriptional regulation.

RNA Processing
The 5′-flanking region of several 5-HT-receptor genes has been cloned, and consensus sequences for transcription factors have been
identified in the promoter region (2 ,3 and 4 ). The identification of these potential regulatory sites sets the stage for investigations
on possible functionally significant regulation of gene transcription in vivo (5 ). A prominent form of post-transcriptional regulation
is alternative RNA splicing, in which the splicing out of intronic sequence varies. Alternative splicing is common and occurs for a
number of 5-HT receptors, including the 5-HT2C, 5-HT4, and 5-HT7 receptors. The two splice variants of the 5-HT2C receptor described
in the literature encode severely truncated proteins with no obvious function (6 ,7 and 8 ). In contrast, the splice variants of the 5-
HT4 receptor (5-HT4(a)-5-HT4(f)) and 5-HT7 receptor (5-HT7(a)-5-HT7(d)) differ in length and composition in the carboxyl terminus (see refs.
9 and 10 for review). Marked species differences and perhaps regional differences lead to different patterns of splicing. Recently,
Claeysen et al. (11 ) showed that the shortest 5-HT4 receptor variants have the highest degree of constitutive activity, suggesting
that the long tail provides structural stability to the molecule. Splice variants of the 5-HT7 receptor have no known functional
differences. In contrast, a second form of post-transcriptional regulation, RNA editing, tends to
 P.16

have marked effects on the functional properties of proteins. For example, RNA editing changes a single amino acid in the β subunit
of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor, which dictates the gating properties of this ligand-
gated ion channel (see ref. 12 for review).
RNA editing in mammalian systems was discovered about a decade ago and is defined as any modification, other than alternative
splicing, that occurs at the level of mRNA. Several mechanisms of RNA editing exist, but mammalian editing generally involves the
conversion of adenosine residues to inosines by the action of a family of adenosine deaminases (13 ). Such editing events have the
potential to alter the genetic code at the level of RNA; the resulting is the formation of multiple protein isoforms with altered
function. The discovery of RNA editing of the 5-HT2C receptor provided the first, and so far only, example of editing of a G protein-
coupled receptor (14 ). Editing of the human 5-HT2C receptor mRNA involves five sites, A through E, where adenosine is converted to
inosine; inosine substitutes for guanosine in the genetic code, thus generating different protein isoforms. Multiple RNA isoforms have
been found for the 5-HT2C receptor in human brain, predicting the formation of protein isoforms with up to three amino acids
changed in the second intracellular loop of the receptor (15 ,16 ). Editing at the A, B, C, and D adenosine residues of human 5-HT2C-
receptor mRNA leads to predicted changes in all three amino acids to yield valine, serine, valine (VSV) at positions 156, 158, and 160
rather than isoleucine, asparagine, isoleucine (INI) at these positions in the unedited receptor isoform (Fig. 2.1 ). Editing at all five
sites predicts the formation of the valine, glycine, valine (VGV) isoform. Because the second intracellular loop has been implicated
in receptor–G protein coupling, initial functional studies have focused on the intracellular signaling properties of 5-HT2C-receptor
isoforms. These studies have shown that edited receptor isoforms couple less efficiently to Gq proteins, evidenced by lowered
agonist potencies to activate phospholipase C (7 ,14 ,15 ) and reduced receptor constitutive activity (16 ,17 ). The discovery that
the 5-HT2C receptor is regulated by RNA editing presents a challenge for pharmacologists because multiple isoforms with potentially
different pharmacologic properties and functions are predicted to exist in brain. It is not clear, for example, which receptor isoform
should be used for in vitro modeling of the receptor and to characterize newly developed drugs. The unedited INI isoform is
predicted to represent less than 10% of the total population of receptors in human brain; the principal isoform is VSV (15 ,16 ). To
date, all studies of function have involved recombinant cells expressing a single receptor isoform. Evaluation of the in vivo
functional consequences of RNA editing of the 5-HT2C receptor awaits the development of experimental methods for isolating the
function of a single, specific isoform in brain. Strategies such as the generation of blocking antibodies that target specific amino
acid combinations in the second intracellular loop or the development of transgenic mice that express a single isoform may be
successful, although they are experimentally challenging and time-consuming. It is not known whether other 5-HT receptors, or for
that matter other G protein-coupled receptors, are subject to RNA editing. It seems likely that the 5-HT2C receptor would not be
unique. However, screening methods for reliably detecting RNA editing are not available; instead, the discovery of edited substrates
depends on comparing genomic and cyclic DNA sequences. Consequently, new edited substrates are slow to emerge.

FIGURE 2.1. RNA editing of the 5-hydroxytryptamine

subtype 2C (5-HT2C) receptor. Editing of the 5-HT2C-receptor
messenger RNA transcript generates multiple receptor
isoforms that differ in one to three amino acids in the
second intracellular loop.

Post-translational Regulation
Receptor desensitization and down-regulation are common adaptive responses to sustained agonist exposure. The most widely
accepted model of desensitization of G protein-coupled receptors is based on extensive studies of the β-adrenergic receptor, a Gs-
linked receptor. In a simplified rendition of the model, agonist binding to a cell surface receptor leads to receptor phosphorylation,
arrestin binding, receptor internalization into endosomes, dephosphorylation of the receptor, and recycling back to the cell surface.
Receptor phosphorylation is thought to mediate desensitization by uncoupling the receptor from G protein. For many receptors, this
phosphorylation event is promoted by a family of G protein-coupled receptor kinases (GRKs). However, second messenger-dependent
kinases and protein kinases C and A, in addition to GRKs, have all been implicated in the desensitization of 5-HT1A receptor (18 ).
Abundant in vivo studies have documented a blunting of 5-HT1A receptor-mediated behavioral responses after long-term treatment
with agonists or serotonin uptake inhibitors that indirectly promote receptor activation. Indeed, desensitization of raphe 5-HT1A
autoreceptors has been proposed to play a role in the delayed therapeutic onset of antidepressant drugs (see ref. 19 for review).
Protein kinase C has also been implicated in 5-HT2A-receptor desensitization (20 ). Subsequent steps in the desensitization–
resensitization cycle have been demonstrated for the 5-HT2A receptor, including arrestin binding to the third intracellular loop of the
receptor (21 ) and internalization into endocytotic vesicles (22 ). Surprisingly, 5-HT2A-receptor antagonists also cause receptor
internalization, which may be related to the earlier findings of antagonist down-regulation of 5-HT2A receptors (see ref. 23 for
review). Importantly, antagonist-mediated 5-HT2A-receptor internalization has been confirmed in cortical pyramidal cells and is
accompanied by an apparent redistribution from dendrites to cell bodies (24 ). The fact that atypical antipsychotic drugs such as
clozapine and olanzapine, but not haloperidol, promote 5-HT2A-receptor internalization has led to speculation that this novel
antagonist property may be related to therapeutic action in schizophrenia.
P.17
 P.18

Receptor Constitutive Activity and Inverse Agonism

In Psychopharmacology: The Fourth Generation of Progress, the concept of inverse agonist properties of serotonin-receptor
antagonists was novel and unique to the 5-HT2C receptor (25 ). Inverse agonism is the ability of certain antagonists to block the
spontaneous (also referred to as constitutive) activity of a G protein-coupled receptor, in addition to blocking the binding of an
agonist. These antagonists are referred to as inverse agonists because their effects are opposite to those of agonists. In contrast,
other antagonists, referred to as neutral antagonists, have no apparent activity when added alone, even though they block the
action of an agonist. To explain receptor constitutive activity and inverse agonism of antagonists, the model of receptor–G protein
coupling was modified to propose spontaneous receptor isomerization to an active form (R*) in the absence of an agonist (26 ).
Antagonists with inverse agonist activity bind to and stabilize the inactive receptor conformation (R), whereas neutral antagonists
were proposed to bind equally well to the active and inactive forms of the receptor. Since 1995, 5-HT1A receptors (27, 28), 5-HT1B
receptors (29 ), 5-HT1D receptors (30 ), mutant 5-HT2A receptors (31 ), 5-HT4 receptors (11 ,32 ), and 5-HT7 receptors (33 ) have been
shown to exhibit constitutive activity; both inverse agonists and neutral antagonists have been described for these receptors.

All these studies demonstrating inverse agonist properties of 5-HT antagonists have been performed in vitro in cells expressing
recombinant receptors. It is not known whether inverse agonism is relevant to the in vivo actions of receptor antagonists, including
their therapeutic properties. Recent in vivo studies of a simple motor reflex have produced convincing evidence for constitutive
activity of the 5-HT2A/2C receptor and differential effects of inverse agonists versus neutral antagonists (34 ,35 ). However, Millan et
al. (36 ) were unable to show differential effects of inverse agonists versus neutral antagonists at the 5-HT1B receptor and concluded
that in vitro demonstrations of inverse agonist activity cannot be extrapolated to the in vivo situation. Recent studies by Berg et al.
(37 ) suggest that even in the absence of measurable effects on basal activity, prolonged treatment with inverse agonists at the 5-
HT2C receptor produces enhanced phospholipase C activation, likely because of increased expression of Gq proteins.

Electrophysiology
Although the electrophysiologic actions of 5-HT may seem quite varied, considerable uniformity is found within each of the major
receptor families. For example, all members of the 5-HT1 family tend to have inhibitory actions either presynaptically or
postsynaptically. Similarly, all members of the 5-HT2 family tend to have excitatory actions. Therefore, the discussion of 5-HT
electrophysiology is organized according to receptor family subtypes.

5-HT Receptors
1

Dense concentrations of 5-HT1A binding sites and high levels of 5-HT1A mRNA expression are found in a number of regions, including
the dorsal raphe nucleus, hippocampal pyramidal cell layer, and cerebral cortex (38 ,39 and 40 ). Studies in these regions have
been useful in delineating the physiologic role of this receptor.

Raphe Nuclei
Serotoninergic neurons of the raphe nuclei are inhibited by the local (microiontophoretic) application of 5-HT to their cell body
region. Thus, the receptor mediating this effect has been termed a somatodendritic autoreceptor (as opposed to the prejunctional
autoreceptor). Early studies in the dorsal raphe nucleus showed that lysergic acid diethylamide (LSD) and other indolamine
hallucinogens are powerful agonists at the somatodendritic 5-HT autoreceptor (41 ,42 ). Functionally, the somatodendritic 5-HT
autoreceptor has been shown to mediate collateral inhibition (43 ). The ionic basis for the autoreceptor-mediated inhibition, either
by 5-HT or LSD, is an opening of K+ channels to produce a hyperpolarization (44 ); these channels are characterized by their inwardly
rectifying properties (45 ). As in the dorsal raphe nucleus, serotoninergic neurons of the lower brainstem are also hyperpolarized by
5-HT via the opening of inwardly rectifying K+ channels (46 ,47 ). Similar findings in acutely isolated (48 ) and individually
microcultured (49 ) dorsal raphe neurons underscore the fact that autoreceptor inhibition is independent of any inputs to the raphe
nucleus. Patch-clamp recordings in the cell-attached and outside-out configuration from such acutely isolated dorsal raphe neurons
show that the increase in K+ current results from a greater probability of opening of unitary K+ channel activity (50 ).

The somatodendritic autoreceptors of serotoninergic neurons in both the dorsal raphe nucleus and the nucleus raphe magnus appear
to be predominantly of the 5-HT1A subtype; a variety of drugs with 5-HT1A selectivity (e.g., 8-OH-DPAT and the anxiolytic drugs
buspirone and ipsapirone) share the ability to inhibit raphe cell firing potently in a dose-dependent manner (47 ,50a ,50b ). Recently,
a highly selective 5-HT1A antagonist (WAY 100635) has been found that potently blocks the direct inhibition of dorsal raphe
serotoninergic neurons both by 5-HT and selective 5-HT1A agonists (51 ,52 ). WAY 100635 also blocks the indirect inhibition of dorsal
raphe neurons induced by selective 5-HT reuptake inhibitors (53 ). Complementing this autoinhibitory role of local 5-HT1A receptors
is a long-loop negative-feedback system activated by postsynaptic 5-HT1A receptors in the medial prefrontal cortex (54 ,55 ).
 P.19

In addition to long-loop feedback systems, short-loop regulatory circuits are found within the dorsal raphe nucleus and the adjacent
periaqueductal gray (PAG). These short-loop circuits involve interactions between 5-HT and local inhibitory GABAergic (γ-
aminobutyric acid) and excitatory glutamatergic neurons (56 ). Interestingly, both the local excitatory and inhibitory inputs to 5-HT
cells are negatively modulated by μ opiate receptors. Local GABAergic neurons are activated by 5-HT via 5-HT2A/2C receptors in a local,
negative feedback loop that complements 5-HT1A-mediated autoinhibition (57 ). Neurokinins such as substance P and neurokinin B,
via NK1 and NK3 receptors, respectively, activate mostly local glutamatergic excitatory inputs to 5-HT cells (58 ). Some of these local
circuits are depicted schematically in Fig. 2.2 .

FIGURE 2.2. Schematic representation of local regulatory

circuitry within the dorsal raphe nucleus (DRN). In addition

to somatodendritic 5-hydroxytryptamine subtype 1A (5-HT1A)

autoreceptors on the 5-HT neurons per se, local GABAergic

(γ-aminobutyric acid) and glutamatergic neurons in the

DRN/ventral periaqueductal gray (PAG) region modulate the

activity of serotoninergic neurons. Note the location of

inhibitory μ opiate receptors on both categories of local

neurons. Also depicted are excitatory 5-HT2A/2C and

inhibitory 5-HT1A receptors on GABAergic neurons and

excitatory NK1 (substance P) and NK3 (neurokinin B)

receptors on glutamate neurons in the DRN/PAG.

Other Subcortical Regions

Inhibitory or hyperpolarizing responses to 5-HT have been reported in a wide variety of neurons in the spinal cord, brainstem, and
diencephalon. In general, such responses have been attributed to mediation by 5-HT1 receptors. In sensory neurons of dorsal root
ganglia, a 5-HT1-like receptor has been reported to reduce the calcium component of action potentials and to produce
hyperpolarizations that can be mimicked by 5-HT1A agonists such as 8-OH-DPAT (59 ). In cerebellar Purkinje cells, 5-HT-induced
inhibition, but not excitation, is mediated through 5-HT1A receptors (60 ). In brain slices of the nucleus prepositus hypoglossi, focal
electric stimulation evokes inhibitory postsynaptic potentials (IPSPs) that are mediated by 5-HT1A receptors to activate an inwardly
rectifying K+ conductance (61 ) and a novel outwardly rectifying K+ conductance (62 ). In the midbrain PAG, a region known to be
involved in pain modulation and fear responses, approximately half the cells are inhibited/hyperpolarized by 8-OH-DPAT, suggesting
mediation by 5-HT1A receptors (63 ). In the ventromedial hypothalamus (64 ) and lateral septum (65 ,66 ), 5-HT and 5-HT1A agonists
produce inhibitory effects, also by activating a K+ conductance. In addition to these postsynaptic effects, 5-HT has been shown to
suppress glutamatergic synaptic transmission via presynaptic 5-HT1B receptors in various regions, including the hypoglossal nucleus
(67 ) and the nucleus accumbens (68 ).

In the rat laterodorsal tegmental nucleus, bursting cholinergic neurons are hyperpolarized by 5-HT via 5-HT1 receptors (69 ). In
freely behaving rats, the direct injection of 5-HT into the laterodorsal tegmental nucleus has been found to suppress rapid-eye-
movement (REM) sleep (70 ). In unanesthetized cats, a corresponding population of neurons that are active selectively during REM
states (REM-on neurons) in the laterodorsal tegmental nucleus has been shown to be inhibited by direct application of the 5-HT1A
agonist 8-OH-DPAT (71 ). It has been proposed that during REM sleep, the removal of a tonic inhibitory 5-HT influence from these
cholinergic neurons may be responsible for the emergence of an activated EEG during this behavioral state.

Hippocampus
Pyramidal cells of the CA1 region express high levels of 5-HT1A-receptor mRNA and 5-HT1A-receptor binding (72 ). Early on,
intracellular recordings in brain slices showed that the 5-HT-induced inhibition was caused by hyperpolarization resulting from an
opening of K+ channels (73 ). Subsequent work, in which various pharmacologic approaches have been used in brain slices, has shown
that the 5-HT-induced inhibition in both CA1 and CA3 pyramidal cells is mediated by the activation of receptors of the 5-HT1A
subtype (74 ,75 ,76 and 77 ). After long-term but not short-term administration of various antidepressant treatments (selective 5-
HT reuptake inhibitors, monoamine oxidase inhibitors,
 P.20

tricyclic drugs, electroconvulsive therapy), disinhibitory responses are seen with the selective 5-HT1A antagonist WAY 100635, which
suggests increased 5-HT1A-mediated inhibitory tone on CA3 hippocampal pyramidal cells (78 ). Interestingly, this increase in 5-HT1A
tone after long-term antidepressant treatment is potentiated by short-term treatment with lithium (79 ).

In addition to the above-mentioned direct effects on pyramidal cells, 5-HT has been shown to depress both excitatory and inhibitory
synaptic potentials in the hippocampus. Relatively high concentrations of 5-HT cause a reduction in electrically evoked excitatory
postsynaptic potentials (EPSPs) in CA1 pyramidal cells (80 ), an effect that is mimicked by 8-OH-DPAT, which suggests mediation by
5-HT1A receptors. Indirect measures indicate that 5-HT acts presynaptically to reduce Ca2+ entry and thereby glutamatergic synaptic
transmission. In addition, a 5-HT1A-mediated inhibitory effect on putative inhibitory interneurons of the hippocampus has been
observed (81 ,82 ). Consistent with an opening of K+ channels, the inhibitory effects of 5-HT on interneurons result from a
hyperpolarization associated with a reduction in input resistance. Functionally, the 5-HT1A-mediated inhibition of GABAergic
interneurons in the hippocampus leads to a disinhibition of pyramidal cells in CA1. Clearly, the effects of 5-HT in the hippocampus
are highly complex, involving both presynaptic and postsynaptic actions that may, to varying degrees, be inhibitory or disinhibitory,
facilitative or disfacilitative.

Cerebral Cortex
Hyperpolarizing/inhibitory responses in pyramidal cells of the cerebral cortex induced by 5-HT1A have been described in a number of
studies. In entorhinal cortex, where the density of 5-HT1A receptors is especially high (and the density of 5-HT2A receptors low),
unopposed 5-HT1A receptor-mediated hyperpolarizing responses are seen (83 ). However, cortical neurons in most other regions
typically display mixed inhibitory and excitatory responses to 5-HT because of expression by the same pyramidal cells of multiple 5-
HT receptor subtypes (e.g., 5-HT1A and 5-HT2/2C) (84 ,85 ,86 and 87 ). Hyperpolarizing responses mediated by 5-HT1A receptors are
often unmasked or enhanced in the presence of 5-HT2 antagonists, consistent with the idea that an interaction occurs between 5-
HT1A and 5-HT2A receptors at an individual neuronal level (84 ,88 ,89 ). A similar suggestion of a shift in the balance between 5-HT-
mediated excitation and inhibition comes from another in vivo study, in which both systemic and local application of 5-HT2
antagonists was shown to prevent an enhancement of the unit activity (and cortical desynchronization) that normally occurs in
response to noxious stimuli (tail compression) in anesthetized rats (90 ).

In addition to the above-mentioned postsynaptic effects, various presynaptic effects are mediated by 5-HT1 receptors in the cerebral
cortex. In cingulate cortex, 5-HT, acting on presynaptic 5-HT1B receptors, reduces the amplitude of electrically evoked EPSPs,
including both N-methyl-D-aspartate (NMDA) and non-NMDA components (87 ). Similar modulations of EPSPs, mediated by 5-HT1A or
5-HT1B receptors, have been reported for several cortical regions, including medial prefrontal (91 ) and entorhinal cortex (92 ).

5-HT Receptors
2

Quantitative autoradiographic studies show high concentrations of 5-HT2 binding sites and mRNA expression in certain regions of the
forebrain, such as the neocortex (layers IV/V), piriform cortex, claustrum, and olfactory tubercle (93 ). With few notable exceptions
(e.g., motor nuclei and the nucleus tractus solitarius), relatively low concentrations of 5-HT2 receptors or mRNA expression are
found in the brainstem and spinal cord. Studies aimed at examining the physiologic role of 5-HT2 receptors in several of these
regions are discussed in the following sections.

Motoneurons
In the facial and other cranial motor nuclei, motoneurons have a high density of 5-HT2-receptor binding sites. Early studies in vivo
showed that 5-HT applied microiontophoretically does not by itself induce firing in the normally quiescent facial motoneurons, but
does facilitate the subthreshold and threshold excitatory effects of glutamate (94 ). Intracellular recordings from facial motoneurons
in vivo or in brain slices in vitro (95 ,96 ) show that 5-HT induces a slow, subthreshold depolarization associated with an increase in
input resistance, indicating a decrease in resting K+ conductance. Ritanserin and other 5-HT2 antagonists are able to block the
excitatory effects of 5-HT in facial motoneurons selectively (97 ). Indolamine (e.g., LSD and psilocin) and phenethylamine (e.g.,
mescaline and DOI) hallucinogens act as 5-HT2 agonists at facial motoneurons. Iontophoretically administered LSD, mescaline, and
psilocin, although having relatively little effect by themselves, produce a prolonged facilitation of facial motoneuron excitability
(98 ). Intracellular studies in brain slices show that the enhancement is in part caused by a small but persistent depolarizing effect
of the hallucinogens (97 ,99 ).

Other Subcortical Regions

In brain slices of the medial pontine reticular formation, 5-HT induces a hyperpolarization in some cells and a depolarization in
other cells (100 ). The hyperpolarizing responses are associated with an increase in membrane conductance and have a 5-HT1
pharmacologic profile. The depolarizing responses have a 5-HT2 pharmacology and are associated with a decrease in membrane
conductance resulting from a decrease in an outward K+ current. These two actions of 5-HT do not appear to coexist in the same
neurons because none of the cells display dual responses to selective agonists.
 P.21

In brain slices of the substantia nigra pars reticulata, a majority of neurons are excited by 5-HT via 5-HT2 receptors (101 ), possibly
of the 5-HT2C rather than 5-HT2A subtype (102 ). Neurons in the inferior olivary nucleus are excited by 5-HT via 5-HT2A receptors, so
that the oscillatory frequency of input to cerebellar Purkinje cells is altered (103 ). In the nucleus accumbens, the great majority of
neurons are depolarized by 5-HT, and they are induced to fire (104 ). This depolarization is associated with an increase in input
resistance secondary to a reduction in an inward rectifier K+ conductance. Pharmacologic analysis shows that the depolarization is
mediated by a 5-HT2- rather than a 5-HT1- or 5-HT3-type receptor.

GABAergic neurons of the nucleus reticularis thalami show marked depolarizing responses to 5-HT, associated with a decrease in a
resting or “leak” K+ conductance; these excitatory responses are blocked by the 5-HT2 antagonists ketanserin and ritanserin (105 ).
The 5-HT-induced slow depolarization potently inhibits burst firing in these cells and promotes single-spike activity. It has been
suggested that the 5-HT-induced switch in firing mode from rhythmic oscillation to single-spike activity, which occurs during states
of arousal and attentiveness, contributes to the enhancement of information transfer through the thalamus during these states.
GABAergic neurons within the medial septal nucleus are also excited by 5-HT via 5-HT2 receptors (106 ). In the dentate gyrus of the
hippocampus, a subpopulation of GABAergic neurons is activated via 5-HT2A receptors, evidenced by an increase in IPSP frequency in
granule cells in the dentate gyrus (107 ). Recently, similar activation of GABAergic neurons via 5-HT2A receptors has been reported in
the CA1 region of the hippocampus (108 ). These observations closely resemble findings in the piriform cortex, where a
subpopulation of GABAergic interneurons is excited by 5-HT via 5-HT2A receptors (see below). Also, indirect evidence suggests that 5-
HT-induced inhibition of dentate/interpositus neurons of the deep cerebellar nuclei is mediated indirectly by the activation of
GABAergic interneurons through 5-HT2 receptors (109 ). Taken together, these findings suggest that in multiple locations within the
central nervous system, excitation of subpopulations of interneurons by 5-HT via 5-HT2 receptors gives rise to indirect inhibitory
effects.

Cerebral Cortex
The electrophysiologic effects of 5-HT have been studied in several cortical regions. In vitro studies in brain slice preparations have
shown that pyramidal cells in various cortical regions respond to 5-HT by either a small hyperpolarization, depolarization, or no
change in potential (84 ,85 and 86 ,110 ). Depending on the region of cortex under study, as described below, the depolarizations
appear to be mediated by 5-HT2A or 5-HT2C receptors.

In addition to these postsynaptic effects, 5-HT induces an increase in “spontaneous” (not electrically evoked) postsynaptic
potentials or currents (PSPs/PSCs) in brain slices from various cortical regions. Originally, recordings were made from pyramidal
cells in a paleocortical region, the piriform cortex. In that region, as in the hippocampus (see above), 5-HT, acting through 5-HT2A
receptors, induces an increase in spontaneous IPSPs (86 ,111 ,112 ,113 ,114 and 115 ). In vivo studies have also provided evidence
for a 5-HT2A receptor-mediated activation of GABAergic neurons in piriform cortex (116 ). As in piriform cortex, 5-HT can increase
IPSCs in pyramidal cells in various layers of the neocortex (117 ,118 ). The IPSCs result from the activation of cortical interneurons
via 5-HT2A/2C or 5-HT3 receptors (117 ). Immunocytochemical evidence has been found in primate cerebral cortex for a segregation of
5-HT2A- and 5-HT3-expressing interneurons; the former project to somatobasilar and the latter to distal apical dendritic regions of
pyramidal cells (119 ).

Quantitatively, in layer V pyramidal cells, synaptic events induced by 5-HT consist largely of EPSPs/EPSCs (118 ). Thus,
approximately 85% of all PSCs are blocked by AMPA/kainate glutamate-receptor antagonists (e.g., LY293558) but not by the GABAA
antagonist bicuculline (118 ). The 5-HT-induced increase in EPSCs is most pronounced in frontal regions, including the medial
prefrontal cortex (118 ). In that region, 5-HT2A receptors are denser than in more posterior regions (40 ,120 ). Recent studies, in
which intracellular labeling with biocytin was used, have confirmed that 5-HT-induced increases in EPSCs occur predominantly in
layer V pyramidal cells, whereas responses are minimal in layer II/III cells and lacking in layer VI cells (121 ).

The 5-HT-induced EPSCs are antagonized competitively by low concentrations of the highly selective 5-HT2A antagonist MDL 100,907
(pA2, 8.8), which indicates mediation by 5-HT2A receptors (118 ,122 ). Norepinephrine, via α1 adrenoceptors, also induces an increase
in EPSPs in layer V pyramidal cells, but (at least in the rat) the increase is only a fraction of that produced by 5-HT (122 ). Changes
in the frequency of synaptic currents or potentials are generally regarded as indicative of a modulation of presynaptic function.
Accordingly, the nonspecific group II/III metabotropic glutamate receptor agonist (1S,3S)-ACPD (118 ) and the selective mGlu II/III
metabotropic agonist LY354740 (123 ), which act at inhibitory autoreceptors and are located on glutamatergic nerve terminals,
suppress the 5-HT-induced increase in the frequency of EPSCs. In addition, the activation of μ receptors, located presynaptically on
thalamocortical inputs, also suppresses 5-HT-induced EPSCs, particularly in the medial prefrontal cortex (124 ). These results are
consistent with the idea that activation of 5-HT2A receptors increases the release of glutamate onto layer V pyramidal cells through a
presynaptic mechanism. 5-HT also produces a small but significant increase in the amplitude of spontaneous EPSCs, an effect that
may involve postsynaptic amplification mechanisms (118 ). Such postsynaptic
 P.22

effects are consistent with the finding of a high level of 5-HT2A-receptor immunoreactivity in the apical dendrites of cortical
pyramidal cells (125 ,126 and 127 ).

The 5-HT-induced EPSCs are blocked by bath application of the slice with the fast sodium channel blocker tetrodotoxin or perfusion
with a solution containing no added calcium (“zero” calcium) (118 ). Ordinarily, tetrodotoxin sensitivity and Ca2+ dependence would
suggest that activation of glutamatergic cells within the slice by 5-HT had led to an impulse flow-dependent release of glutamate.
However, several lines of evidence argue against this conventional interpretation. First, rarely were neurons within the confines of
the brain slice induced to fire by bath application of 5-HT. Second, none of the pyramidal cells (a potential source of intracortical
excitatory inputs) was depolarized sufficiently by 5-HT as recorded under our conditions to reach the threshold for firing. Third,
EPSCs can be induced by the microiontophoresis of 5-HT onto “hot spots” along the trajectory of apical dendrites of layer V
pyramidal cells (118 ). Together, these experiments suggest that the increase in spontaneous EPSCs induced by 5-HT in neocortical
pyramidal cells occurs through a focal action involving a Ca2+-dependent mechanism that is not based on an increase in impulse flow
in excitatory afferents.

As an alternative to a conventional impulse flow-related mechanism, it has been hypothesized that the 5-HT-induced EPSCs result
from an activation of the “asynchronous” release pathway (128 ). One of several distinguishing characteristics of this alternative
mechanism of transmitter release is that Sr2+ can substitute for Ca2+ in the asynchronous, but not synchronous, release (129 ). This
feature appears to be the result of the differential involvement of two isoforms of the calcium-sensing protein synaptotagmin in the
two alternative release mechanisms (130 ). Consistent with this idea, Sr2+ is highly effective in enabling 5-HT to induce an increase in
the frequency of EPSCs in the absence of Ca2+ (128 ).

Recently, it has been found that LSD and other hallucinogenic drugs, acting as partial agonists at 5-HT2A receptors, promote a late
component of electrically evoked EPSPs (131 ). It is possible that this late component, rather than representing conventional
polysynaptic transmission, is mediated through the mechanism of asynchronous transmitter release, possibly involving a release of
intraterminal Ca2+ stores via the phospholipase C, inositol triphosphate (IP3) pathway. An enhancement of asynchronous evoked EPSPs
via 5-HT2A receptors would provide a possible synaptic mechanism for the hallucinogenic effects of these drugs. In contrast, 5-HT
itself does not promote the late component of electrically evoked release except during the washout phase, presumably because of
opposing actions at 5-HT1 or other non-5-HT2A receptors (132 ). Figure 2.3 depicts the proposed location of various 5-HT-receptor
subtypes and their interactions with other neurotransmitter receptors within cortical circuitry.

FIGURE 2.3. Modulation of excitatory and

inhibitory transmission by multiple 5-

hydroxytryptamine (5-HT) receptors in the

cerebral cortex. 5-HT2A receptors are depicted as

enhancing glutamate release from a

glutamatergic terminal onto a layer V pyramidal

cell; the same terminal is seen to be negatively

modulated by various Gi/Go-coupled receptors

(e.g., μ-opiate, 5-HT1B, and mGluR II/III). In

addition, 5-HT2A receptors are shown to have a

direct postsynaptic excitatory effect that is

opposed by postsynaptic 5-HT1A receptors.

Finally, 5-HT2 and 5-HT3 receptors are shown on

anatomically distinct GABAergic inputs to the

somatobasilar and apical regions, respectively, of

the pyramidal cell.

5-HT Receptors
3

Excitatory responses to 5-HT have been found in various central neurons that have many of the characteristics of peripheral 5-HT3
responses, including rapid onset and rapid desensitization, features typical of ligand-gated ion channels rather than G protein-
coupled receptor responses (133 ,134 ). In cultured NG108-15 cells, the permeation properties of the 5-HT3 channel are indicative of
a cation channel with relatively high permeability to Na+ and K+ and low permeability to Ca2+ (134 ). A 5-HT-gated ion channel has
been cloned that has physiologic and pharmacologic properties appropriate for a 5-HT3 receptor (135 ). In the oocyte expression
system, this receptor shows rapid desensitization and is blocked by 5-HT3 antagonists (e.g., ICS 205–930 and MDL 72222). Its
sequence homology with the nicotinic acetylcholine receptor (27%) and the β1 subunit of the GABAA receptor (22%) indicates that this
5-HT3-receptor clone is a member of the ligand-gated ion channel superfamily. Typically, members of this superfamily are comprised
of multiple subunits; however, only one 5-HT3-receptor subunit and an alternatively spliced variant have been cloned to date (136 ).
 P.23

In hippocampus slices, 5-HT has been reported to increase spontaneous GABAergic IPSPs, most likely through a 5-HT3 receptor-
mediated excitation of inhibitory interneurons; these responses also show fading with time (137 ,138 ). A similar 5-HT3 receptor-
mediated induction of IPSCs has been reported in the neocortex (117 ). Whole-cell patch-clamp recordings have confirmed a direct
5-HT3 receptor-mediated excitatory effect on hippocampal interneurons independent of G-protein activation (139 ). Although fast,
rapidly inactivating excitation has generally become accepted as characteristic of 5-HT3 receptors, nondesensitizing responses have
also been reported. In dorsal root ganglion cells, a relatively rapid but noninactivating depolarizing response has been described that
has a 5-HT3 pharmacologic profile (140 ). In neurons of nucleus tractus solitarius brain slices, a postsynaptic depolarizing response to
5-HT3 agonists has been observed that is not rapidly desensitizing (141 ). In addition to these postsynaptic effects, a 5-HT3 receptor-
mediated increase in Ca2+ influx has been described in a subpopulation of striatal nerve terminals (142 ).

5-HT , 5-HT , and 5-HT Receptors

4 6 7

The first known protein Gs-coupled 5-HT receptor, the 5-HT4 receptor, was identified on the basis of pharmacologic and biochemical
criteria (e.g., positive coupling of 5-HT responses to adenylyl cyclase) (9 ). Subsequently, a receptor with matching pharmacologic
and other properties was cloned and found to be expressed in various regions of the brain (143 ). Two other 5-HT receptors
positively coupled to adenylyl cyclase have been cloned. Because their pharmacology differed from that of the previously described
5-HT4 site, they were designated as 5-HT6 and 5-HT7 receptors (144 ,145 and 146 ). At this time, electrophysiologic studies are
available only for the 5-HT4 and 5-HT7 receptors and are described below.

5-HT Receptors
4

Binding studies using a selective 5-HT4 ligand indicate that 5-HT4 receptors are present in several discrete regions of the mammalian
brain, including the striatum, substantia nigra, olfactory tubercle, and hippocampus (147 ). Because these regions also express 5-
HT4-receptor mRNA, it appears likely that the receptors function postsynaptically to mediate certain actions of 5-HT. The best
studied of these regions is the hippocampus, in which both biochemical and electrophysiologic studies have provided a detailed
picture of the actions of 5-HT at 5-HT4 receptors. Electrophysiologic studies show that 5-HT4 receptors mediate an inhibition of a
calcium-activated potassium current that is responsible for the generation of a slow after-hyperpolarization in hippocampal
pyramidal cells of the CA1 region (74 ,148 ,149 ). A suppression of the after-hyperpolarization would enhance the ability of these
cells to respond to excitatory inputs with robust spike activity.

5-HT Receptors
7

The circadian rhythm in mammals is set by a pacemaker located primarily in the suprachiasmatic nucleus of the hypothalamus. This
pacemaker activity can be maintained in hypothalamic slices, in which suprachiasmatic neurons display diurnal changes in neuronal
firing rate. Administration of 5-HT appears to produce a phase shift in this activity (150 ) by acting on a receptor that may be of the
5-HT7 subtype (144 ). This shift is mediated by stimulation of adenylate cyclase because it is mimicked by increasing intracellular
cyclic adenosine monophosphate (cAMP) and blocked by inhibiting protein kinase A (151 ). However, the precise mechanism by
which 5-HT7 receptors act is not presently known because it is unclear whether suprachiasmatic neurons themselves express the 5-
HT7 receptors (144 ). Furthermore, the effect of 5-HT on the membrane properties of these cells has not been examined. 5-HT7
receptor activation has been reported to inhibit GABAA currents on suprachiasmatic neurons in culture (152 ), but the relationship, if
any, between these observations and 5-HT changes in circadian activity remains to be determined.

Another electrophysiologic effect that may be mediated through 5-HT receptors that are positively coupled to adenylate cyclase is
the enhancement of the hyperpolarizing-activated nonselective cationic current Ih. The Ih channels, which are homologous to cyclic
nucleotide-gated channels in specialized sensory neurons, are positively modulated by cAMP (153 ,154 ). An increase in Ih tends to
prevent excessive hyperpolarization and increase neuronal excitability. In a number of regions of the brain, including the thalamus
(155 ), prepositus hypoglossi (156 ), substantia nigra zona compacta (157 ), and hippocampus (158 ), 5-HT has been shown to
enhance Ih through a cAMP-dependent mechanism. Results of a pharmacologic analysis with multiple nonselective drugs suggested
that the increase in Ih induced by 5-HT in dorsal root ganglion cells is mediated by 5-HT7 receptors (159 ). Recently, the first drug
with selectivity toward the 5-HT7 receptor was shown to block activation of adenylyl cyclase by 5-HT agonists in guinea pig
hippocampus (33 ). The increasing availability of such selective drugs should greatly enhance the electrophysiologic evaluation of Gs-
coupled 5-HT receptors.

INTRACELLULAR SIGNAL TRANSDUCTION PATHWAYS

Part of "2 - Serotonin "

Multiple Signaling Pathways: G Proteins and Second Messengers

Multiple intracellular signaling pathways constitute a common theme for G protein-coupled receptors, and the 5-HT receptor family
is not unique. Inhibition of adenylate cyclase
 P.24

was the first intracellular pathway to be described for Gi/o protein-coupled receptors, such as the 5-HT1A receptor. However, it is now
clear that these receptors regulate multiple signaling pathways and effector molecules (Fig. 2.2 ), including activation of G protein-
gated inwardly rectifying K+ (GIRK) channels, inhibition of voltage-sensitive Ca2+ channels, activation of phospholipase Cβ, and
activation of mitogen-activated protein kinase (see ref. 18 for review). Although all these signals are sensitive to pertussis toxin, so
that Gi/o proteins are implicated, they may be mediated by distinct G protein complexes. For example, coupling to GIRK channels is
mediated by βγ subunits released from Gi (and possibly Go) proteins, whereas inhibition of Ca2+ channels is mediated by βγ subunits
released from Go proteins. The profile of signaling molecules varies from cell to cell, offering diverse signaling possibilities and
contributing additional complexity. For example, 5-HT1A receptor activation of phospholipase C is cell-type dependent; this signal is
mediated by G protein βγ subunits and thus requires the presence of a βγ-regulated phospholipase C isoform. The βγ subunits,
generated by dissociation of the heterotrimeric Gi protein, also activate the type 2 isoform of adenylate cyclase. This activation is
conditional, dependent on the coactivation by Gαs (i.e., Gαi potentiates the action of Gαs). The obvious question is why the opposing
actions of Gαi and Gβγ do not offset each other. The answer may lie in the details. In addition to the large family of G proteins (21 α
subunits, 5 β subunits, and 11 γ subunits), the adenylate cyclase family comprises at least nine members, each regulated by distinct
inputs. Most of these molecules are found in the central nervous system. The G protein that contributes βγ activation of type 2
adenylate cyclase is Gαi1 or Gαi2 heterotrimer (160 ), whereas all three Gαi subunits (αi3 > αi2 > αi1) have the ability to inhibit adenylate
cyclase types 5 and 6 (161 ). Thus, in cells in the brain in which Gαi1 or Gαi2 heterotrimer is coexpressed with type 2 adenylate cyclase,
5-HT1A-receptor activation may potentiate Gs-mediated increases in cAMP. This type of interaction has been shown to occur in brain,
in which Gi-linked receptors enhance β-adrenergic responses (162 ); a similar interaction may take place in cells that coexpress a 5-
HT1A receptor family member with one of the 5-HT receptors (5-HT4, 5-HT6, or 5-HT7) linked to activation of adenylate cyclase.

Although a 5-HT receptor-mediated increase in cAMP formation in superior colliculus was one of the earliest second messenger
pathways defined in brain, the 5-HT4 receptor was one of the last 5-HT receptors to be cloned (143 ). This receptor and the 5-HT6
and 5-HT7 receptors have in common the ability to activate adenylate cyclase via Gαs (Fig. 2.2 ). In transfected cells, the 5-HT6
receptor couples to adenylate cyclase type 5, the typical Gαs-sensitive isoform (163 ). In contrast, the 5-HT7 receptor increases
intracellular calcium, which activates calmodulin-stimulated adenylate cyclase type 1 or 8. A recent characterization of rat
hippocampal homogenates suggests that both the 5-HT4 and 5-HT7 receptors are involved in cAMP formation (adenylate cyclase
isoform unknown) in the hippocampus (164 ). Interestingly, the 5-HT1A receptor produces a slight increase in cAMP formation,
perhaps reflecting Gβγ potentiation of Gs activation of adenylate cyclase type 2 mediated by the 5-HT4 or 5-HT7 receptor.

Receptors that couple to the Gq family members (Gq, G11, G14, and G15/16) activate phospholipase C in a pertussis toxin-insensitive
manner. Activation of phospholipase C was the first signal transduction mechanism identified for the 5-HT2-receptor family and is
essentially universal. This probably reflects the wide distribution of Gq/11 and the functional redundancy of these two G proteins. The
5-HT2C receptor has been shown to couple in a pertussis toxin-sensitive manner to Gi/o in Xenopus oocytes (e.g., see ref. 165 ) and in
some transfected cell lines (166 ). In contrast, recent evidence suggests that phospholipase C activation in a native setting (choroid
plexus) is mediated entirely by Gq/11 coupling (167 ). Coupling of the 5-HT2C receptor to G13 with subsequent cytoskeletal
rearrangement has been recently described in a transfected cell line (168 ). Extensive evidence suggests that 5-HT2A and 5-HT2C
receptors couple to other effector pathways, in addition to phospholipase C (Fig. 2.4 ). Phospholipase A2 is a well-characterized
independent signal transduction pathway that leads to arachidonic acid, with subsequent prostaglandin and leukotriene formation
(169 ). 5-HT2A-receptor activation of mitogen-activated protein kinase has been extensively characterized in vascular smooth muscle
and is also thought to be independent of phospholipase C activation (170 ,171 ). The 5-HT2A receptor increases phospholipase D
activity via a small G-protein ARF (adenosine diphosphate ribosylation factor) pathway, with protein kinase C activation being the
principal consequence (172 ). 5-HT2A receptors differentially regulate brain-derived neurotrophic factor in hippocampus and cortex
and play a role in stress-induced down-regulation of brain-derived neurotrophic factor expression in hippocampus (173 ,174 ). In
addition, a 5-HT2A receptor-mediated increase in transforming growth factor-α1, secondary to protein kinase C activation, has been
described (175 ). The 5-HT2A and 5-HT2C receptors elicit region-specific increases in immediate early genes c-fos and Arc in rat brain
(176 ), which are likely downstream of phospholipase C activation. Extensive, complex cross-talk between the 5-HT2A and 5-HT2B
receptor and the 5-HT1B/D receptor has been demonstrated in immortalized serotoninergic cells, in which the 5-HT2B receptor, via a
phospholipase A2 product, attenuates 5-HT1B/D receptor-mediated adenylate cyclase inhibition (177 ). Coactivation of the 5-HT2A
receptor blocks this interaction by an unknown mechanism. These examples of parallel, interacting, and converging intracellular
signaling pathways illustrate the complexity of receptor signaling, even within a single receptor subclass.

FIGURE 2.4. Examples of potential converging

and interacting signaling pathways for 5-

hydroxytryptamine-receptor subtypes. This

figure illustrates only a few of the nearly

unlimited possibilities, depending on the cell

phenotype. Also listed are additional effectors

activated by one or another of these receptors

with pathways of activation that have not yet

been determined.
 P.25

Physiologic Correlates
In general, the electrophysiologic effects of 5-HT correspond well to the G-protein and second messenger coupling of the various
receptor subtypes. The Gi/Go-coupled 5-HT1 receptors generally mediate inhibitory effects on neuronal firing through an opening of
inwardly rectifying K+ channels or a closing of voltage-gated Ca2+ channels. Inhibitions mediated by 5-HT1 receptors have been
observed in neurons located in diverse regions of the central nervous system, ranging from pyramidal cells of the cerebral cortex
and hippocampus to serotoninergic neurons of the brainstem raphe nuclei. The Gq/11-coupled 5-HT2 family of receptors generally
mediates slow excitatory effects through a decrease in K+ conductance or an increase in nonselective cation conductance. Slow
excitatory effects mediated by 5-HT2 receptors have been observed in a number of regions, including the spinal cord and brainstem
(e.g., motoneurons), subcortical regions (e.g., nucleus accumbens), and cerebral cortex, where these receptors are most
concentrated. The 5-HT3 receptors, which are ligand-gated channels with structural homology to nicotinic cholinergic receptors,
mediate fast excitatory effects of 5-HT. Specific examples are given below for 5-HT1, 5-HT2, and 5-HT4 receptors, for which
intracellular transduction pathways have been studied most intensively.

5-HT Receptors
1

The opening of K+ channels via 5-HT1A receptors in dorsal raphe neurons is mediated by pertussis toxin-sensitive G proteins
(178 ,179 ). The molecular mechanisms underlying the opening of K+ channels are most likely common to all neurotransmitter
receptors that couple through the Gi/Go family of G proteins. As in the dorsal raphe, these receptors activate a pertussis toxin-
sensitive G protein that couples to the opening of inwardly rectifying K+ channels through a membrane-delimited pathway (74 ,180 ).
It is widely accepted that the βγ rather than α subunits regulate the channels (181 ,182 and 183 ). The effector mechanism that
ultimately mediates the inhibitory effect signaled by 5-HT1A receptors is the inwardly rectifying K+ channel. Interestingly, at least
one of the potassium K+ subunits identified in heart, GIRK-1, is expressed at high levels in hippocampus (184 ), which suggests that it
might be involved in mediating the 5-HT1A receptor-induced hyperpolarization in this region.
 P.26

Consistent with this possibility, the K+ current activated by 5-HT1A receptors in the CA1 region does show the characteristic signature
of this potassium channel family-namely, inward rectification (74 ).

5-HT Receptors
2

The role of G proteins in mediating the 5-HT2-induced slow inward current that results from K+ channel closure has been evaluated in
facial motoneurons by using the hydrolysis-resistant guanine nucleotide analogues GTPγS and GDPβS (185 ). The 5-HT-induced
inward current becomes largely irreversible in the presence of intracellular GTPγS. Mediation by G proteins is also suggested by the
fact that the inward current is reduced by intracellular GDPβS, which prevents G-protein activation. Although the identity of the G
protein(s) mediating the electrophysiologic responses has not yet been determined directly, the 5-HT2 family of receptors is known
to be coupled to phospholipase C. Thus, a member of the Gq/11 family may be involved because the latter can directly activate
phospholipase C (186 ).

5-HT Receptors
4

Initially, it was shown that 5-HT suppresses a calcium-activated potassium current that is responsible for the generation of a slow
after-hyperpolarization in hippocampus pyramidal cells of the CA1 region (see above). Subsequent studies have implicated 5-HT4
receptors, acting via cAMP and protein kinase A, in mediating this action (187 ). A similar activation of a cAMP-dependent protein
kinase has been implicated in the suppression of a voltage-activated K+ current in cultured neurons from the superior colliculus
(188 ). More recently, it has been shown that 5-HT4 receptors reduce after-hyperpolarization in hippocampus pyramidal cells by
inhibiting calcium-induced calcium release from intracellular stores (189 ).

Pharmacologic Significance
The pharmacologic significance of a single receptor regulating multiple signaling pathways is only just beginning to be defined. The
most explicit studies of promiscuous coupling of receptors to multiple G-protein signaling pathways have involved transfection of a
recombinant receptor into various cell models that do not normally express the receptor. Powerful genetic strategies involving
antisense techniques, overexpression of signaling molecules, and expression of constitutively active and dominant negative mutants
have exposed a multitude of fascinating possibilities for a single receptor to sculpt multiple signals depending on the properties of
the cell. In addition, theoretical arguments (190 ) and experimental evidence (191 ,192 and 193 ) have appeared in support of the
novel concept of agonist-directed trafficking of the intracellular signal. This model proposes that when a single receptor interacts
with multiple signaling pathways, the pattern of intracellular signaling may differ depending on the agonist. Although the
mechanism of agonist-specified signaling is not known, one possibility is that different agonists promote distinct receptor
conformations, thereby exposing interfacial domains with altered protein–protein interaction properties. All these studies in
artificial conditions tell us only what can occur, not what does occur in vivo. Techniques for studying the role of multiple signaling
pathways in native preparations are needed to tease out the significance of the various signaling molecules in normal physiology and
in pathologic states. Transgenic and knockout strategies have some utility; however, targeting signaling molecules will have a
multitude of unwanted consequences because of their universal role in cell physiology. Another strategy was recently described that
has significant potential for teasing out signaling pathways downstream of receptor activation (167 ). Synthetic blocking peptides
targeting specific protein–protein interactions in a signaling pathway are rendered membrane-permeable by a novel conjugation
reaction, so that the function of a particular signaling step in native systems can be defined.

BEHAVIORAL CORRELATES
Part of "2 - Serotonin "

5-HT Neuronal Activity and Behavioral State

In a variety of mammalian species, serotoninergic neurons of the raphe nuclei have been found to have a slow, tonic pattern of
firing (approximately one to two spikes per second). The maintenance of rhythmic firing under a wide variety of conditions has
suggested that serotoninergic neurons possess intrinsic tonic pacemaker mechanisms. Intracellular recordings from dorsal raphe
neurons show that spikes arise from gradual depolarizing ramps (pacemaker potentials) rather than synaptic potentials. The
pacemaker rhythm of serotoninergic neurons results from a complex interplay of intrinsic ionic currents (e.g., a voltage-dependent
transient outward potassium current, a low-threshold inward calcium current, and a calcium-activated outward potassium current)
(194 ). Also modulating the activity of serotoninergic neurons are various neurotransmitters, including norepinephrine and 5-HT
itself. Norepinephrine, acting via α1 adrenoceptors, accelerates pacemaker activity of serotoninergic neurons by closing potassium
channels. Conversely, 5-HT itself, acting on 5-HT1A autoreceptors, opposes excessive activity of serotoninergic neurons.

The highly regulated pacemaker activity of serotoninergic neurons suggests that the 5-HT system serves an important homeostatic
function. Through its effects on neuronal excitability in diverse regions of the brain and spinal cord, the serotoninergic system is in a
strategic position to coordinate complex sensory and motor patterns during different
 P.27

behavioral states. Recordings from serotoninergic neurons in unanesthetized animals have shown that activity is highest during
periods of waking arousal, reduced in quiet waking, reduced further in slow-wave sleep, and absent during REM (dream) sleep (195 ).
It can be hypothesized that the function of the 5-HT system, by its coordinated fluctuations in activity, is to promote a given
behavioral state. This concept is illustrated in the following scenario. When serotoninergic neurons are in a tonic firing mode, the
following conditions would prevail: (a) Motoneurons would be in a relatively depolarized, excitable state (via 5-HT2 receptors) and
thus receptive to the initiation of movement; (b) neurons of the nucleus reticularis thalami would be in a depolarized, single-spike
mode (via 5-HT2 receptors) and thus conducive to thalamocortical sensory information transfer (105 ,155 ); (c) GABAergic neurons of
the septohippocampal pathway would be activated (in part via 5-HT2A receptors), potentially enhancing long-term potentiation by
inhibiting GABAergic neurons of the hippocampus (106 ,196 ); (d) neurons of the laterodorsal tegmental nucleus would be
hyperpolarized (via 5-HT1 receptors) and therefore not able to generate the bursting activity of REM sleep (69 ,70 and 71 ).
Conversely, with a reduction in serotoninergic activity during various stages of sleep, the above conditions would switch such that
motoneurons would become less excitable, thalamocortical sensory information transfer would be diminished, hippocampal function
would be reduced, and sleep spindles and pontogeniculo–occipital (PGO) waves would emerge.

Molecular Genetics (Including Genetic Polymorphisms)

5-HT-Receptor/Transporter Knockouts
New drugs are beginning to appear that show considerable selectivity for a particular serotonin receptor subtype; however, many
are not yet readily available to the general scientific community. Genetically modified mice that fail to express a specific receptor
provide a powerful means to complement pharmacologic tools for evaluating the behavioral consequences of a particular serotonin-
receptor protein (see ref. 197 for review). The first 5-HT-receptor knockout mouse was described in 1994 (198 ), in which the 5-HT1B
receptor was eliminated by homologous recombination technique. These original studies showed markedly enhanced aggression in 5-
HT1B-receptor knockout mice. Since then, altered responses to drugs of abuse, including enhanced alcohol consumption (199 ) and
sensitization to cocaine (200 ), in addition to impaired paired-pulse inhibition (201 ) and paradoxical sleep (202 ), have been shown
to be prominent phenotypic traits. In 1995, a “knockout” mouse line expressing a mutant, nonfunctional 5-HT2C receptor was
described (203 ). Subsequently, enhanced seizure susceptibility (204 ), obesity and late-onset diabetes (205 ), and a specific deficit
in dentate gyrus long-term potentiation (206 ) have been reported. Mouse lines have recently been generated that are null for other
important 5-HT-related molecules; these including the 5-HT1A receptor, which is associated with enhanced anxiety (207 ,208 and
209 ), the serotonin transporter, with enhanced cocaine sensitivity (210 ,211 ), and the 5-HT5A receptor, with reduced sensitivity to
LSD (212 ). Although monoamine oxidase A-null mice have general alterations in biogenic amine dynamics, evidence suggests that
the enhanced levels of 5-HT found in these mice are associated with neurodevelopmental abnormalities (213 ,214 ). Innovative
technologies such as inducible, conditional knockouts, which have the potential for temporally and spatially controlling gene
manipulation, hold great promise for the future. This is illustrated in a recent study in which localized rescue of knocked-out genes
was used to study the differential sorting of the 5-HT1A and 5-HT1B receptor in striatal neurons (215 ). In these transgenic mice, but
not transfected neurons in culture, reproduction of the normal targeting of the 5-HT1B receptor to axon terminals set the stage for
mutagenesis studies of molecular determinants of receptor targeting to axon terminals in vivo.

Genetic Polymorphisms
Molecules involved in brain 5-HT pathways have been favorite targets for candidate gene studies, and the number of publications
dealing with genetic variations in 5-HT systems has increased dramatically during the past few years. Recent population studies have
probed for single nucleotide polymorphisms in synthetic enzymes, inactivation molecules, and receptors for 5-HT. The list of human
diseases studied is extensive and includes obsessive-compulsive disorder, major depression, bipolar depression, schizophrenia,
Alzheimer’s disease, eating disorders, anxiety, neuroticism, fibromyalgia, alcoholism, suicide, homicide, substance abuse,
pathologic gambling, and responses to psychotherapeutic agents. Despite the abundance of publications, no definitive, reproducible
links between allelic variants of 5-HT-related molecules have been found in human populations with behavioral disorders or brain
diseases. More often than not, results are not reproducible from study to study, in large part because of the heterogeneous nature
of psychiatric diseases, the absence of a specific diagnostic laboratory test, and the modest numbers of patients in many studies.
The most extensively studied genetic polymorphism in a 5-HT-related molecule is the insertion/deletion polymorphism in the
promoter region of the 5-HT transporter gene (216 ). These variable-length polymorphisms are biologically significant because in
vitro studies have shown that the short form reduces the expression of transporter mRNA, with subsequently reduced uptake
capacity (217 ). Although many studies suggest that the short form is associated with affective disorders, others have failed to
replicate these findings (218 ).
 P.28

Some commonly studied polymorphisms, such as the C103T variant in the 5-HT2A receptor, are silent (i.e., do not change the genetic
code), whereas other polymorphisms, such as the 5-HT2C receptor Cys23Ser allele (219 ), produce mutant proteins with no apparent
alterations in functional properties. The clinical importance of such a subtle genetic variant may require analysis of other related
genes in tandem. Methods for detecting genetic polymorphisms are advancing rapidly and now allow simultaneous genotyping of
several nucleotide polymorphisms; for example, a method was recently described to detect multiple single-nucleotide
polymorphisms of 5-HT-related genes (220 ).

OVERVIEW AND CONCLUSIONS

Part of "2 - Serotonin "
This review has emphasized recent developments in molecular, transductional, and cellular aspects of the 5-HT system. Molecular
topics that were hardly mentioned in the previous edition of this book include RNA editing, post-translational processing, genetic
polymorphisms, and the use of selective 5-HT-receptor and transporter gene knockouts. Notable developments in cellular physiology
since the previous edition include growing numbers of studies on the more recently cloned 5-HT-receptor subtypes (e.g., 5-HT3–7) and
refinements in the analysis of 5-HT1- and 5-HT2-receptor function. Examples of the latter include the following: (a) the recognition
that the well-known 5-HT1A autoreceptor feedback regulation of 5-HT neurons occurs within the context of a complex set of local
and long-loop regulatory circuits; (b) the finding that 5-HT2 receptors have a dramatic influence on cortical information processing,
which has allowed new insights into the mechanism of action of hallucinogenic and atypical antipsychotic drugs. In turn, advances in
molecular and cellular research on individual receptor subtypes have provided new experimental tools for the behavioral analysis of
the 5-HT system (e.g., pharmacologic agents with more precisely defined actions and gene knockouts). The question remains of
whether the diverse cellular and molecular actions of 5-HT mediated by the various receptor subtypes can be incorporated into a
holistic scheme that can define the overall function of the 5-HT system. Selected examples have been given of how the 5-HT system
can be seen as modulating, in a complex but coordinated fashion, a number of motor, sensory, and other systems to promote a given
behavioral state or function. The recent molecular and cellular advances, by enabling a more comprehensive analysis of the
elementary actions of individual 5-HT-receptor subtypes, have set the stage for a more precise analysis of overall function.

ACKNOWLEDGMENTS
Part of "2 - Serotonin "
This work was supported by grants from the National Institute of Mental Health, the National Institute on Drug Abuse, and the state
of Connecticut.

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3
Opioid Peptides and Their Receptors: Overview and Function in Pain
Modulation
Gavan P. McNally
Huda Akil
Gavan P. McNally and Huda Akil: Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan.
Few neurotransmitter systems have fascinated the general public as much as the endorphins, otherwise known as the endogenous
opioid peptides. They have been termed the “heroin within” and endowed with the power to relieve pain and allow one to
experience “runner’s high” or enjoy the taste of chocolate. Although these powers may or may not withstand close scientific
scrutiny, there is little question that endogenous opioid systems play a critical role in modulating a large number of sensory,
motivational, emotional, and cognitive functions. As inhibitory neuropeptide transmitters, they fine-tune neurotransmission across a
wide range of neuronal circuits, setting thresholds or upper limits. In addition, they have served as prototypes for understanding
many structural and functional features of peptidergic systems. Thus, the first neuronal receptor binding assays were conducted on
opioid receptors. The first peptides to be discovered and identified after the hypothalamic neurohormones (oxytocin and vasopressin)
were the endogenous opioids. The first mammalian cyclic DNA (cDNA) to be cloned was an opioid precursor (proopiomelanocortin),
which also served as the prototype for genes that encode multiple active substances and process them in a tissue-specific and
situation-specific manner.
Scientific studies of these systems during the last 30 years have uncovered a complex and subtle system that exhibits impressive
diversity in terms of the number of endogenous ligands (more than a dozen) yet amazing convergence at the level of receptors (only
three major types). Based on the results of these studies, the endogenous opioids have been implicated in circuits involved in the
control of sensation, emotion, and affect, and a role has been ascribed to them in addiction—not only to opiate drugs, such as
morphine and heroin, but also to other highly abused drugs, such as alcohol. This chapter cannot do justice to the rich body of
information we possess on the endogenous opioid system. However, we attempt to give the reader key information about the
biochemical nature of the system, along with an update on our understanding of the recently cloned receptors and their functions.
Finally, we describe the regulation of pain responsiveness as one example of a function mediated by opioids to illustrate the
complexity of their role.

 OPIOID PEPTIDES AND THEIR RECEPTORS

 RECEPTOR AND LIGAND KNOCKOUTS: INSIGHTS, ISSUES, AND COMPLEXITIES
 AN EXAMPLE OF OPIOID FUNCTION AND ITS CLINICAL IMPLICATIONS: OPIOIDS AND PAIN CONTROL
 CONCLUSIONS AND FUTURE DIRECTIONS

OPIOID PEPTIDES AND THEIR RECEPTORS

Part of "3 - Opioid Peptides and Their Receptors: Overview and Function in Pain Modulation "

Genes and Proteins

The opioid peptide precursors are encoded by three genes: pre-proopiomelanocortin, pre-proenkephalin, and pre-prodynorphin.
Each precursor is subject to complex post-translational modifications that result in the synthesis of multiple active peptides. These
peptides share the common N-terminal sequence of Tyr-Gly-Gly-Phe-(Met or Leu), which has been termed the opioid motif; this is
followed by various C-terminal extensions yielding peptides ranging from 5 to 31 residues in length. The major opioid peptide
encoded by pre-proopiomelanocortin is β-endorphin. In addition to β-endorphin, the proopiomelanocortin precursor encodes the
nonopioid peptides adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and β-lipotropic pituitary
hormone (β-LPH). Pre-proenkephalin encodes multiple copies of Met-enkephalin, including two extended forms of Met-enkephalin (a
heptapeptide and an octapeptide), and a single copy of Leu-enkephalin. Pre-prodynorphin encodes three opioid peptides of various
lengths that all begin with the Leu-enkephalin sequence: dynorphin A, dynorphin B, and neoendorphin (Fig. 3.1 ).

FIGURE 3.1. The opioid-peptide precursors. (From Akil H, Owens C, Gutstein H, et al. Endogenous opioids: overview and
current issues. Drug Alcohol Depend 1998;51:127–140, with permission.)

The μ-opioid receptors (MORs), δ-opioid receptors (DORs), and κ-opioid receptors (KORs) have been isolated and cloned. The mouse
DOR receptor was the first opioid receptor cloned (1 ,2 ), and this initial cloning facilitated the rapid cloning of MOR and KOR from
various rodent species (3 ,4 ,5 ,6 ,7 ,8 and 9 ). The coding regions of human genes for these
 P.36

receptors were subsequently isolated and chromosomally assigned (10 ,11 and 12 ). These studies confirmed earlier pharmacologic
data indicating that all three receptors belong to the superfamily of seven transmembrane-spanning G protein-coupled receptors. A
high degree of structural similarity exists between the three opioid receptors, which is highest in transmembrane domains 2, 3, and
7 and the first and second intracellular loops. The extracellular loops diverge considerably among the three receptor classes, and
this divergence may explain differences in ligand selectivity among the opioid receptors (Fig. 3.2 ).

FIGURE 3.2. The opioid receptors display a high degree of structural similarity. Numbers refer to the percentages of amino
acid identity between the cloned μ-, δ-, and κ-opioid receptors.

The relationship between the opioid peptides and their receptors is complex. This has been reviewed in detail elsewhere (13 ), and
we will note only some salient features. It is clear from studies of the cloned receptors that high-affinity interactions between each
of the precursor and receptor families are possible (14 ). For example, the proenkephalin peptide Tyr-Gly-Gly-Phe-Met-Arg-Phe binds
with subnanomolar affinity to each of the cloned receptors. Similarly, although binding with greater affinity to the KOR, several of
the shorter prodynorphin peptides bind with reasonable affinity to the MOR and DOR. By contrast, the binding of shorter
proenkephalin peptides Leu-Enk (Tyr-Gly-Gly-Phe-Leu) and Met-Enk (Tyr-Gly-Gly-Phe-Met) readily discriminates between the three
receptor families. Overall, the KOR displays the greatest selectivity across the endogenous ligands, with an approximately 1000-fold
difference in affinity between the most preferred (Dyn A 1–7) and least preferred (Leu-Enk) ligand, whereas the MOR and DOR differ
only across a 10-fold range (14 ). These differences in selectivity could indicate the existence of distinct mechanisms for ligand
recognition, such that MOR and DOR recognize the common Tyr-Gly-Gly-Phe core, whereas the KOR discriminates among the larger
variation in C-terminal regions. Indeed, elegant studies in which receptor chimeras were used have identified the critical domains in
the three opioid receptors that help discriminate among the endogenous ligands (13 ).

Further attempts to detect novel opioid receptors resulted in the isolation of a clone with high structural homology to the opioid
clones but little or no binding affinity for the opioid ligands (15 ,16 ). The structural similarity between this orphan (or opioid
receptor-like) opioid receptor
 P.37

(ORL-1) and the opioid receptors is highest in the transmembrane regions and cytoplasmic domain and lowest in the extracellular
domains critical for ligand selectivity (see below). A ligand for the receptor was subsequently identified by two groups using
chromatographic fractionation techniques coupled to ORL-1-mediated inhibition of adenylyl cyclase (17 ,18 ). This 17-amino acid
peptide is identical in length and C-terminal sequence to dynorphin A. Curiously, the N-terminal is slightly modified (Phe-Gly-Gly-
Phe) from the opioid core described above. It has been termed orphanin-FQ or nociceptin because of its putative ability to lower
pain thresholds. The orphanin-FQ/nociceptin (OFQ/N) precursor has been cloned from mouse, rat, and human and has been
localized on the short arm of human chromosome 8 (19 ,20 ). In addition to OFQ/N, evidence suggests that this precursor may
encode other biologically active peptides. Immediately downstream to OFQ/N is a 17-amino acid peptide (OFQ-2) that also starts
with phenylalanine and ends with glutamine but is otherwise distinct from OFQ/N, and a putative peptide upstream from OFQ/N
may be liberated on post-translational processing (nocistatin). The OFQ/N system is a distinct neuropeptide system with a high
degree of sequence identity to the opioids. This slight change in structure results in a profound alteration in function. Thus, OFQ/N
is motivationally neutral, as indexed by conditioned place preference (21 ), and has pain modulators distinct from those of the
opioid peptides (see below). However, changes in as few as four amino acids endow the ORL-1 receptor with the ability to recognize
prodynorphin products while still retaining recognition of OFQ/N (22 ). These findings suggest that unique mechanisms may have
evolved to ensure selectivity against the opioids versus selectivity for OFQ/N.

Issues and Complexities Revealed by Multiple Pharmacologic Forms, Splice Variants,

and Receptor Dimers
The molecular cloning studies described above identified only a single gene encoding each of the opioid receptors. These findings
contrast with results of pharmacologic studies indicating the existence of two subtypes of MOR and DOR and up to four subtypes of
KOR, and suggesting additional receptor families (23 ,24 ,25 and 26 ). For example, the DOR1 subtype is held to display greater
affinity for the agonist DPDPE, whereas the DOR2 subtype displays greater affinity for the agonist deltorphin 2 (26 ). These two
subtypes may also make independent contributions to DOR antinociception (27 ). It is possible that further molecular cloning will
identify unique genes encoding these receptor subtypes. However, several authors have suggested that if multiple opioid-receptor
subtypes exist, they could be derived from a single gene, and multiple mechanisms might exist to achieve these distinct
pharmacologic profiles. In this section, we consider two such pathways to opioid-receptor diversity: alternative splicing of receptor
RNA and dimerization of receptor proteins.

Alternative splicing of receptor heteronuclear RNA (e.g., exon skipping and intron retention) has been accorded an important role in
producing in vivo diversity within many members of the superfamily of seven transmembrane-spanning receptors (28 ). For example,
alternative splicing of the coding region for the N-terminus of the corticotropin-releasing hormone CRH-2 receptor results in α, β,
and γ variants, each with a unique tissue distribution (see ref. 28 for review). It follows that splice variants may exist within each
of the three opioid-receptor families and that this alternative splicing of receptor transcripts may be critical for the diversity of
opioid receptors. A technique used extensively to identify potential sites of alternative splicing is antisense oligodeoxynucleotide
(ODN) mapping. The ability of antisense ODNs to target specific regions of cDNA allows systematic evaluation of the contribution of
individual exons to the observed properties of a receptor. Antisense ODN targeting of exon 1 of the cloned rat and mouse MOR
prevents morphine analgesia in these species (29 ,30 and 31 ). By contrast, administration of antisense ODNs targeting exon 2,
which are inactive against morphine analgesia, prevents the analgesia produced by heroin, fentanyl, and the morphine metabolite
morphine-6-β-glucuronide (M6G) (29 ,30 and 31 ). A similar disruption of M6G but not morphine analgesia is observed following
administration of antisense ODNs targeting exon 3 (29 ). These results suggest that unique MOR mechanisms may mediate the
analgesic effects of a variety of opiate alkaloids, and are consistent with the claim that these unique receptor mechanisms could be
achieved via alternative splicing. The use of antisense ODNs has also resulted in the identification of potential sites for splice
variation in the KOR and DOR (32 ). Central to the claim that these results reflect the existence of opioid-receptor splice variants is
the in vivo isolation of such variants. A splice variant of the MOR has been identified that differs considerably within its C-terminus
(33 ). As might be expected on the basis of the location of the alternative splicing, this variant exhibits a binding profile similar to
that of the cloned MOR but does not readily undergo the desensitization frequently observed following agonist exposure. Thus,
although it differs in composition, the existence of this splice variant cannot explain the results described above. However, just such
a variant was detected in mice subjected to targeted disruption of exon 1 (34 ). Thus, transcripts of the MOR that contained exons 2
and 3 were identified in exon 1-deficient mice. Moreover, whereas morphine analgesia was abolished, heroin and M6G analgesia was
retained in these mice (see below).

The physical interaction of receptors to form a unique structure (dimerization) has also been accorded an important role in
regulating receptor function. For example, dimerization of GABABR1 (γ-aminobutyric acid subtype B receptor 1) and GABABR2
subunits is required for the formation
 P.38

of a functional GABAB receptor (35 ). Both the cloned KORs and DORs have been found to exist in vitro as homodimers (36 ). However,
the most important demonstrations of this kind are those showing dimerization between different functional opioid-receptor types.
Jordan and Devi (37 ) coexpressed tagged KOR and DOR or tagged KOR and MOR and used coprecipitation techniques to show that
KOR and DOR can exist as heterodimers in vitro. Dimerization of these receptors profoundly altered their properties. The affinity of
the heterodimers for highly selective KOR and DOR agonists and antagonists was greatly reduced. Instead, the heterodimers showed
greatest affinity for partially selective agonists such as bremazocine. This pharmacologic profile is similar to that claimed for the
KOR2-receptor subtype, which suggests that receptor dimerization may explain at least part of the discrepancy between the
molecular and pharmacologic properties of opioid receptors. Heterodimerization thus offers a mechanism for the formation of novel
receptor forms and a possible explanation for the in vivo diversity of opioid receptors. It will be of particular interest to identify
those factors governing formation of opioid-receptor heterodimers, to determine if and how frequently opioid receptors dimerize in
vivo, and to generate ligands that selectively recognize the dimerized form of the receptors.

Signal Transduction Mechanisms and Their Adaptation after Chronic Stimulation

The opioid receptors couple to G proteins inhibiting adenylyl cyclase, activating inwardly rectifying K+ channels and decreasing the
conductance of voltage-gated Ca2+ channels (38 ). These mechanisms of signal transduction have been verified by studies of the
cloned receptors expressed in a variety of host cells (see ref. 39 for review). Studies with the cloned receptors have also indicated
that the opioid receptors may couple to an array of other second messenger systems, which include activation of the mitogen-
activated protein (MAP) kinases and the phospholipase C-mediated cascade leading to the formation of IP3 (inositol-1,4,5-
triphosphate) and diacyl glycerol (see ref. 40 for review). Prolonged exposure to opiates results in adaptations at multiple levels
within these signaling cascades. The significance of these adaptations rests, at least in part, in the causal relationship that may exist
between them and those seen at the organismic level following iterated exposure to opiates, such as tolerance, sensitization, and
withdrawal. We consider two such adaptations: those related to cyclic adenosine monophosphate (cAMP) signaling and those related
to receptor densensitization and internalization.

In an elegant series of experiments using rat locus ceruleus (LC) neurons, Nester and co-workers identified increased levels of
protein Giα and protein Goα, adenylyl cyclase, and protein kinase A following prolonged in vivo exposure to morphine (41 ,42 and 43 ).
This up-regulation of the cAMP signaling pathway mediates the increased excitability of LC neurons observed after prolonged
exposure to morphine and has been invoked as a causal mechanism for the increased or “rebound” activity of LC neurons frequently
observed when the drug is withdrawn (44 ,45 ). It follows that these compensatory adaptations in cAMP signaling could be causal to
the opiate withdrawal syndrome observed at the organismic level. Consistent with this possibility, infusions of a protein kinase A
inhibitor into the LC reduced the severity of the antagonist-precipitated withdrawal syndrome in rats (46 ,47 ). Importantly, these
adaptations are not unique to opioid receptors in the LC. Increased levels of adenylyl cyclase and protein kinase A in response to
chronic morphine exposure have also been detected in the nucleus accumbens and amygdala (48 ). However, the changes in levels of
G-protein subunits induced by this treatment are more complex, with decreased levels of Giα detected in the nucleus accumbens and
increased levels of Giα and Gio in the amygdala. These widespread changes are also of significance at the organismic level. For
example, infusions of a protein kinase A inhibitor into the periacqueductal gray (PAG) reduced the severity of the antagonist-
precipitated withdrawal syndrome (47 ), and inactivation of Gi and Go proteins in the nucleus accumbens reduced heroin self-
administration in rats (49 ).

The desensitization, internalization, and sequestration of opioid receptors following their activation may also constitute mechanisms
for adaptation in signaling relevant for understanding alterations in the physiologic impact of opiates. For example, phosphorylation
of MOR and DOR via protein kinase C results in a transient desensitization that could subserve acute tolerance to opiates (50 ,51 ,52
and 53 ). Similarly, the internalization of opioid receptors via a classic endocytic pathway may have important implications for the
physiologic impact of opiates. The internalization of opioid receptors occurs in a ligand-specific manner. For example, DAMGO and
methadone promote internalization of the MOR, but morphine does not (53 ,54 ). This ligand-specific internalization is determined,
at least in part, by differences in the conformational changes induced by the ligand and is independent of its ability to stimulate G-
protein signaling (55 ). These findings may offer a novel explanation of differences in the efficacy and abuse potential of various
opiates. However, at the time of this writing, few attempts have been made to study the relevance of these alterations in signaling
to the adaptations seen in response to opiate exposure in vivo. Perhaps the most interesting are demonstrations that acute
morphine analgesia is enhanced in mice in which the gene encoding β-arrestin 2 was disrupted (56 ). Opioid-receptor internalization
is mediated, at least in part, by the actions of the G protein-receptor kinases (GRKs). The GRKs selectively phosphorylate the
agonist-bound receptor promoting interactions with β-arrestins, which interfere with G-protein coupling and promote receptor
internalization (56 ).
 P.39

Demonstrations that acute morphine analgesia is enhanced in mice lacking β-arrestin 2 are consistent with a role for the GRKs and
arrestins in regulating alterations in responsivity to opiates in vivo. This finding is even more intriguing given the inability of
morphine to support arrestin translocation and receptor internalization in vitro (57 ).

RECEPTOR AND LIGAND KNOCKOUTS: INSIGHTS, ISSUES, AND

COMPLEXITIES
Part of "3 - Opioid Peptides and Their Receptors: Overview and Function in Pain Modulation "

Advances in understanding the molecular biology of the opioid family, coupled with developments in recombinant technology, have
resulted in the generation of mice with targeted disruptions of various opioid genes. The study of these animals offers unique
insights into opioid-receptor function. The initial study of these mice has allowed evaluation of the critical receptor subtypes
mediating the effects of a variety of opiate alkaloids and the selective peptide agonists. In addition, they have identified potential
interactions between receptor subtypes and suggested novel functions for opioids (e.g., reproductive function).

MOR Knockouts
The MOR gene has been disrupted via targeted deletion of exon 1 (34 ,58 ,59 ), exon 2 (34 ), or exons 2 and 3 (60 ). Disruption of
exons 2 and 3 had no detectable effect on development, health, and fertility (60 ), whereas disruptions of exon 1 impaired sexual
function in male mice, manifested by reduced mating activity, decreased sperm count and motility, and smaller litter size (59 ).
Evidence was also found for alterations in hematopoiesis—specifically, increased proliferation of granulocyte-macrophages and
erythroid and multipotential progenitor cells—in exon 1 knockout mice (59 ). Assessment of these mice has revealed that the MOR is
absolutely necessary for the analgesic effects of morphine. Thus, systemic, intracerebal ventricular, and intrathecal administration
of morphine failed to produce analgesia as assayed by tail flick, hot plate, and paw withdrawal tests across a wide dose range. For
example, doses of morphine as high as 56 mg/kg failed to produce analgesia in exon 1 knockout mice (58 ), and the median effective
dose (ED50) for morphine analgesia in exon 2 knockout mice exceeded 100 mg/kg (a potency shift of two orders of magnitude) (34 ).
The MOR is also required for the rewarding (indexed by levels of conditioned place preference) and immunosuppressive effects of
injections of morphine, and for the physical dependence induced by such injections (indexed by somatic signs of morphine
withdrawal) (60 ,61 ). By contrast, the analgesic efficacy of heroin and the major morphine metabolite M6G remains intact in exon
1-deficient mice (34 ). This result is consistent with the antisense mapping studies described above. Although successfully
identifying the critical receptor substrate for the therapeutic and recreational uses of morphine, these experiments have failed to
address the involvement of MOR in basal pain sensitivity convincingly. For example, considerable controversy has surrounded the
ability of an injection of naloxone to produce hyperalgesia in otherwise intact animals, and this has not been resolved by studies of
the MOR knockout mice. Sora et al. (58 ) reported that MOR knockout mice displayed increased sensitivity to noxious stimulation,
but this hyperalgesia was not readily detected by others (60 ). This difference could be related to differences in the impact of
specific exon deletion, as in measurements of reproductive function. Alternatively, a stress-induced analgesia, such as that
provoked by exposure to novel handling procedures or contextual cues, may have decreased basal pain sensitivity among control
animals.

In addition to providing insight into the mechanisms of actions of the opiate alkaloids, studies of MOR knockout mice have allowed
systematic investigation into the potential interactions between the three opioid-receptor families in vivo. Studies of DOR function
in MOR knockout mice have failed to detect compensatory changes in either the number or localization of DORs (62 ). Similarly, no
significant alteration in DOR signal transduction, as indexed by G-protein and adenylyl cyclase activity, has been observed (63 ). By
contrast, the analgesic efficacy of DOR agonists in these mice may be slightly reduced. Specifically, a reduction in DPDPE analgesia
appears most robust, whereas the analgesic effects deltorphin 2 have been found intact or slightly attenuated (63 ,64 ). This
evidence for MOR-mediated effects of DOR agonists is intriguing and consistent, at least in part, with the possibility of interactions
between MOR and DOR in vivo. However, it is worth bearing in mind that these studies uniformly indicate the preservation of a large
component of DOR function in MOR knockout mice. Studies of KOR function in MOR knockout mice have also failed to detect
significant alterations in receptor number, distribution, and signal transduction (62 ,63 ). However, no evidence has been found of a
reduction in the analgesic efficacy of KOR agonists, unlike that of DOR agonists, in MOR knockout mice (64 ).

DOR Knockouts
The DOR gene has been disrupted in mice via targeted disruption of exon 2 (65 ). This deletion had no detectable effects on the
health or reproductive function of the mice. Deletion of exon 2 completely abolished [3H]DPDPE and [3H]deltorphin 2 binding in the
brain, which indicates that the putative subtypes of the DOR are encoded by the same gene product. Studies of pain sensitivity in
these mice indicate that basal pain sensitivity is unaffected by disruption of the DOR gene. Spinal DPDPE and deltorphin 2 analgesia
is significantly reduced in the DOR knockout mice. By contrast, the analgesic efficacy of intracerebral ventricular infusions of DPDPE
and deltorphin 2 remains intact. The
 P.40

retention of supraspinal but not spinal DOR analgesia in DOR knockout mice is surprising. This could be evidence for a novel receptor
mechanism because this residual supraspinal analgesia is reduced by naltrexone but not by selective MOR or KOR antagonists.
Disruption of the DOR gene has no significant effect on the levels and distribution of either MOR or KOR, nor is any effect noted on
the levels and distribution of proenkephalin, prodynorphin, and proopiomelanocortin. Similarly, no significant alterations occur in
the analgesic effectiveness of morphine, M6G, and the κ agonist U50,488H.

KOR Knockouts
The KOR gene has been disrupted in mice via targeted deletion of the initiation codon and N-terminal coding region (66 ). This
disruption had no detectable effects on the health of the mice but increased litter size. The deletion completely abolished [3H]CI-
977 binding in the brain. Studies of pain sensitivity revealed that KOR knockout mice are hyperalgesic when assayed by the acetic
acid writhing test but not the formalin, tail pressure, tail flick, and hot plate tests. This finding is consistent with the important role
accorded KOR in the regulation of visceral nociception. Systemic injection of the KOR agonist U50,488H failed to produce an
analgesic response as assayed by the tail flick and hot plate tests. Similarly, the locomotor depressive effects and aversive
motivational effects of the injection (indexed by conditioned place aversion learning) were abolished. These results indicate that
the analgesic and motivational effects of the prototypical KOR agonist are mediated via actions at the receptor(s) encoded by the
KOR gene and are consistent with results of antisense mapping studies indicating that ODNs directed against each of the three exons
of the KOR gene disrupt the analgesic efficacy of U50,488H. The effects of disruption of the KOR gene on the activity of dynorphin B
and α-neodynorphin, whose selectivity in antisense mapping studies differs considerably from that of U50,488H (67 ), remains
unclear. Disruption of the KOR gene had no significant effect on the levels and distribution of either MOR or DOR (68 ), nor was any
effect noted on the level and distribution of proenkephalin, prodynorphin, and proopiomelanocortin (67 ). Interestingly, the
analgesic efficacy of morphine was retained, but the aversive motivational effects of the dependence induced by iterated exposures
to morphine were reduced. This finding supports demonstrations of a role for dynorphin and the KOR in opiate withdrawal (69 ).

Pre-proenkephalin Knockouts
Mice with targeted deletions of exon 3 of the pre-proenkephalin gene have been created (70 ). Although this disruption had little
effect on levels of prodynorphin- and proopiomelanocortin-derived peptides, a large up-regulation of MOR binding in the striatum
was observed (71 ). Neither fertility nor gross abnormalities developed in the enkephalin knockout animals. These mice displayed
increased anxiety and fear-related behaviors (indexed by freezing, hiding, and performance in an open field and elevated O maze).
These results suggest that enkephalins are important in the negative feedback control of anxiety and aversive motivation.
Enkephalin knockout mice appeared hyperalgesic when tested with the hot plate, but not the tail flick, test. However, because the
procedure for this test involved repeated exposure to the hot plate apparatus, it is again unclear whether the experimental mice
were hyperalgesic or whether the control mice were hypoalgesic as a consequence of repeated testing in the hot plate apparatus.
The enkephalin knockout mice also showed altered sensitivity when assayed by the formalin test. Specifically, a decrease in
recuperative behaviors (lifting and licking the injected paw) could be mimicked by injection of naloxone (10 mg/kg) in wild-type
control mice, which suggests that the proenkephalin-derived peptides may regulate responding in the formalin test. This result is
also difficult to interpret because naloxone does not modulate formalin pain in rats under resting conditions. In short, across three
measures of pain sensitivity, three different influences of the deletion of the pre-proenkephalin gene were detected: no effect in
the tail flick test, increased sensitivity (hyperalgesia) in the hot plate test, and decreased sensitivity (indexed by recuperative
responding) in the formalin test. Although dissociations between these measures are not uncommon, the pattern of responding
across the three measures is difficult to interpret and underscores the complexity of pain modulation by aversive motivational states
such as anxiety and fear. Indeed, these mice displayed intact analgesic responses to stressors (swim stress) that produce naloxone-
reversible analgesia. This result is consistent with the binding studies reviewed above, indicating the potential for high-affinity
interactions between peptides derived from the proopiomelanocortin and prodynorphin precursors and each receptor class.

Orphanin-FQ-Receptor and Ligand Knockouts

The gene encoding the ORL-1 receptor has been disrupted via targeted deletion of exon 1 (72 ), and the OFQ/N precursor has been
disrupted via targeted deletion of exon 2 (73 ). Studies of these mice have proved particularly interesting. First, they have
confirmed that the role of OFQ/N in pain modulation is quite distinct from that of the other opioids. Thus, disruption of the ORL-1
receptor had no effect on basal pain sensitivity in the tail flick test but prevented the development of tolerance to morphine
analgesia (72 ), whereas disruption of the OFQ/N precursor consistently decreased pain sensitivity on the same measure. This
discrepancy between the effects of receptor and precursor disruption could be interpreted to mean that post-translational
 P.41

processing of the OFQ/N precursor may result in the presence of multiple active peptides that interact with unique receptors to
produce different physiologic effects (see above). However, these studies uniformly indicate that if OFQ/N and the ORL-1 receptor
have any role in pain modulation, it is facilitative (or pronociceptive) rather than inhibitory (or antinociceptive). Second, these
studies have confirmed that OFQ/N serves an important role in the regulation of emotional responsiveness. Specifically, OFQ/N
knockout mice display increased anxiety (indexed by performance in the elevated plus maze, open field, and light–dark box) and
enhanced basal and post-stress glucocorticoid levels. Interestingly, these findings contrast with the effects of administration in rats.
Devine et al. (74 ) have shown that infusions of OFQ/N increase plasma ACTH and glucocorticoid levels in the unstressed animal and
prolong the stress response in the stressed rat. The reasons for this discrepancy are unclear. Finally, these studies have suggested an
important role for the OFQ/N system in learning and memory processes. Thus, OFQ/N-receptor knockout mice show enhanced
hippocampal long-term potentiation and a moderately enhanced performance in tests of spatial learning (75 ). However, the OFQ/N-
precursor knockout mice do not show enhanced performance in the same test of spatial learning (73 ). Regardless of the reason for
the discrepancy between the OFQ/N and ORL-1 knockout mice, the interpretation of these effects on learning and memory is
difficult. For example, the spatial task used in these experiments can be mediated by several learning strategies. Clearly, a more
sophisticated characterization of the nature of the potential learning and memory deficits in these mice is required, and the results
described above provide an important starting point.

Summary
Studies of mice with targeted disruptions of opioid-receptor and peptide genes have enabled important insights into the function of
the opioid family. Chiefly, they have made possible the identification of the critical receptor substrates for a variety of opiate
alkaloids and opioid peptides. These studies have also provided insights into the functional diversity of each receptor class. For
example, it is clear that the two subtypes of DOR identified in pharmacologic studies are encoded by the single cloned DOR gene.
Furthermore, the retention of MOR- and KOR-independent supraspinal DPDPE analgesia in these mice raises the possibility that
further, unidentified opioid-receptor variants may exist. A similar possibility is raised by the retention of heroin and M6G analgesia
in mice with targeted deletions of the MOR. These results suggest that complex post-transcriptional modifications play an important
role in producing the in vivo diversity of opioid-receptor pharmacology. At the time of this writing, only OFQ/N-receptor and OFQ/N-
precursor knockout mice have been studied in more complex behavioral tasks. However, the widespread distribution of opioid
peptides and their receptors in the central nervous system, in addition to their critical role in controlling an animal’s interaction
with its environment, ensure that it is only a matter of time before mice are studied with more behaviorally sophisticated and
ecologically relevant measures of attention, learning, memory, and motivation. Finally, it is worth noting that this first generation
of genetic manipulations are neither tissue-specific nor conditional. Compensatory adaptation within the opioid-peptide and
receptor family following the targeted disruption of one of its members appears to be minimal. Indeed, in the studies reviewed
above, the only evidence for such compensation has been obtained for measures of receptor binding in pre-proenkephalin knockout
animals. Nonetheless, the possibility of widespread adaptation in nonopioid systems cannot be discounted. Thus, the application of
tissue-specific and inducible knockout techniques to the opioid receptors and their peptides remains an exciting area of research.

AN EXAMPLE OF OPIOID FUNCTION AND ITS CLINICAL IMPLICATIONS:

OPIOIDS AND PAIN CONTROL
Part of "3 - Opioid Peptides and Their Receptors: Overview and Function in Pain Modulation "

Understanding the role of opioids in pain modulation is not only of clinical importance but also of historical interest. Demonstrations
that microinjections of morphine into various brainstem regions are analgesic (76 ), and that injections of naloxone partially reverse
the analgesia produced by focal electric stimulation in these regions (77 ,78 ), provided the first physiologic evidence for an
endogenous opioid system. In this section, we briefly review the neural circuits subserving opioid analgesia and discuss recent
findings relevant to these actions. Many excellent reviews of this topic are available (79 ,80 ).

Functional Anatomy of Opioids in Descending Pain Control Circuits

It is well established that the analgesic effects of opioids arise from their ability to inhibit directly the ascending transmission of
nociceptive information from the spinal cord dorsal horn, and from their ability to activate pain control circuits that descend from
the midbrain, via the rostral ventromedial medulla (RVM), to the spinal cord dorsal horn. Opioid peptides and their receptors are
distributed throughout these descending pain control circuits (81 ,82 ). MOR messenger RNA (mRNA) or binding has been detected
throughout the PAG, pontine reticular formation, median raphe, nucleus raphe magnus and adjacent gigantocellular reticular
nucleus in the RVM, and spinal cord. Inspection of the discrepancies between levels of receptor binding and mRNA expression
provide important insights into the mechanisms of MOR analgesia. For example, the
 P.42

presence of significant MOR binding in the superficial dorsal horn but scarcity of mRNA expression suggests that the majority of
these spinal MOR binding sites are located presynaptically on the terminals of primary afferent nociceptors. This conclusion is
consistent with the high levels of MOR mRNA expression in dorsal root ganglia (DRG). A similar mismatch between MOR binding and
mRNA expression can be found in the dorsolateral PAG (strong binding vs. sparse mRNA). DOR mRNA and binding have been detected
in the ventral and ventrolateral quadrants of the PAG, pontine reticular formation, and gigantocellular reticular nucleus, but only at
low levels in the median raphe and nucleus raphe magnus. Like MOR binding sites, DOR binding sites are present in significant
numbers in the dorsal horn without detectable mRNA expression, which suggests an important role for presynaptic actions of DOR in
spinal analgesia. Finally, KOR mRNA and binding are widely distributed throughout the PAG, pontine reticular formation, median
raphe, and nucleus raphe magnus and adjacent gigantocellular reticular nucleus. Again, significant levels of KOR binding but sparse
levels of mRNA have been found in the dorsal horn. Although all three receptor mRNAs are found in the DRG, they are localized on
different classes of primary afferent nociceptors. Thus, MOR mRNA has been detected in medium- and large-diameter DRG cells,
DOR mRNA in large-diameter cells, and KOR mRNA in small- and medium-diameter cells. This differential localization could be linked
to functional differences in pain modulation.

The distribution of opioid receptors in descending pain control circuits indicates substantial overlap between MOR and KOR. The
largest differentiation between these two receptors and DOR is in the PAG, median raphe, and nucleus raphe magnus (82 ). A similar
differentiation of MOR and KOR from DOR is observed in the thalamus, which suggests that interactions between KOR and MOR may
be important for modulating nociceptive transmission from the dorsal horn as well as in higher nociceptive centres. The actions of
MOR agonists are invariably antinociceptive, whereas those of KOR agonists can be either antinociceptive or pronociceptive.
Consistent with the anatomic overlap between the MOR and KOR, the pronociceptive actions of the KOR appear to be mediated by a
functional antagonism of the actions of the MOR. The MOR produces antinociception within descending pain control circuits, at least
in part, via the removal of GABAergic inhibition of RVM projecting neurons in the PAG and spinally projecting neurons in the RVM
(79 ). Pan et al. (83 ) have presented evidence from both in vitro slice preparations and in vivo pain responding that the pain
modulatory effects of the KOR in the brainstem oppose those of the MOR. Thus, activation of the KOR hyperpolarized the same RVM
neurons hypopolarized by the MOR, and microinjections of a κ agonist into the RVM antagonized the analgesia produced by
microinjections of DAMGO into this region. These data are among the strongest that opioids can have pronociceptive in addition to
antinociceptive effects and could explain behavioral evidence for a reduction in hyperalgesia following injections of naloxone.

As described above, significant opioid-receptor binding, little detectable expression of receptor mRNA in the spinal cord dorsal horn,
but large levels of this mRNA in DRG have been observed. The anatomy of spinal opioid receptors suggests that their actions relevant
to analgesia at this level are predominantly presynaptic. At least one presynaptic mechanism viewed as having clinical significance is
the inhibition of spinal tachykinin signaling. Indeed, it is well established that opioids decrease the noxious stimulant-evoked release
of tachykinins from primary afferent nociceptors (84 ,85 ). Recently, the significance of this action has been questioned. Measuring
the internalization of neurokinin receptors following noxious stimulation, Trafton et al. (86 ) demonstrated that at least 80% of
tachykinin signaling remains intact after the intrathecal administration of large doses of opioids. These results indicate that
although opioid administration may reduce tachykinin release from primary afferent nociceptors, the reduction has little functional
impact on the actions of tachykinins on postsynaptic nociceptive neurons. The obvious implication of this finding is that either
tachykinin signaling is not central to nociception and/or opioid antinociception at the spinal level, or that, contrary to the
conclusions suggested by anatomic studies, the presynaptic actions of opioids are of little analgesic significance.

Just as important insights have been made into brainstem and spinal mechanisms for opioid analgesia, so too have insights been
made into forebrain mechanisms for such analgesia. It is well established that the actions of opioids in bulbospinal pathways are
critical to their analgesic efficacy. It has been less clear what role should be accorded forebrain actions and whether these actions
are independent of those in bulbospinal pathways. There can be little doubt that opioid actions in the forebrain contribute to
analgesia because decerebration prevents analgesia when rats are tested for pain sensitivity with the formalin test (87 ), and
microinjections of opioids into the several forebrain regions are analgesic in this test (88 ). However, because these manipulations
frequently leave intact the analgesic efficacy of opioids in measures of phasic nociception, such as the tail flick test, a distinction
has been drawn between forebrain-dependent mechanisms for morphine analgesia in the presence of tissue injury and bulbospinal
mechanisms for this analgesia in the absence of tissue injury. In an important series of experiments, Manning and Mayer (89 ,90 )
have shown that this distinction is not absolute and that opioid actions in the forebrain are also important to analgesia, both in
measures of tissue damage and in acute, phasic nociception. Thus, systemic morphine analgesia in both the tail flick and formalin
tests was disrupted by either lesioning or reversible inactivation of the central nucleus of the amygdala. The involvement
 P.43

of the amygdala in morphine analgesia is particularly interesting because this structure has been implicated in the environmental
activation of pain control circuits, and it projects extensively to those brainstem regions involved in descending pain control (80 ).

Role of OFQ/N and ORL-1 in Pain Modulation

OFQ/N mRNA and peptide are present throughout the descending pain control circuits described above. For example, OFQ/N-
containing neurons are present in the PAG, the median raphe, throughout the RVM, and the superficial dorsal horn (91 ) This
distribution overlaps with that of the opioid peptides, but the degree of colocalization remains unclear. ORL-1 binding and mRNA
can be detected in the PAG, median raphe, and RVM (92 ). In the spinal cord, ORL-1 mRNA expression is stronger in the ventral than
in the dorsal horn, but levels of binding are higher in the dorsal horn. High levels of ORL-1 mRNA are also found in the DRG. Despite
this clear anatomic evidence for a role of the orphanin system in pain modulation, its function remains unclear. As reviewed above,
targeted disruption of the ORL-1 receptor had little effect on basal pain sensitivity according to several measures, whereas targeted
disruption of the OFQ/N precursor consistently elevated pain sensitivity in the tail flick test, findings that suggest an important role
for OFQ/N in regulating basal pain sensitivity. Intrathecal injections of OFQ/N have been reported to be analgesic as assayed by the
tail flick and formalin tests (93 ,94 ). Similarly, these injections attenuated the hyperalgesia produced by constriction injury of
sciatic nerve (95 ). However, the profound motor effects of these injections render interpretation of changes in response latency
difficult. The effects of supraspinally administered OFQ/N are also difficult to interpret; hyperalgesia has been detected across a
variety of measures, but failures to detect hyperalgesia have also been reported.

Three interesting results may explain at least part of the variations noted in the effects of the orphanin opioid system in modulating
pain. First, Rossi et al. (95 ,96 ) reported a biphasic effect of OFQ/N administration, characterized initially by hyperalgesia and later
by analgesia. Second, Grisel et al. (97 ) reported that OFQ/N does not affect basal pain sensitivity but does reduce analgesia
according to the site of administration. Finally, Heinricher et al. (98 ) reported that OFQ/N exerts an inhibitory effect on several
classes of RVM neurons whose activity has been implicated in producing analgesia and hyperalgesia at the spinal level. These results
suggest that the effects on pain modulation observed following administration of OFQ/N in the intact animal are influenced by route,
time since administration, the presence of stressors that provoke analgesia (e.g., novel handling or test procedures), and the
current balance of activity in pain modulatory neurons in the RVM. The development of specific ORL-1-receptor antagonists will
undoubtedly enable a rapid clarification of the role of the orphanin opioid system in pain modulation.

CONCLUSIONS AND FUTURE DIRECTIONS

Part of "3 - Opioid Peptides and Their Receptors: Overview and Function in Pain Modulation "

The interplay between the orphanin system and the endogenous opioids represents a prime example of evolutionary changes that
have led to subtle diversity in structure and significant alteration in function. Indeed, this entire peptidergic family exemplifies the
way in which an increase in genetic diversity can lead from simple on/off signaling to a complex pattern of signaling wherein
multiple, coordinately secreted peptides interact with multiple receptors to effect a complex regulation of functions as diverse as
pain responsiveness, stress regulation, control of feeding, and modulation of development, learning, and memory. Many questions
remain to be answered in the context of the opioid family. At the most basic level is the question of whether additional members of
the family exist. The completion of sequencing of the human genome and the rat or mouse genome should help answer this question.
We should be able to lay to rest the questions of whether additional opioid-receptor types or subtypes exist, and whether other
endogenous ligands that are uniquely selective for a particular receptor type exist. In particular, endomorphin 1 (Tyr-Pro-Trp-Phe)
and endomorphin 2 (Tyr-Pro-Phe-Phe) have been proposed by Zadina et al. (99 ) to be endogenous, highly selective μ ligands.
However, their precursor remains uncloned, although the genome project should help clarify the matter. Further, as we obtain full
sequences of the genomes of other species, we should be able to track the fascinating evolutionary history of this peptide family.

At functional levels, many questions remain, especially concerning the exact role of endogenous opioids in addictive and emotional
behavior and psychiatric disorders. Because these disorders are typically of a complex genetic nature, involving the interaction of
multiple genes with one another and with the environment, it is likely that the endogenous opioid genes are involved in vulnerability
to certain brain-related illnesses. Here again, progress in genomics and complex genetics should open new avenues for investigating
the likely role of the opioid molecules in a range of psychiatric disorders.

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4
Norepinephrine

Gary Aston-Jones

G. Aston-Jones: Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

This chapter reviews findings from basic research concerning brain norepinephrine (NE) systems. The focus is on work that is
relevant to the mechanisms of psychiatric disorders, or the actions of drugs used to treat such disorders. The locus ceruleus (LC)
system receives most of the attention here, but recent findings concerning the role of the A2/A1 medullary cell groups in drug abuse
are also reviewed. Emphasis is placed on studies published since the last version of this volume. Space limitations prevent a
thorough review of the involvement of any brain NE system in mental function and dysfunction, so that only a fraction of the
relevant research can be covered. Apologies are offered to those whose work could not be included.

 MOLECULAR–GENETIC STUDIES
 NEUROANATOMY
 NEUROPHYSIOLOGY
 BEHAVIOR
 PSYCHOPATHOLOGY
 CONCLUSIONS
 ACKNOWLEDGMENTS

MOLECULAR–GENETIC STUDIES
Part of "4 - Norepinephrine "

Previous studies have revealed molecular properties of NE neurons and their effector systems that have extended our understanding
of the function and pharmacology of this system. For example, Duman et al. (1 ) have shown that acute opiate administration
decreases cyclic adenosine monophosphate (cAMP) and adenylate cyclase activity in LC neurons, whereas long-term use of opiates or
opiate withdrawal results in elevated activity in this second messenger mechanism. Continuing studies in this vein have resulted in a
more complete picture of molecular events and properties within LC neurons that help regulate their discharge activity. Thus, the
adenylate cyclase/cAMP system is up-regulated with chronic stress but down-regulated with long-term antidepressant treatment (2 ).
Additional studies indicate that impulse activity of LC neurons may be regulated in part by a nonspecific cation current that is
activated by this second messenger system (2 ). These findings suggest a molecular mechanism whereby the overall excitability of LC
neurons may be modulated in accordance with long-term environmental or pharmacologic conditions and may be involved in the
mechanisms of action of antidepressant and other psychopharmacologic agents.

Recent genetic studies have also revealed important aspects of NE systems relevant to their role in psychopharmacology. Xu et al.
(3 ) studied the brains of mice with a knockout of the NE transporter (3 ). These mice exhibited characteristics of animals treated
with antidepressants (i.e., prolonged clearance of NE and elevated extracellular levels of this catecholamine). In a test for
antidepressant drugs, the NE transporter knockouts behaved like antidepressant-treated wild-type mice, being hyperresponsive to
locomotor stimulation by cocaine or amphetamine. Importantly, these animals also exhibited dopamine D2/D3-receptor
supersensitivity. Thus, NE transporter function can alter midbrain dopaminergic systems, an effect that may be an important
mechanism of action of antidepressants and psychostimulants.

NEUROANATOMY
Part of "4 - Norepinephrine "

Chemoanatomy of the LC
The neuroanatomy of the major brain NE systems has been recently reviewed in detail (4 ), and only the most salient features are
described here. In the rat and primate (but not cat, guinea pig, and most other species), virtually all neurons located within the
compact LC nucleus are noradrenergic. It is notable that LC neurons also often contain other possible neurotransmitters (e.g.,
neuropeptides), and subsets of rat NE neurons can be distinguished by neurotransmitter molecules that they co-localize (see ref. 4
for review). Additional work is needed to determine the functional significance of co-localization of other transmitter molecules
within LC neurons.

Peri-LC Dendritic Shell

A prominent feature of LC neurons in all species is that their dendrites typically extend hundreds of micra from the parent cell body.
Our recent studies have revealed that these dendrites in rat are organized into two prominent collections that project outside the
nuclear core in the caudodorsal
 P.48

and rostroventromedial directions (5 ). This work has also demonstrated that these dendrites receive numerous synaptic contacts,
indicating that the extranuclear peri-LC processes serve as a substantial receptive surface for LC neurons.

Afferents to the LC
Prior studies indicated that prominent afferents to the LC include the nucleus paragigantocellularis (PGi) and the ventromedial
aspect of the prepositus hypoglossi (PrH) in the rostroventrolateral and dorsomedial medulla, respectively (6 ,7 ). These nuclei
provide strong excitatory and inhibitory influences on LC neurons, respectively, and are also sources of several neurotransmitter
inputs to the LC nucleus (see below) (4 ,8 ). However, as previously stated, LC dendrites that extend outside the LC nucleus proper
provide a prominent receptive surface for inputs to LC neurons (5 ). Studies of inputs to these peri-LC dendritic zones indicate
several additional possible strong inputs to LC neurons, including the periaqueductal gray, medial preoptic nucleus, prefrontal
cortex, and hypothalamus (4 ,8 ). Recent work has confirmed some of the proposed inputs, showing direct contacts onto peri-LC
dendrites from amygdala (9 ) and nucleus tractus solitarius (NTS) (10 ). Additional work is needed to test some of the other possible
inputs to LC distal dendrites. These dendritic inputs are important in revealing additional functional circuitry linked to the LC
system (e.g., limbic, autonomic, and cognitive functions).

A host of immunohistochemically defined fibers have been found in LC afferents (see ref. 4 for review). The sources of some of
these inputs have been determined. Strong glutamate (11 ) and epinephrine inputs (12 ) originate in the PGi, γ-aminobutyric acid
(GABA) inputs arise from the PrH (13 ), and strong enkephalin projections to the LC originate in both the PGi and the PrH (14 ).
Histamine fibers innervate the LC, presumably originating in the tuberomammillary nucleus (15 ). A particularly dense innervation by
serotonin fibers also exists; the origin of this projection has not been determined. Ultrastructural analyses have shown that several
of these inputs directly innervate LC neurons (16 ,17 ,18 ,19 and 20 ).

Most recently, the novel neuropeptide hypocretin (synonymous with orexin) has been shown to innervate the LC densely in rats and
monkeys (21 ,22 ,23 and 24 ) (Fig. 4.1 ). This projection presumably originates in the hypothalamus (the sole location of hypocretin-
producing cells) and is mirrored by dense projections to other nuclei associated with sleep and arousal functions (e.g., the raphe
serotonin neurons, tuberomammillary histamine cells, and cholinergic neurons of the brainstem). Initial studies of this peptide
suggested a role in feeding (24 ,25 ). However, more recent work has stimulated considerable interest in this neurotransmitter by
closely linking its function to sleep regulation. Specifically, mutations of the gene that makes a hypocretin receptor (26 ), or of
another gene that makes hypocretin itself (27 ), produced narcolepsy symptoms in animals. This finding supports the long-standing
belief that the LC system is important in sleep–waking processes (28 ) and indicates that sleep disorders may involve anomalies in
this hypocretin projection to the LC. These findings also offer a novel target for pharmacologic manipulation of the LC and other
systems involved in sleep function.

FIGURE 4.1. Photomicrograph showing dense innervation of the locus ceruleus (LC) by hypocretin/orexin Fibers. Low-power
(A) and high-power (B) photographs of frontal sections through the rat LC after staining with antibodies for hypocretin and
tyrosine hydroxylase (TH). Note the proximity of numerous black, punctate hypocretin fibers and brown TH-positive NE somata
and dendrites. (From Horvath TL, Peyron C, Sabrina D, et al. Strong hypocretin (orexin) innervation of the locus coeruleus
activates noradrenergic cells. J Comp Neurol 1999;415:145–159, with permission.) See color version of figure.

The functions of the different inputs to LC neurons or their dendrites are being revealed in behavioral and neurophysiologic studies.
Stimulation of the PGi strongly excites LC neurons (11 ). The PGi has strong autonomic functions, an observation consistent with the
marked parallel found between LC and sympathetic activities (29 ). These findings, together with the strong cortical projections of
LC neurons, suggest that the LC acts as a cognitive component of a global sympathetic system (8 ). In contrast, strong inhibition is
produced by PrH stimulation (13 ); the functional significance of this input is unclear. That inhibitory adrenergic input also arises
from the PGi is revealed when the strong glutamate input is antagonized pharmacologically (30 ). Inputs to distal LC dendrites from
the amygdala (9 ) or NTS (10 ) may convey limbic/emotional or autonomic information to the LC, respectively, although an influence
of activity in these afferents on LC activity has not yet been found (8 ,31 ). Our unpublished studies in monkey indicate that the
anterior cingulate cortex strongly innervates the LC (32 ). Some of our other recent results suggest that this input may modulate the
mode of LC activity and thereby its influence on cognitive performance (described below) (33 ). Finally, our recent studies using
transsynaptic retrograde tracing reveal that the suprachiasmatic nucleus is a prominent indirect afferent to the LC (34 ,35 and 36 ).
This is the first demonstration of a circuit that links the circadian suprachiasmatic nucleus mechanism with the arousal/alerting LC
system. Inasmuch as other studies have linked circadian disturbances with
 P.49

depression (37 ), and the LC system is also associated with depression and other mood disorders (38 ), this pathway may also be
important for affective function.

Topography of LC Efferents
It is well-known that LC axons are highly branched and have extensive efferents that ramify throughout the central nervous system,
providing NE innervation at all levels of the neuraxis (see ref. 4 for review). Previous studies have found topography among these
efferent projections (39 ), but the degree of specificity for projections of different LC neurons appears to be quite limited. Recent
studies by Simpson et al. (40 ) have revealed topography of a novel type. They report that LC neurons selectively collateralize to
different nuclei of the somatosensory system, so that individual neurons are more likely to send branches to thalamic and cortical
areas within the somatosensory system than to, e.g., a somatosensory thalamic nucleus and a visual cortical area. This “functional
topography” for projections of individual LC neurons provides a new dimension for the anatomic organization of this ubiquitous brain
system and may indicate a means for coordination or synchronization of NE release along relays in serial functional pathways.

A2 NE Neurons of the Caudal Medulla

Norepinephrine neurons in the A2 group (caudal NTS) have recently been implicated in behavioral functions of psychiatric
importance. Previously relegated solely to autonomic and visceral control (e.g., see ref. 41 ), the strong ascending projections of
these NE cells to forebrain areas such as the hypothalamus (42 ), bed nucleus of the stria terminalis (BNST) (43 ), nucleus accumbens
(44 ), and amygdala (45 ,46 ) have now been shown also to be important in affective and cognitive processes (43 ,47 ). As described
below, these findings identify new circuits for understanding affective and mnemonic functions.

NEUROPHYSIOLOGY
Part of "4 - Norepinephrine "

Several recent findings regarding the neurophysiology of LC neurons have extended our understanding of this system. Notably,
integration of studies at the cellular and behavioral levels indicates a potentially important role of coupling among LC neurons.

Electrotonic Coupling
Experiments by Christie and Williams and colleagues (48 ,49 and 50 ) showed that LC neurons may be regulated by electrotonic
coupling, not only during development but also in adults. Additional studies by these workers indicate that such coupling may be
modulated by inputs to LC neurons that alter cAMP (51 ). This is significant because electrotonic coupling allows rapid, powerful
cell-to-cell communication (electrically and biochemically) via large transmembrane channels between neurons (called gap
junctions). Once relegated to the domain of the esoteric but unimportant, electrotonic coupling is now being demonstrated in an
increasing number of central neurons. Of great interest is the fact that such coupling is readily modulated by other inputs to coupled
cells—for example, in the retina, coupling is strongly attenuated by dopamine inputs in a cAMP/protein kinase A manner. This line of
work is very promising in neuropsychopharmacology because it suggests a novel set of targets (receptors that regulate electrotonic
coupling) that could be used to develop new drugs to modulate the function of systems important in mental function and
dysfunction (such as the LC). Our recent work (described below) shows how modulation of such coupling can have profound
influences on behavior and cognitive performance (33 ). It is noteworthy that electrotonic coupling has been reported among striatal
neurons in a dopamine-modulated manner (see Chapter 9 , this volume), as well as among interneurons in the cerebral cortex
(52 ,53 ).

LC Activity, Electrotonic Coupling, and Cognitive Performance in Behaving Monkeys

A possible role for electrotonic coupling among LC neurons in cognitive performance was revealed by combining our recordings of LC
neurons in monkeys performing a signal detection task with neural network modeling (33 ). In these recordings, LC neurons exhibited
two modes of activity during task performance: a phasic mode, in which LC cells responded phasically to target stimuli, and a tonic
mode, in which the tonic baseline activity of LC neurons was high but responses to target cues were absent. Moreover, the phasic
mode corresponded closely to focused attention and good task performance, whereas the tonic mode was associated with scanning
attentiveness and poor performance in this task, which requires focused attention. Task performance could be improved by systemic
or local (intra-LC) injection of clonidine during poor performance, which indicates a causal influence of these patterns of LC activity
on performance. A neural network model was constructed to investigate mechanisms involved in generating these modes of LC
activity and the corresponding task performance. Space limitations prohibit a full discussion of the findings, which are reported and
reviewed in recent publications (33 ,54 ). In brief, the model showed that modulated electrotonic coupling among LC neurons could
produce the patterns of LC firing observed in the monkeys, and that known modulatory effects of NE could then translate these
modes of LC activity into corresponding levels of task performance, also observed in the monkeys (Fig. 4.2 and Fig. 4.3 ). These
findings have a number of implications for neuropsychopharmacology.
 P.50

First, they support the view that the LC has an important role in attentional processes, and that pathology in LC function could
contribute to mental disorders with attentional components [e.g., attention-deficit/hyperactivity disorder (ADHD), stress disorders,
schizophrenia]. These results also indicate that alterations in coupling among widely projecting neurons can have profound mental
and behavioral consequences, offering a new dimension for analyzing the function of highly divergent modulatory brain systems.
Finally, these results, in view of other findings that electrotonic coupling can be rapidly modulated by neurotransmitter inputs (55 ),
indicate that coupling may be a valuable new target for pharmaceutical development in neuropsychopharmacology.

FIGURE 4.2. Simulation of locus ceruleus (LC) activity by modulated electrotonic coupling. Upper: Post-stimulus time
histograms (PSTHs) for LC activity during the visual discrimination task. A,B: Response for targets. C,D: Response for
distractors. A,C: Periods of good performance (phasic LC mode). B,D: Poor behavioral performance (false alarm rate typically >
7%; tonic LC mode). Stimuli occur at time zero. All histograms are normalized to a standard of 100 trials. Note that the phasic
LC mode is found during periods of good performance, and that the tonic mode corresponds to poor performance on this task.
Bin width, 10 ms. Lower: Simulation of LC responses. A,B: Response to targets. C,D: Response to distractors. A,C: Coupling
among LC neurons. B,D: No coupling among LC neurons. These simulation PSTHs are normalized for 100 trials, as for the
empiric data. Note that coupling reduces tonic (baseline) LC activity but increases phasic (transient) response to target
stimuli, capturing the phasic mode of LC neurons in our recordings. See Fig. 4.3 for corresponding behavioral simulation
results. (From Usher M, Cohen JD, Rajkowski J, et al. The role of locus coeruleus in the regulation of cognitive performance.
Science 1999;283:549–554, with permission.)

FIGURE 4.3. Simulation of behavioral performance by modulated coupling among locus ceruleus (LC) neurons. Left: Graphs
showing higher rate of false alarm errors (% FA) during epochs of poor versus good performance by monkeys in the visual
discrimination task (33). No differences were noted in the percentage of hit responses during the various levels of
performance, as misses were rare. Right: Graphs showing higher % FA in the simulated data from our model (33) during epochs
of low versus high coupling among LC neurons. Note similarity to empiric data at left. See Fig. 4.2 and ref. 33 for further
details.

Opiate Withdrawal
A long series of studies has implicated the LC system in opiate withdrawal (see ref. 56 for review). Recent work has
 P.51

shed light on molecular and cellular changes that occur in LC neurons during long-term opiate exposure that may underlie their
strong activation during withdrawal (reviewed above). It is generally acknowledged that the bulk of this hyperactive LC response is
mediated by glutamate inputs from the PGi (11 ,57 ,58 ). However, a possible intrinsic source of withdrawal-induced hyperactivity in
LC neurons has been somewhat controversial. Although some studies find no evidence for withdrawal-induced activation of LC
neurons in slices taken from morphine-dependent rats (59 ,60 ), others have presented evidence for such intrinsically mediated
withdrawal responses in LC (61 ,62 ,63 and 64 ). Our study of local intra-LC microinfusion of opiate antagonists in morphine-
dependent rats has confirmed the likelihood that intrinsic changes with dependence contribute to the hyperactivity of these neurons
during withdrawal (65 ). Different studies have suggested different mechanisms for this locally mediated withdrawal effect. Lane-
Ladd et al. (62 ) and Nestler and Aghajanian (66 ) have presented evidence from slice experiments consistent with the possibility
that long-term morphine exposure causes a sustained increase in a tetrodotoxin-insensitive Na+ current, linked to the increase in
cAMP, adenylate cyclase activity, and cAMP response element-binding protein (CREB) that occurs in the LC during withdrawal. In
their view, this inward current causes LC hyperactivity when the inhibitory influence of morphine is removed during withdrawal. Our
recent in vitro studies suggest a different mechanism. These results indicate that long-term opiate administration produces a
decrease in K+ conductance in LC cells that leads to a state of increased excitability when the inhibitory influence of morphine is
removed during withdrawal (63 ,64 ). The decreased K+ conductance during long-term morphine administration may be a direct
compensatory response to the increased K+ conductance evoked by acute opiates (49 ). In either case, it seems clear that the local
component of withdrawal-induced activation of LC neurons is small compared with the strong excitation evoked by the increased
glutamate input from the PGi (see above).

Hypocretin/Orexin
As discussed above, the hypothalamic neuropeptide hypocretin, which is strongly implicated in sleep regulation, densely innervates
the LC in rat and monkey (21 ). Recent studies have revealed that this peptide activates LC neurons both in vitro (21 ,67 ) and in
vivo (68 ). The activation is associated with a mild depolarization but is independent of tetrodotoxin and Ca2+ (67 ). The results have
led to the tentative conclusion that hypocretin activates LC neurons by decreasing a resting potassium conductance (67 ). Overall,
the results are important because they indicate a possible pathway and transmitter mechanism by which the LC becomes activated
during arousal from sleep, which may in turn help to drive a sleep-to-waking transition. This pathway could be involved also in the
psychiatric disorders associated with sleep dysfunction (e.g., depression, stress disorders, ADHD).

Cortical Influences on LC Activity

Tract-tracing studies have revealed that the prefrontal cortex may directly innervate LC neurons. Our retrograde and anterograde
studies in rat find a projection from the medial prefrontal cortex to the extranuclear peri-LC dendritic zone (69 ). Another of our
studies confirms a projection from the cingulate cortex to the LC in the monkey (32 ). In line with these findings, additional
experiments have revealed prominent effects of cortical stimulation on LC activity. As shown in Fig. 4.4 , we found that electric
stimulation of the medial prefrontal cortex in rats activates LC neurons; similar results were obtained with chemical stimulation
(70 ). We also found this activation to be mediated by glutamate release within the LC, as would be expected for a direct cortical
(presumably glutamatergic) input (71 ). In contrast, Sara and Herve-Minvielle (72 ) reported that medial prefrontal stimulation in
rats results in inhibition of LC activity. Procedural differences may underlie the different results. In particular, the study by Sara and
Herve-Minvielle used ketamine anesthesia, a potent glutamate antagonist. Thus, the results may indicate an underlying inhibitory
effect of prefrontal activation on LC activity when the more potent glutamate-mediated excitation is antagonized. In any case, the
results reveal that the prefrontal cortex can strongly influence activity of LC neurons.

FIGURE 4.4. Activation of locus ceruleus (LC) neuron by stimulation of medial prefrontal cortex (PFC) in rat. A: Cumulative
post-stimulus time histogram (PSTH) for single-pulse electric stimulation of the PFC. Stimulation presented at arrow. B: PSTH
for train stimulation (20 Hz for 0.5 s) given during the epoch designated by small dots. Bin width in each PSTH, 5 ms. C:
Response of an LC cell to stimulation of PFC with 100-mM glutamate (at bar below). D: Response of an LC neuron to
stimulation of PFC with 10-mM D,L-homocysteic acid plus 50-μM bicuculline (DLH + bic; 60-nL injection). (From Jodo E, Chiang
C, Aston-Jones G. Potent excitatory influence of prefrontal cortex activity on noradrenergic locus coeruleus neurons.
Neuroscience 1998;83:63–80, with permission.)

Postsynaptic Actions of NE
The proposed role of the NE–LC system in arousal was confirmed by Berridge and Foote (73 ), who showed that local activation of LC
neurons by microinjection of bethanechol produces EEG activation in the halothane-anesthetized rat. Similar studies demonstrated
that LC inactivation by local microinfusion of clonidine decreases EEG arousal (74 ). Additional experiments revealed that the
arousing effects of LC stimulation are mimicked by stimulation of β adrenoceptors within the medial septum and are blocked by β-
receptor antagonists infused into this area (75 ). Continuing studies along these lines confirmed that local LC stimulation in waking
animals increases EEG and behavioral indices of arousal (76 ). Additional studies found, however, that septal infusion of β
antagonists in unanesthetized animals does not decrease arousal (77 ). Thus, in the waking rat, actions at other NE or non-NE
receptors may also be necessary for arousal. Together, these studies indicate that LC activity is an important regulator of EEG
arousal, and that these effects are mediated, at least in part, by β receptors in the medial septum area. Additional studies are
needed to determine the precise location of these actions and what other systems and receptors may be important for maintaining
the alert state.
 P.52

Studies in intact animals have shown that β-receptor activation from the LC can induce plasticity in hippocampal responses. Chaulk and Harley (78 ) found that in vivo or in vitro
administration of β- or α-receptor agonists significantly potentiates the population spike amplitude recorded in the dentate gyrus in response to perforant path stimulation. Because
the LC is the sole source of NE in the hippocampus, these findings confirm previous results that LC stimulation also potentiates such dentate gyrus responses (79 ,80 ). These results
indicate a role for NE from the LC in plasticity in hippocampal activity, and may provide evidence for a role of this system in memory consolidation (described below).

BEHAVIOR
Part of "4 - Norepinephrine "

Opiate Withdrawal and the LC

Several recent studies in which behavioral pharmacologic techniques were used have reexamined the role of the LC system in opiate withdrawal and abuse. The results of lesion studies
by Chieng and Christie (81 ), Caille et al. (82 ), and Delfs et al. (43 ), in which different methods and approaches were used, all agree that the LC system is not necessary for physical
signs of morphine withdrawal (Fig. 4.4 ). This finding contrasts with previous ideas and represents a significantly changed view of the role of the LC system in withdrawal. Although
some studies involving microinjection of agents that alter LC activity (83 ) or molecular events within LC neurons (62 ,84 ) implicate the LC in withdrawal responses, their results must
be viewed with caution because diffusion of injected substances from the small LC nucleus to adjacent areas that have been implicated in withdrawal, such as the periacqueductal
gray (85 ), difficult to rule out. Further studies are needed to determine the behavioral consequences of LC hyperactivity during opiate withdrawal.

Critical Role of A2 NE Neurons Innervating the BNST in Aversion Induced by Opiate

Withdrawal
Our recent work has demonstrated that NE innervation of the BNST from A2 noradrenergic neurons is critical for affective responses to opiate withdrawal (43 ,86 ). We demonstrated
that antagonists of β receptors injected into the BNST, or lesions of the ventral NE bundle that carries fibers from the A2 group to the BNST, eliminate aversive responses to withdrawal
(Fig. 4.5 ). Interestingly, these same manipulations had almost no effect on the physical withdrawal response. These findings, and other results showing that aversive responses to
withdrawal can occur in the absence of somatic responses (87 ,88 ), indicate that withdrawal aversion is not simply a consequence of physical symptoms, and that separate pathways
are involved in physical and affective withdrawal responses (Fig. 4.5 and Fig. 4.6 ). This is important for neuropsychopharmacology because the affective response during withdrawal is
the most potent motivator of further drug seeking (89 ). Thus, studies to develop pharmacotherapies for opiate abuse should focus on aversive withdrawal responses specifically, rather
than examining only physical signs. Lesions of the LC system had no effect on aversive or physical signs of withdrawal, findings that corresponded to other recent results (discussed
above).

FIGURE 4.5. Effects of intra-BNST (bed nucleus of the stria terminalis) injection of
noradrenergic drugs on conditioned place aversion and somatic signs of opiate
withdrawal. A–D: Effects of the β-antagonist cocktail betaxolol/ICI 118,551 (A,B) or
propranolol isomers (C,D) on place aversion and somatic signs. E,F: Effects of ST-
91 on place aversion and somatic signs. TC, teeth chatter; ET, eye twitch; WDS,
wet dog shakes; JUMP, jumping; WR, writhing; PG, penile grooming; PT, paw
tremor. All data are expressed as mean ± standard error of the mean (n = 6 to 8
animals per dose). For A–D, p < .05, analysis of variance followed by Fisher’s PLSD
test for multiple comparisons. For E,F, p < .05, Student’s t-test. (From Delfs J, Zhu
Y, Druhan J, et al. Noradrenaline in the ventral forebrain is critical for opiate
withdrawal-induced aversion. Nature 2000;403:430–434, with permission.)

FIGURE 4.6. Effects of dorsal (DNAB) and ventral (VNAB) noradrenergic bundle
lesions on aversive and somatic signs of opiate withdrawal. A,C: Aversion scores.
Aversion score equals time in the naltrexone-paired side on the test day minus the
preconditioning day. B,D: Number of somatic counts in 30 minutes. See Fig. 4.5
legend for details and abbreviations. Nondependent lesioned animals exhibited
neither aversion nor somatic signs following naltrexone (data not shown). All data
are mean ± standard error of the mean (n = 6 to 8 control, 10 to 11 lesioned
animals per group). p < .05, analysis of variance followed by Fisher’s PLSD test for
multiple comparisons. (From Delfs J, Zhu Y, Druhan J, et al. Noradrenaline in the
ventral forebrain is critical for opiate withdrawal-induced aversion. Nature
2000;403:430–434, with permission.)

Memory and the LC

Recent studies by Clayton and Williams (90 ) have indicated new evidence for involvement of the NE–LC system in memory. Inactivation of the PGi (a major input to the LC, described
above) with either lidocaine or the GABA agonist muscimol immediately after acquisition in a one-trial inhibitory avoidance task produced marked deficits on a retention test given 48
hours later. Conversely, chemical stimulation
 P.53

of the PGi with glutamate following training in either an inhibitory avoidance or spatial delayed matching to sample radial maze task
enhanced retention performance when assessed 48 or 18 hours later, respectively (91 ). Given the excitatory connections between
PGi and LC, these findings suggest that pharmacologic manipulation of PGi neuronal activity may affect memory formation via
influences on LC and subsequent NE release in brain systems involved in the encoding of new information.

Recent studies by Przybyslawski et al. (92 ) have also indicated a role for the LC–NE system in memory. These experiments indicate
that memories are normally reconsolidated each time they are reactivated by relevant cues. They found that blockade of β
adrenoceptors after memory reactivation, during the consolidation process, produced impairment on future tests of the same
memory. These results indicate that reactivation of memory produces a β receptor-dependent intracellular cascade that reenacts
the consolidation process responsible for the initial memory acquisition. This NE-dependent lability of active memory traces
indicates a novel mechanism to target in pharmacologic manipulation of memory-related disorders, such as posttraumatic stress
disorder and Alzheimer’s disease.

Studies by Mao et al. (93 ) have found a role for α1 and α2 NE receptors in the dorsolateral prefrontal cortex in memory. Infusion of α1
agonists into the monkey prefrontal cortex produced deficits in working memory (93 ), whereas similar treatments with α2 agonists
improved memory performance (94 ).

Memory and the A2 NE System

Studies by McGaugh (95 ) during the last several years have established a role for NE stimulation of β receptors in the amygdala in
the strong memories that are established for emotionally salient events. Recently, this line of work has shown that the NTS is
involved in this process, as lidocaine anesthesia of the NTS prevents the memory-enhancing effects of peripheral epinephrine (47 ).
Because the A2 neurons of
 P.54

the NTS strongly innervate the amygdala, this finding indicates that the A2 neurons may be importantly involved in memory
modulation. These studies suggest that a “central nervous system–periphery–central nervous system long-loop” circuit may be
involved, in which descending activity in response to emotional events produces a peripheral response (e.g., epinephrine release);
this response in turn stimulates receptors on vagal afferents that then stimulate the NTS to release NE in its hypothalamic and
forebrain targets. This possible route for enhancement of emotional memories and other cognitive processes has received little
attention previously. Such a loop may also be involved in the activation of A2 neurons during opiate withdrawal that leads to the
corresponding aversive response (described above) (43 ). This is potentially important clinically and psychopharmacologically
because peripheral receptors on visceral afferent fibers that may be involved in mental disorders represent a novel mechanism and
target for new pharmacotherapies.

PSYCHOPATHOLOGY
Part of "4 - Norepinephrine "

Depression
Recent work by Miller et al. (96 ) has increased our understanding of the role of NE systems in depression. In their studies, reduction
of NE metabolites (presumably reflecting decreased NE turnover) after treatment with α-methyl-p-tyrosine (AMPT) caused no change
in scores on the Hamilton Depression Rating Scale in normal human subjects. In contrast, AMPT administration and reductions in NE
turnover in patients in remission from depression after treatment with desipramine or mazindol significantly increased the Hamilton
Depression Rating Scale measures of depressive symptoms (97 ). This change was not seen in patients under treatment with
serotonin antidepressants (fluoxetine or sertraline). The results indicate that monoamine deficiency alone may not produce
depressive symptoms, but that different types of depression exist that respond to manipulations of different monoamine systems.

Advances in understanding the actions of antidepressant drugs have highlighted the possible role of NE systems in depression. New
drugs such as venlafaxine, which inhibits reuptake of both serotonin and NE, have been found to be effective, particularly in
refractory depression (98 ). In addition, the highly effective antidepressant paroxetine, which was previously thought to act
selectively to block serotonin reuptake, has recently been found also to inhibit NE reuptake (99 ,100 ). These findings confirm long-
held beliefs that NE is importantly involved in depression, and indicate that blockers of NE uptake, including drugs that selectively
act at the NE transporter, such as reboxetine (101 ,102 ), may be effective in treating at least certain types of affective illness
(103 ).

Anxiety
Brain NE has long been implicated in anxiety disorders (104 ). Our studies with cocaine- and morphine-dependent animals have
provided new evidence for a role of central NE systems in anxiety. By means of a place-conditioning paradigm, we found that
withdrawal from long-term administration of morphine or cocaine is associated with strong anxiety, measured by the conditioned
burying paradigm (105 ). Importantly, the anxiogenic response to drug withdrawal is strongly attenuated by administration of the β-
receptor antagonist propranolol, and by similar doses of the lipophobic β1 antagonist atenolol, which is believed to act primarily
peripherally. These findings indicate that at least some types of anxiety involve stimulation of peripheral β adrenoceptors.

ADHD
The firing patterns of LC neurons in behaving monkeys indicate that this system plays an important role in attention and
performance (reviewed above) (33 ,54 ,106 ). In particular, one mode of LC activity, characterized by elevated tonic discharge,
corresponds to poor performance on a continuous performance task that requires focused attention, with a high rate of false alarm
errors. These and other results have led us to propose that this tonic mode of LC activity promotes high behavioral flexibility and
disables focused or selective attention (33 ,54 ). This view also implies that attentional disorders may be associated with LC
dysregulation in which the proper mode of activity is not engaged adaptively for the context at hand. Specifically, several parallels
have been noted between behaviors in monkeys during the tonic mode of LC activity and symptoms of ADHD, including
hypervigilance, irritability, poor focused attentiveness, and a high false alarm rate in continuous performance tasks. These findings
indicate that the LC may play an important role in ADHD, and that drugs that modulate LC mode, or switching between modes, may
be helpful in treating this disorder. In fact, many of the stimulants that are effective in treating ADHD decrease tonic LC activity.

A role for the LC–NE system in attentional disorders is also indicated by behavioral pharmacology experiments by Arnsten and
colleagues (107 ). These investigators have found that overstimulation of α1 receptors in the prefrontal cortex produces deficits in
behaviors that depend on prefrontal function (107 ). Because ADHD includes symptoms of prefrontal dysfunction, these findings raise
the possibility that an overactive LC system may contribute to ADHD by overstimulation of α1 receptors in prefrontal areas (108 ).

CONCLUSIONS
Part of "4 - Norepinephrine "

An impressive amount of research on NE systems has been performed since the previous edition of this volume was
 P.55

published. This work is revealing an increasingly important role for brain NE in mental function and dysfunction. Mechanisms by which NE systems are involved in cognitive, addictive,
stress-related, and other behavioral functions are being elucidated. This progress not only reinforces the importance of this system for neuropsychopharmacology, but also indicates
that NE systems represent a promising area for discovering new and fruitful approaches to developing treatments for psychiatric disorders.

ACKNOWLEDGMENTS
Part of "4 - Norepinephrine "

This work was supported by PHS grants NS24698, DA06214, DA10088, MH55309, and MH59978. Comments on the manuscript by Drs. Glenda Harris and Jon Druhan are greatly
appreciated.

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5
Regulation of G Protein-Coupled Receptors by Phosphorylation and
Endocytosis

Mark Von Zastrow

Mark von Zastrow: Department of Psychiatry, Department of Cellular and Molecular Pharmacology, and Program in Cell Biology,
University of California, San Francisco, California.

G protein-coupled receptors (GPCRs) comprise a large superfamily of heptahelical integral membrane proteins that mediate
transmembrane signal transduction in response to a wide variety of hormones, neurotransmitters, and neuromodulators. GPCRs are
extremely important targets for neuropsychopharmacology. Indeed, the vast majority of clinically relevant neuropsychiatric drugs
either bind directly to specific GPCRs (e.g., many antipsychotic drugs) or function indirectly via GPCRs by influencing the amount of
available native agonist (e.g., many antidepressant drugs).

A general feature of GPCRs is that they are extensively regulated in cells (1 ,2 ,3 and 4 ). Regulation of GPCRs is thought to play a
fundamental role in maintaining physiologic homeostasis in the face of fluctuating internal and external stimuli. A number of
pathologic states are associated with disturbances in the number or functional activity of certain GPCRs (5 ). In addition, many
clinically important drugs influence the physiologic regulation of GPCRs (6 ). Together, these observations suggest that mechanisms
of GPCR regulation may be of fundamental importance to neuropsychiatric disorders and to the actions of clinically relevant drugs.

The physiologic and biomedical importance of GPCR regulation has motivated an enormous amount of study into underlying
molecular mechanisms of regulation. Progress in this area has been facilitated enormously by molecular and cell biological
approaches applied to a variety of experimental model systems. Our understanding remains at an early stage and is limited, in most
cases, to studies of a small number of GPCRs. Nevertheless, great progress has been made in elucidating certain mechanisms of
GPCR regulation, to the extent that it is possible to begin to discern fundamental principles that control the number and functional
activity of GPCRs in individual cells.

The present chapter discusses some of this progress, with an emphasis on developing a unified view of GPCR regulation. We have
restricted our scope to a limited number of regulatory mechanisms that have been elucidated by detailed study of the some of the
most extensively characterized GPCRs. First, we survey classic studies describing the general properties of the physiologic and
pharmacologic regulation of receptor-mediated signaling; these have established a terminology and conceptual framework for our
later focus on specific mechanisms of receptor regulation. Second, we discuss pioneering studies of “prototypic” GPCRs that have
established paradigms for understanding the role of receptor phosphorylation in mediating rapid desensitization of GPCRs. Third, we
focus on a specific mechanism mediating regulated endocytosis of certain GPCRs, and discuss how this endocytic mechanism can
promote rapid desensitization and resensitization of receptor-mediated signal transduction. In this section, we also highlight the
close interdependence between mechanisms of GPCR phosphorylation and membrane trafficking in mediating rapid regulation of
receptor function. Finally, we discuss the functions of both phosphorylation and endocytic membrane trafficking in mediating
longer-term regulation of the number of GPCRs present in cells, focusing on recent studies into mechanisms that control down-
regulation of receptors via proteolytic degradation in lysosomes.

 GENERAL PROCESSES OF GPCR REGULATION

 SPECIFIC MECHANISMS OF GPCR REGULATION
 CONCLUSIONS AND FUTURE PERSPECTIVES

GENERAL PROCESSES OF GPCR REGULATION

Part of "5 - Regulation of G Protein-Coupled Receptors by Phosphorylation and Endocytosis "

Rapid Desensitization and Resensitization

It has been known for many years that multiple mechanisms can contribute to GPCR regulation (1 ,7 ). Early studies,
 P.60

which preceded the elucidation of any of the biochemical machinery involved, distinguished general processes of receptor regulation
according to differences in kinetics and reversibility. This is well illustrated by classic studies of the β2-adrenergic receptor (B2AR),
reviewed in detail elsewhere (2 ,3 and 4 ). Agonist-induced activation of the B2AR stimulates adenylyl cyclase via coupling to the Gs
heterotrimeric G protein (Fig. 1A ). Receptor-mediated signaling via this pathway occurs within seconds after agonist binding.
However, after more prolonged activation, the ability of receptors to activate adenylyl cyclase via Gs diminishes greatly. This
diminution of signal transduction is generally called desensitization and is mediated, at least in part, by regulation of the receptor
itself. A process of rapid desensitization was so named because it occurs within seconds to minutes after agonist-induced activation.
Rapid desensitization of the B2AR can be reversed within several minutes after removal of agonist in a process called resensitization.
Rapid desensitization of the B2AR is not associated with a decrease in the total number of receptors present in cells or tissues, and
resensitization does not require biosynthesis of new receptor protein. Therefore, rapid desensitization is thought to reflect a change
in the functional activity, rather than absolute number, of receptors (Fig. 5.1B ).

FIGURE 5.1. Desensitization and down-regulation of G protein-coupled receptors. Panel A: Within milliseconds to seconds
after agonist binding, receptors present in the plasma membrane mediate signal transduction to effectors by functionally
coupling (promoting guanine nucleotide exchange on) a heterotrimeric G protein. Panel B: Within several minutes after agonist
binding, rapid desensitization occurs by functional uncoupling of the receptor from G protein. This represents a change in the
functional activity of receptors, which inhibits signal transduction to the effector without changing the number of receptors in
the cell. Panel C: After more prolonged agonist-induced activation of receptors (typically several hours to days), the number
of receptors present in cells is greatly reduced, so signal transduction via G proteins to effectors is strongly attenuated. This
process is called receptor down-regulation because it is thought to represent primarily a reduction in the number, rather than
functional activity, of receptors.

Down-regulation and Up-regulation of Receptors

The kinetics of rapid desensitization and resensitization may be relevant to the physiologic action of catecholamines (endogenous
agonist ligands for the B2AR), as these molecules can be released intermittently by vesicular exocytosis and are rapidly removed
from the extracellular milieu by membrane transport, enzymatic degradation, or both. However, many clinically important drugs
that activate GPCRs have a more prolonged duration of action. Studies of these drugs established the existence of a distinct process
of receptor regulation that occurs much more slowly, typically within several hours to days after prolonged or repeated exposure of
tissues to ligand. This process is called down-regulation because (in contrast to rapid desensitization) it is associated with a
pronounced decrease in the total number of receptors present in cells or tissues, as typically detected by means of radioligand
binding techniques. Further distinguishing the process of down-regulation from rapid desensitization, recovery of signaling activity
after down-regulation is generally a slow process that requires biosynthesis of new receptor protein (8 ). Therefore, down-regulation
is thought to reflect primarily a change in the number, rather than functional activity, of receptors present in cells or tissues (Fig.
5.1C ). In most cases, down-regulation of GPCRs (like rapid desensitization) is induced by agonists, but not by antagonists. Moreover,
certain antagonists can induce an opposite process of increased receptor number called up-regulation (9 ,10 ). These observations
are consistent with a fundamental role of down-regulation and up-regulation as a negative feedback mechanism that seeks to
maintain physiologic homeostasis of receptor signaling. However, in some cases, processes associated with GPCR down-regulation
may be induced by antagonists (11 ,12 ). In other cases, up-regulation of receptors can be induced by drugs with partial agonist
activity (10 ). These observations suggest that certain clinically relevant drugs may not simply mimic or block the effects of
endogenous agonists. Indeed, it is proposed that such “paradoxical” regulatory effects may contribute to the pathologic or
therapeutic actions of certain clinically relevant
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neuropsychiatric drugs, including opiate analgesics and atypical antipsychotic agents (10 ,13 ,14 ,15 and 16 ).

Distinguishable Processes of Homologous and Heterologous Desensitization

Another important observation leading to our present view of GPCR regulatory mechanisms has come from studies investigating the
pharmacologic specificity of receptor regulation (1 ,17 ). Many cell types express multiple types of GPCR (Fig. 5.2A ). It has been
observed that in some cases, prolonged activation of one type of GPCR causes attenuated signal transduction only by that receptor,
without any detectable effect on signaling by other types of GPCR present in the same cell. In this case, the regulation of receptors
is said to be homologous (Fig. 5.2B ). The existence of homologous processes of regulation provided early evidence, before specific
regulatory mechanisms were elucidated, that signal transduction can be modulated by modification of the receptor itself. In other
cases, activation of one type of GPCR attenuates signaling not only by that receptor but also by other type(s) of GPCR present in the
same cell. Regulation of this kind is said to be heterologous (Fig. 5.2C ). Heterologous regulation of receptor signaling is consistent
with modification of a “downstream” component in the signal transduction pathway that is involved in signaling by more than one
type of GPCR. However, as discussed below, important examples of heterologous regulation mediated by modification of the
receptor protein itself also exist. Homologous processes of receptor regulation are capable of modulating signal transduction in a
highly specific manner, whereas heterologous processes of regulation may play an important role in facilitating functional “cross-
talk” between pharmacologically distinct signaling pathways (18 ).

FIGURE 5.2. Homologous and heterologous desensitization.

Panel A: Receptors 1 and 2 can signal via the same G

protein-mediated pathway. Panel B: Agonist 1 causes

uncoupling only of receptor 1; signaling induced by agonist

2 binding to receptor 2 is not affected. This is homologous

desensitization of receptor 1. Panel C: Agonist 1 causes

functional uncoupling of both receptor 1 and receptor 2, so

that signaling induced by both agonists is blocked. This is

heterologous desensitization.

SPECIFIC MECHANISMS OF GPCR REGULATION

Part of "5 - Regulation of G Protein-Coupled Receptors by Phosphorylation and Endocytosis "

Rapid Desensitization Mediated by Phosphorylation-Dependent Uncoupling of

Receptor from Heterotrimeric G Protein
It is well established that many GPCRs are regulated by phosphorylation. Classic studies of rhodopsin (a light-activated GPCR) and
the B2AR (a ligand-activated GPCR) provide examples of illustrating distinct molecular mechanisms that mediate homologous and
heterologous desensitization of receptors. Because the principles behind these mechanisms have proven to be widely applicable to
other GPCRs, rhodopsin, and the B2AR are often considered “prototypic” GPCRs that have established general paradigms for
understanding phosphorylation-dependent regulation of GPCRs (19 ).

Phosphorylation of Rhodopsin: a Model for Functional Inactivation of GPCRs

Elegant studies of the vertebrate visual system identified a critical role of phosphorylation in inactivating rhodopsin following light-
induced activation (20 ). Light-activated rhodopsin is a good substrate for phosphorylation by a cytoplasmic enzyme called rhodopsin
kinase, whereas rhodopsin that has not been activated by light is a poor substrate (20 ). Phosphorylation of the carboxyl-terminal
cytoplasmic domain of rhodopsin is sufficient to attenuate the ability of light-activated rhodopsin to couple functionally to its
cognate heterotrimeric G protein (transducin). Studies of rhodopsin function in isolated membrane fractions indicated that
rhodopsin kinase-mediated phosphorylation can strongly attenuate rhodopsin signaling, but it does so to a lesser extent than
observed in the intact rod cell. A second cytoplasmic protein was identified that, when added to membrane preparations in
combination with rhodopsin kinase, greatly accelerates the attenuation of rhodopsin signaling (21 ). This protein was proposed to
act as a protein
 P.62

cofactor that “arrests” signal transduction by phosphorylated rhodopsin and was therefore called arrestin (Fig. 5.3A ).

FIGURE 5.3. Paradigms for phosphorylation-dependent desensitization. A: GRK1-mediated inactivation of rhodopsin. B: GRK2-
mediated desensitization of the B2AR. C: PKA-mediated desensitization of the B2AR.

GRK-Mediated Phosphorylation of the B2AR: a Model for Homologous Desensitization

Studies of functional reconstitution of B2AR-mediated activation of adenylyl cyclase provided compelling evidence for a role of
phosphorylation in mediating rapid desensitization of a ligand-activated GPCR (22 ). Biochemical purification of the cytoplasmic
activity responsible for this phosphorylation identified a kinase called β-adrenergic receptor kinase (BARK), and it was noted that
this kinase preferentially phosphorylates agonist-occupied receptors (23 ). Protein sequencing and cyclic DNA (cDNA) cloning later
indicated that this kinase is closely similar in structure to rhodopsin kinase (24 ). Furthermore, cDNA-cloning experiments in which
reduced stringency hybridization was used identified additional kinases homologous to rhodopsin kinase and BARK (19 ). Collectively,
these observations led to the discovery of the family of G protein-coupled receptor kinases (GRKs). According to this nomenclature,
rhodopsin is denoted GRK-1, the originally identified BARK enzyme is denoted GRK-2, and other members of this family of protein
kinases are numbered sequentially thereafter. Six members of the GRK family of receptor kinases have been identified to date.

Biochemical reconstitution studies indicated that increasingly purified fractions of BARK exhibit a reduced ability to attenuate B2AR-
mediated signal transduction. Further analysis of this effect led to the identification of a distinct protein component that copurifies
with BARK in initial stages of purification but is resolved from BARK in more highly purified fractions. This protein component
reconstitutes strong attenuation of B2AR-mediated activation of adenylyl cyclase when added back to highly purified fractions of
BARK (25 ,26 ). The protein cofactor involved in desensitization of the B2AR turned out to be a protein similar to visual arrestin and
was therefore named β-arrestin or barrestin. cDNA cloning has identified additional proteins with similar structure, thus defining a
family of protein cofactors for phosphorylation-dependent regulation of GPCR function (4 ). Two nomenclatures are currently in
common use for these molecules. In one, the originally identified β-arrestin is denoted barrestin 1, and additional homologues are
named sequentially barrestin 2, and so on. In another nomenclature, all members of this protein family are referred to as arrestins,
with visual arrestin denoted arrestin 1, β-arrestin as arrestin 2, and subsequently identified family members numbered sequentially
thereafter. Four members of the arrestin family of protein cofactors have been identified to date.

As noted above, an important feature of many GRKs is that their kinase activity is highly sensitive to the conformation of the
receptor that they phosphorylate. This property of GRKs facilitates specific phosphorylation of only those receptors that are
activated by ligand, whereas other receptors present in the same cells (but not activated by agonist) are not phosphorylated. Thus,
GRK-mediated phosphorylation is generally considered to be a paradigm for homologous desensitization (Fig. 5.3B ).

Protein Kinase A-Mediated Phosphorylation of the B2AR: a Model of Heterologous

Desensitization
Other kinases, such as the so-called second messenger-regulated kinases, are also implicated in mediating desensitization of GPCRs.
For example, the B2AR can be phosphorylated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). PKA-
mediated phosphorylation of a single residue located in the third intracellular loop of the B2AR impairs the ability of the receptor to
couple to Gs and subsequently activate adenylyl cyclase (27 ,28 and 29 ). Phosphorylation of this residue is thought to impair
receptor–G protein coupling directly, without a requirement for any known protein cofactor such as an arrestin. An important
feature of PKA is that this kinase can phosphorylate B2ARs whether or not they have been activated
 P.63

by ligand, in contrast to the preferential phosphorylation of agonist-activated receptors by GRK-2. Because PKA is activated by cAMP,
a signaling intermediate produced as a result of B2AR activation, PKA-mediated phosphorylation of the B2AR can be viewed as an
example of feedback inhibition by a second messenger. In addition, because activation of any other receptor that stimulates
adenylyl cyclase can also activate PKA, phosphorylation of the B2AR by PKA is generally considered to be a paradigm for
heterologous desensitization (Fig. 5.3C ).

Rapid Desensitization and Resensitization Mediated by Regulated Endocytosis of

Receptors

Endocytosis of GPCRs by Clathrin-Coated Pits

Studies conducted in cultured cells indicate that many GPCRs can be regulated by ligand-induced endocytosis. The B2AR is perhaps
the most extensively studied GPCR with respect to endocytic membrane trafficking. Early evidence for agonist-induced endocytosis
of the B2AR was suggested by observations from subcellular fractionation and radioligand binding assays conducted with membrane-
impermeant antagonists (30 ,31 ). These studies indicated that the number of B2AR binding sites detected in the plasma membrane
can be reduced within several minutes after agonist-induced activation, a process called sequestration.

The development of receptor-specific antibodies allowed the application of immunocytochemical methods to visualize the
subcellular localization of the B2AR and directly demonstrate agonist-induced internalization of the receptor protein. Internalization
of the B2AR was observed to represent a steady state of a highly dynamic process involving continuous endocytosis and recycling of
receptors through an endocytic pathway similar to that mediating constitutive (ligand-independent) endocytosis of nutrient
receptors (32 ). This dynamic cycling of the B2AR was also suggested by elegant studies in which subcellular fractionation and
radioligand binding techniques were used (33 ).

Regulation of B2AR endocytosis was shown to be mediated by a ligand-dependent lateral redistribution of receptors in the plasma
membrane, from a relatively diffuse distribution throughout the plasma membrane to a pronounced concentration of agonist-
activated receptors in structures resembling clathrin-coated pits when examined by immunoelectron microscopy. Furthermore, this
process of ligand-regulated concentration of B2ARs in coated pits of the plasma membrane was shown to be mechanistically distinct
from the subsequent endocytosis of receptors by membrane fission, which can occur even in the absence of continued ligand-
induced activation of receptors (34 ). A protein that is required for this latter step of endocytic membrane fission is the cytoplasmic
guanosine triphosphatase dynamin (35 ,36 ). Consistent with this, agonist-induced endocytosis of the B2AR is inhibited by
overexpression of certain “dominant-negative” mutant forms of dynamin (37 ,38 ). Subsequent studies have demonstrated that
regulated endocytosis of several other GPCRs is also mediated by a dynamin-dependent mechanism, which suggests a conserved role
of clathrin-coated pits in the endocytosis of many GPCRs (Fig. 5.4 ).

FIGURE 5.4. Regulated endocytosis of the β2-adrenergic receptor (B2AR) by clathrin-coated pits. G protein-coupled receptor
kinase-mediated phosphorylation of the B2AR promotes receptor interaction with nonvisual arrestins, which cause uncoupling
of heterotrimeric G proteins and also promote interaction of arrestin–receptor complexes with clathrin coats. Once
concentrated in clathrin-coated pits by this mechanism, receptors undergo endocytosis rapidly (even if agonist is removed from
the receptor in the coated pit) via a constitutive (ligand-independent) mechanism of endocytic membrane fission that requires
the cytoplasmic guanosine triphosphatase dynamin.

Role of GPCR Phosphorylation in Promoting Endocytosis by Clathrin-Coated Pits

Studies elucidating the mechanism mediating the key step of regulated concentration of GPCRs in clathrin-coated pits were initiated
by the observation that GRK-mediated phosphorylation of the M2 muscarinic acetylcholine receptor can promote agonist-induced
endocytosis of the receptor, whereas a kinase-defective mutant form of GRK inhibits this process (39 ). Similar observations were
made for other GPCRs, including the B2AR (37 ,40 ). In an elegant series of
 P.64

experiments, it was shown that certain arrestins can directly promote B2AR concentration in clathrin-coated pits by physically
linking phosphorylated receptors with clathrin (40 ). This endocytic “adapter” function of arrestins can be distinguished from the
function of arrestins as cofactors for functional uncoupling of receptor from G protein because the latter process can occur in the
absence of endocytosis. Further distinguishing these functions, visual arrestin (arrestin 1) was shown to be unable to serve as an
adapter for B2AR endocytosis even though it can serve as a cofactor for desensitization mediated by functional uncoupling of G
protein (41 ). This distinction between visual and nonvisual arrestins led to the identification of a carboxyl-terminal clathrin-binding
domain, present specifically in nonvisual arrestins, that is necessary for endocytosis of GPCRs but not for phosphorylation-dependent
uncoupling of receptors from heterotrimeric G proteins (42 ,43 ).

Functional Role of Endocytosis in the Processes of Rapid Desensitization and

Resensitization of GPCRs
Physiologic ligands are generally thought to bind to GPCRs in the plasma membrane. Biochemical and immunocytochemical studies
suggest that certain GPCRs, such as the B2AR, interact with heterotrimeric G proteins primarily in the plasma membrane but not in
intracellular membranes after endocytosis (44 ,45 ). Together, these observations suggest that endocytosis may, by itself, mediate
desensitization of GPCR-mediated signal transduction by directly reducing the number of receptors present in the plasma membrane.
Indeed, in some cases, endocytosis may be a principal mechanism of rapid desensitization (46 ). However, as discussed above, rapid
desensitization by phosphorylation-dependent uncoupling of receptor from G protein does not require endocytosis of the receptor.
This is consistent with the ability of certain GPCRs, such as the α2A-adrenergic receptor, to desensitize in the absence of detectable
endocytosis (47 ,48 ). Studies of μ-opioid receptors expressed in transfected cells suggest that the effectiveness of endocytosis as a
means of attenuating signal transduction is inversely proportional to the number of “spare receptors” present in cells (46 ). Thus,
the precise role of endocytosis in contributing to desensitization of GPCR-mediated signal transduction probably varies among
systems and may be particularly important in cells expressing relatively low numbers of receptors.

Strong evidence is available to indicate that endocytosis of certain GPCRs serves a distinct function in promoting resensitization,
rather than desensitization, of signal transduction. The most thoroughly studied example of this mechanism derives from elegant
studies of the B2AR (2 ,49 ,50 ). As discussed above, agonist-induced phosphorylation of the B2AR by GRKs causes rapid
desensitization by promoting receptor interaction with arrestins and functional uncoupling from heterotrimeric G proteins (Fig. 5.5 ,
step 1). This initial desensitization of receptors occurs in the plasma membrane and does not require endocytosis of the receptor
protein. Within several minutes after this initial uncoupling of receptor from heterotrimeric G protein, arrestins promote the
concentration of receptors in clathrin-coated pits and subsequent endocytosis (Fig. 5.5 , steps 2 and 3). Endocytic membranes
containing internalized
 P.65

B2ARs are associated with activity of a protein phosphatase (PP2A) that can catalyze dephosphorylation of receptors (51 ). Based on these observations, it is proposed that endocytosis
of receptors promotes dephosphorylation of receptors, after which receptors can be recycled back to the plasma membrane in a dephosphorylated, fully active state (Fig. 5.5 , steps 4
and 5). Supporting this hypothesis, inhibitors of B2AR endocytosis do not block agonist-induced desensitization but strongly inhibit resensitization of receptor-mediated signal
transduction following removal of agonist from the culture medium (52 ). Thus, agonist-induced regulation of the B2AR appears to involve two linked regulatory cycles: a biochemical
cycle mediating phosphorylation and dephosphorylation of receptors, and a membrane trafficking cycle mediating endocytosis and recycling of receptors.

FIGURE 5.5. Linked cycles of G protein-coupled receptor phosphorylation

and endocytosis mediating rapid desensitization and resensitization of the
β2-adrenergic receptor (B2AR). Agonist-induced activation of the B2AR
causes G protein-coupled receptor kinase-mediated phosphorylation,
which promotes receptor interaction with nonvisual arrestins and
uncoupling of heterotrimeric G protein (step 1). Arrestin binding to the
phosphorylated receptor also promotes receptor concentration in
clathrin-coated pits (step 2), promoting rapid endocytosis of receptors by
dynamin-dependent fission of coated pits from the plasma membrane and
subsequent formation of endocytic vesicles (step 3). Together, these
steps cause profound functional desenstization of signal transduction.
Endocytic membranes containing internalized B2ARs are associated with a
protein phosphatase (PP2A) that can catalyze dephosphorylation of
receptors (step 4). Dephosphorylation of receptors followed by recycling
to the plasma membrane (step 5) mediates the return of receptors to the
plasma membrane in a fully functional state, promoting functional
resensitization of the signal transduction system.

Down-regulation of GPCRs by Regulated Proteolysis

Evidence for Regulated Proteolysis of GPCRs
Down-regulation of GPCRs is defined from saturation binding analysis by a decrease in total specific binding sites (Bmax) without a change in apparent affinity (Kd), which suggests that
down-regulation reflects a decreased number of total receptors present cells or tissues (1 ,53 ). In principle, this process could be mediated by modulation of receptor biosynthesis or
degradation. In practice, evidence suggests that both classes of mechanism contribute to the GPCR down-regulation observed physiologically. The role of transcriptional regulatory
mechanisms in controlling biosynthesis have been characterized in some detail (see Chapter 17 ). As discussed in greater detail below, there is also considerable evidence supporting
the importance of proteolysis of the receptor itself in mediating down regulation of a number of GPCRs (8 ).

Mechanisms of GPCR Proteolysis

Multiple mechanisms can mediate GPCR proteolysis. Proteolysis of endocytosed receptors in lysosomes is perhaps the best-established mechanism contributing to GPCR down-regulation
in mammalian cells, including certain neurally derived cell types (54 ,55 ). However, it is apparent that other mechanisms of GPCR proteolysis also exist, some of which may not
involve membrane trafficking of receptors at all. For example, the V2 vasopressin receptor can undergo ligand-induced proteolysis at the cell surface by a nonendocytic mechanism
mediated by a plasma membrane-associated metalloprotease (56 ). Recent studies of B2AR down-regulation support the idea that regulated proteolysis of GPCRs can occur without
endocytosis in some cell types (57 ).

In mammalian cells, ubiquitination is well established to promote degradation of various cytoplasmic proteins by a nonlysosomal mechanism mediated by proteasomes (58 ). Emerging
evidence also suggests a role of ubiquitination in promoting endocytosis and proteolytic degradation of certain membrane proteins, including GPCRs in yeast (59 ,60 ). The role of such
a mechanism in mediating down-regulation of mammalian signaling receptors comes from studies of receptor tyrosine kinases (61 ). Alternate mechanisms of GPCR proteolysis in
mammalian cells have been reported to be mediated by a distinct, nonproteasomal mechanism (56 ) or have been shown to be insensitive to inhibitors of proteasome-mediated
proteolysis (57 ). Thus, to our knowledge, it is not yet clear to what extent ubiquitination or proteasomes may contribute to down-regulation of GPCRs in mammalian cells.

Membrane Pathway Mediating Receptor Delivery to Lysosomes

The delivery of membrane proteins from the plasma membrane to lysosomes is a multiple-step process that is mediated by endocytosis of receptors from the plasma membrane
followed by shuttling to lysosomes via a specific series of membrane transport reactions (62 ,63 ). It is well-known that many GPCRs undergo ligand-induced endocytosis. However,
specific mechanisms and pathways mediating subsequent stages of receptor trafficking to lysosomes are poorly understood.

Early studies demonstrated that ligand-induced sequestration and down-regulation of the B2AR can be differentially affected by pharmacologic manipulations and receptor mutation,
which suggests that these processes may be mediated by distinct mechanisms (64 ,65 ,66 and 67 ). Furthermore, naturally occurring subtypes of α2-adrenergic receptor down-regulate
with similar rates (68 ) despite significant differences in their ability to undergo rapid endocytosis (47 ,48 ). Analogous processes of rapid sequestration and more gradual down-
regulation have also been observed in studies of opioid receptors, where pharmacologic differences between the effects of individual agonists are very pronounced (69 ,70 and 71 )
and appear to be relevant to the physiologic effects of opiate drugs in native neurons (14 ,72 ). Compelling evidence for the existence of distinguishable membrane trafficking
mechanisms comes from recent studies of mutant thrombin and substance P receptors, in which divergent residues in the carboxyl-terminal cytoplasmic domain specify differences in
receptor trafficking between lysosomal and recycling pathways (73 ). Analyses based on kinetic modeling techniques are consistent either with completely separate pathways
mediating rapid endocytosis and proteolytic degradation of GPCRs or with the operation of partially overlapping pathways that differ in their rate-limiting step (53 ). These models
differ in whether sorting of GPCRs is proposed to occur before or after endocytosis (Fig. 5.6 ).

FIGURE 5.6. Models for sorting of G protein-coupled receptors

(GPCRs) between pathways mediating reversible internalization
(“sequestration”) and proteolytic down-regulation of GPCRs. Panel
A: This model proposes that distinct GPCRs are segregated in the
plasma membrane and are subsequently endocytosed by different
membrane vesicles that either mediate reversible internalization or
deliver receptors to lysosomes. Panel B: This model proposes that
receptors can be endocytosed by the same membrane mechanism
and delivered to the same early endocytic vesicles. Receptors are
sorted after endocytosis between distinct pathways that mediate
either recycling or lysosomal delivery of receptors.
 P.66

Molecular Sorting of GPCRs after Endocytosis

Extensive previous studies of receptor-mediated uptake of extracellular ligands indicate that sorting of receptors between recycling
and lysosomal pathways can occur after endocytosis (74 ). Recent studies of adrenergic and opioid receptors suggest that this is also
true for certain GPCRs. For example, it has been shown recently that both agonist-induced sequestration and down-regulation of the
B2AR are specifically inhibited by a dominant-negative mutant form of dynamin, which suggests that endocytosis of receptors by
clathrin-coated pits is an obligate first step common to membrane pathways leading to recycling of endosomes and lysosomes (75 ).
Recent studies of membrane trafficking of opioid receptors, which are also endocytosed by a dynamin-dependent mechanism
involving clathrin-coated pits (76 ), support this conclusion. These studies demonstrate that distinct GPCRs can be sorted in a type-
specific manner between recycling and degradative pathways after endocytosis by the same mechanism (Fig. 5.6B ). These studies
also suggest that the machinery that sorts GPCRs can function very rapidly (within several minutes) after the initial endocytosis of
receptors (77 ).

Insight into a Mechanism Controlling Endocytic Sorting of the B2AR

The membrane machinery controlling postendocytic sorting of internalized GPCRs to lysosomes remains poorly understood. A recent
study of B2AR trafficking has provided initial insight into a specialized mechanism that controls sorting of a specific GPCR between
recycling and lysosomal pathways. As noted above, the B2AR is capable of recycling rapidly to the plasma membrane following
endocytosis in cultured cells. It was observed that a series of mutations in the distal part of the carboxyl-terminal cytoplasmic tail
strongly inhibit recycling of receptors and cause receptors to be “mistargeted” to lysosomes (78 ). All the receptor mutations that
cause this phenotype disrupt a specific interaction of the B2AR with NHERF/EBP50/E3KARP-family proteins (78 ,79 and 80 ), a
previously described
 P.67

family of proteins that interact with the B2AR via PDZ (PSD95/Discs large/ZO-1-homologous) domains and also associate with the
cortical actin cytoskeleton (81 ). Overexpression of a mutant form of EBP50/NHERF that cannot interact with the cortical actin
cytoskeleton, or chemical disruption of the actin cytoskeleton itself, also inhibits recycling of internalized B2ARs. These
observations suggest that sorting of internalized B2ARs between a distinct recycling and degradative pathway can be mediated by a
protein complex associated with the cortical actin cytoskeleton. Moreover, these studies suggest that phosphorylation of a specific
serine residue (Ser411), a potential substrate for a subset of GRKs (82 ), can modulate the sorting of receptors by this mechanism
(78 ). Thus, sorting of receptors after endocytosis, like the initial endocytosis of receptors from the plasma membrane, may be
closely linked to the cycle of receptor phosphorylation and dephosphorylation involved in mediating desensitization and
resensitization of signal transduction. A proposed model summarizing our current understanding of distinct stages of B2AR
endocytosis and sorting is summarized in Fig. 5.7 .

FIGURE 5.7. Model for endocytic sorting of the

β2-adrenergic receptor (B2AR) by
phosphorylation-regulated association of
receptors with the cortical actin cytoskeleton.
B2ARs undergo rapid desensitization and
endocytosis (step 1). If receptors are
dephosphorylated (step 2), they are able to
interact with a cytoskeleton-associated protein
complex via a PDZ domain-mediated protein
interaction with the carboxyl-terminal
cytoplasmic domain of the receptor, which
promotes rapid recycling of receptors to the
plasma membrane (step 3a). If serine 411 in the
receptor tail remains phosphorylated, this
protein interaction does not occur, and
recycling of receptors is inhibited. Receptors
that fail to recycle can later be delivered to
lysosomes, with down-regulation of receptors
accomplished by proteolysis (step 3b).

CONCLUSIONS AND FUTURE PERSPECTIVES

Part of "5 - Regulation of G Protein-Coupled Receptors by Phosphorylation and Endocytosis "
In this chapter, we have surveyed general processes of GPCR regulation and focused on selected mechanisms that are understood in
molecular detail. A general principle emerging from this approach is that phosphorylation and membrane trafficking mechanisms are
of fundamental importance to multiple processes of GPCR regulation. A second important principle is that distinct mechanisms of
regulatory phosphorylation and membrane trafficking are closely interdependent, which leads to an appreciation of linked cycles
coordinating the functional activation and regulation of receptors. An interesting corollary of this principle, suggested by certain
recent studies of atypical antipsychotics and opiate analgesics (15 ), is that the linkage between specific mechanisms of receptor
activation and regulation may be modified or disrupted by certain drugs. Therefore, it may be possible to target specific
mechanisms of GPCR regulation, or the biochemical linkages between these mechanisms, to develop novel therapeutics that may
influence the regulation of GPCRs in a manner quite different from that of classic agonists or antagonists.
We have restricted our focus to a subset of mechanisms that are, arguably, among the best established experimentally. It is
important to note that these studies are in large part limited to model cell systems rather than native tissues. Nevertheless, the
available data derived from transgenic and homologous recombination methodologies, and studies based on immunocytochemical
localization of endogenously expressed GPCRs, suggest that phosphorylation (83 ,84 ) and endocytosis of receptors do occur in native
tissues in response to physiologic stimulation (85 ,86 ) and certain drugs (14 ,72 ). Thus, it is reasonable to propose that mechanisms
elucidated with model cell systems may indeed be relevant to receptor regulation in vivo. However, it is not yet possible to predict
precisely how specific mechanism(s) of regulation modulate the central nervous system under physiologic or pathophysiologic
conditions, or how specific mechanisms of regulation may contribute to the in vivo effects of clinically important drugs. Thus, an
important future goal is to examine the regulation of GPCRs in native cell types and tissues, and to determine how specific
mechanisms of regulation contribute to physiologic and pathophysiologic states.
Finally, it is important to note that by restricting our scope to a limited subset of GPCR regulatory mechanisms, we have
underrepresented the diversity of GPCR regulation and the high degree of specificity with which individual receptors are regulated
in various cell types. For example, although we have discussed only endocytosis of GPCRs by clathrin-coated pits, substantial
evidence indicates that other mechanism(s) also can mediate regulated endocytosis of receptors (37 ,87 ,88 and 89 ). Moreover, it
is increasingly apparent that mechanisms of GPCR phosphorylation and endocytosis discussed in this chapter serve additional
important functions in signal transduction, such as controlling the interaction of “classic” GPCR signaling pathways with mitogenic
kinase cascades (see also Chapter 16 and Chapter 22 ) (90 ,91 and 92 ). Indeed, we anticipate that our present understanding of the
array of GPCR signaling and regulatory mechanisms is at a relatively early stage of rapid development, a view consistent with the
results of recent studies identifying an unexpected diversity of cellular proteins that interact with specific GPCRs (93 ). Future
studies of these unexplored mechanisms of GPCR regulation may lead to important new insights relevant to neuropsychiatric disease
and may identify exciting new targets for the development of novel therapeutic drugs.

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6
The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction

Joseph T. Coyle

Michael L. Leski

John H. Morrison

Joseph T. Coyle: Department of Psychiatry, Harvard Medical School, Boston, Massachusetts.

Michael Leski: Department of Psychiatry, McLean Hospital, Belmont, Massachusetts.

John H. Morrison: Mount Sinai School of Medicine, New York, New York.

L-Glutamic acid (Glu) is accepted as the major excitatory neurotransmitter in the nervous system, although other acidic amino acids
such as L-aspartic acid and L-homocysteic acid may also participate (1). Nevertheless, ongoing research reveals that the functions of
Glu are much more diverse and complex than simply generating excitatory postsynaptic currents (EPSCs). It plays a major role in
brain development, affecting neuronal migration, neuronal differentiation, axon genesis, and neuronal survival (2, 3 and 4). In the
mature nervous system, Glu is central to neuroplasticity, in which there are use-dependent alterations in synaptic efficacy as well as
changes in synaptic structure. These latter actions are intimately implicated in memory and related cognitive functions. Finally,
persistent or overwhelming activation of glutamate-gated ion channels can cause neuronal degeneration (5) depending on the
circumstances, this occurs by means of necrosis or apoptosis (6). Known as “excitotoxicity,” this phenomenon has been linked to the
final common pathway of neuronal death in a range of disorders including Huntington’s disease, Alzheimer’s disease, amyotrophic
lateral sclerosis (ALS), and stroke (7, 8).

This chapter provides an overview of the physiology and pharmacology of brain glutamatergic systems. There is a special emphasis
on glutamate receptors because their rich diversity confers physiologic and pharmacologic specificity for this single neurotransmitter,
which is used by up to 40% of all brain synapses. Finally, the potential role of glutamatergic system dysfunction in the
pathophysiology of neuropsychiatric disorders is addressed.

 AMPA-KAINATE RECEPTORS
 NMDA RECEPTORS
 METABOTROPIC GLUTAMATE RECEPTORS
 GLUTAMATE TRANSPORTERS
 GLUTAMATE AND GLIA
 GLUTAMATE AND NEURODEGENERATION
 GLUTAMATE AND BRAIN DISORDERS
 SCHIZOPHRENIA
 CONCLUSION
 ACKNOWLEDGMENT

AMPA-KAINATE RECEPTORS
Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

Glutamate receptors mediating fast EPSCs have been distinguished from the voltage-dependent NMDA receptors through the effects
of conformationally restricted agonists. The former glutamate-gated ion channels (iGluRs) have been segregated into two types: the
α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and KA receptors; however, cloning of the genes that
encode the proteins comprising these iGluRs and the analysis of their pharmacology and biophysics in various expression systems
indicate that each family of receptors represents complex heteromeric proteins consisting of multiple subunits with differential
representation resulting in diverse functional attributes (9).

The AMPA receptor family consists of four genes that encode proteins of approximately 900 amino acids with 70% homology among
themselves (GluR1-4) (1); however, cell-specific exon splice variants and posttranscriptional editing result in a complex range of
physiologic responses. Two exon splice variants of GluR1-4 known as “flip” and “flop” affect desensitization, with the flip form
associated with larger, more sustained currents (10). Splice variants truncated at the carboxy terminus have been described for
GluR2 and GluR4 as well as the kainate subunits, GluR5–7 (9). Furthermore, nuclear editing of the mRNA encoding GluR2 transforms
this receptor channel from one permeable to Ca2+ to one impermeable to the cation. Double-stranded RNA adenosine deaminase
converts the adenosine in a CAG codon for glutamine (Q) to an inosine, thereby creating a CIG codon for arginine (R) (11, 12). The
regulatory site for the editing process is located downstream in an intron that aligns with the exon to form the secondary structure
recognized by the enzyme. Although the other AMPA receptor subunits are generally Ca2+ permeable, the presence of edited GluR2
dominates a heteromeric receptor
 P.72

complex in that the multisubunit AMPA receptor behaves like GluR2 (i.e., has low calcium permeability). In the mature brain, the
vast majority of GluR2 is edited and the majority of AMPA receptors have low calcium permeability, suggesting that GluR2 is
reasonably ubiquitous (see the following). Mice in which the GluR2 editing process has been inactivated by a null mutation exhibit
increased Ca2+ permeability with AMPA receptor activation, epilepsy, and early death (13). Stroke is associated with the suppression
of GluR2 expression in the penumbra, resulting in increased Ca2+ permeability through the remaining AMPA receptors, thereby
causing delayed neuronal degeneration (14).

Five separate genes encode the components of the kainate receptor: GluR5–GluR7 and KA1–KA2 (1). Homomeric complexes of GluR5,
GluR6, and GluR7 form ion channels in the Xenopus expression system that are activated by KA but not by AMPA. However,
homomeric complexes of KA1 or KA2 do not generate functional ion channels, although they exhibit high-affinity binding for kainic
acid. It appears that KA1 or KA2 in conjunction with GluR5, GluR6, or GluR7 form functional KA receptors. GluR5 and GluR6 possess
the Q/R editing site in their second transmembrane (M2) domain, which modulates Ca2+ permeability as for GluR2. Additional editing
sites on GluR5–7 have complex effects on the receptor-channel function (15, 16). GluR5–7 are also widely represented in the brain,
particularly in neocortex and hippocampus.

A number of modulators of AMPA/KA receptors have been identified that act by attenuating their rapid and profound desensitization.
Cyclothiazide selectively inhibits desensitization of AMPA receptors, whereas the lectin, concanavalin A, blocks desensitization at KA
receptors (17). “Ampakines” also enhance AMPA receptor activity by attenuating desensitization and have been shown to facilitate
glutamatergic neurotransmission in vivo, thereby improving performance in several cognitive tasks (18). Polyamines, first noted for
their effects on NMDA receptors, mediate rectification of Ca2+-permeable AMPA and KA receptors by inducing a voltage-dependent
channel blockade (19).

A number of conformationally restricted analogues have assisted in characterizing the activity of subsets of AMPA/KA receptors.
Prior to development of AMPA, quisqualic acid was used as a non–NMDA receptor agonist; however, its specificity for AMPA receptors
is poor as it also activates mGluR 1 and 5 metabotropic receptors and inhibits glutamate carboxypeptidase II (GCP II) (20, 21). The
conformational rigidity of AMPA provides good specificity for AMPA receptors, whereas the more flexible kainic acid interacts with
KA receptors as well as other types of iGluRs. The orientation, length, and saturation of the side chain of kainic acid and its
analogues play a critical role in their binding to the receptor site with domoic acid exhibiting higher affinity and dihydrokainic acid
having much lower affinity than kainate (22). The first potent selective antagonists at AMPA/KA receptors with negligible effects at
NMDA receptors to be developed were the dihydroxyquinoxalines: CNQX, DNQX, and NBQX. With the exception of NBQX, which has a
somewhat higher selectivity for AMPA receptors, they do not distinguish between AMPA and KA receptors. More recently 2,3-
benzodiazepines have been demonstrated to act as selective noncompetitive AMPA receptor antagonists. The most potent in this
family, GYKI53655, blocks AMPA receptors with an IC50 of 1 μM and has a 200-fold lower affinity for KA receptors (23). The recently
developed LY294486 has tenfold specificity for inhibiting GluR5, indicating that iGluR subtype specific ligands can be developed.

Most of our initial knowledge concerning the regional brain expression of KA/AMPA receptors was based on autoradiographic studies.
Specific binding of [3H]-KA is relatively enriched in the hippocampal CA 3, striatum, deep layers of the neocortex, the reticular
nucleus of the thalamus, and the cerebellar granular cell layer. When considered in aggregate, immunohistochemical staining for
the five KA receptor subunits shares this ligand binding distribution (26).

Audioradiographic studies of the distribution of [3H]-AMPA binding show high density in the CA 1 stratum radiatum, the dentate gyrus,
superficial layers of the neocortex, and the molecular layer of the cerebellum (25). Consistent with its role in mediating EPSCs,
moderate levels of binding are observed throughout the rest of the central nervous system (CNS). The distribution of [3H]-AMPA
binding corresponds with the regional expression of GluR1 and GluR2, because levels of GluR3 and GluR4 are much lower in the adult
rat brain and have a more restricted distribution (27).

Although the autoradiographic approaches have been very informative with respect to general distribution of the KA/AMPA receptors,
they have been less informative with respect to cellular and synaptic distribution and differential subunit distribution. It has
recently become possible to overcome these limitations on both spatial and biochemical resolution, and begin the arduous process
of delineating the GluR subunit profile of identified neurons and circuits in specific brain structures such as the neocortex and
hippocampus. This capacity is a direct result of molecular neurobiological analyses that have fostered a detailed understanding of
the subunit protein constituents of the three classes of inotropic GluRs, and thus allowed for in situ hybridization to be used to
localize specific mRNAs (28) and with the use of class and subunit-specific antibodies for immunocytochemistry, it is now feasible to
analyze GluR distribution at the highest levels of cellular and synaptic resolution (29, 30).

AMPA receptor subunit distribution in the hippocampus and neocortex offer an instructive example of such an approach. Although
early studies demonstrated a wide distribution of AMPA subunits GluRs 2/3 in the brain and spinal cord (31, 32, 33 and 34), it
became clear early on that the relationship
 P.73

between GluRs and specific circuits needed to be analyzed at a high level of resolution; colocalization studies directed at subsets of
neurons (29, 35, 36, 37 and 38) and ultrastructural dissection of the synapse (39, 40, 41, 42, 43 and 44). A key theme that emerges
from these studies is that regional distribution and cellular colocalization patterns should not be extended to a synaptic
interpretation: Such interpretations must be founded on ultrastructural data as seen in the following examples.

The cellular distribution of GluR2 has been linked to heterogeneity in calcium permeability of AMPA receptors (45, 46). For example,
electrophysiological analyses have demonstrated that pyramidal cells have AMPA receptors with low calcium permeability, and
interneurons have AMPA receptors with relatively high calcium influx (47, 48), and these properties are linked to the relative
abundance of GluR1 versus GluR2 mRNAs (45, 46). In addition, early reports using polyclonal antisera that did not differentiate
among GluR2, 3, and 4c, obtained results implying that GABAergic interneurons might not contain GluR2, 3, and 4C (49, 50 and 51).

Definitive conclusions regarding selective distribution of GluR2-specific protein were not possible until 1996, when an exclusively
GluR2-specific monoclonal antibody (52), followed by a rabbit polyclonal (53) were developed. The GluR2 antibodies showed that
virtually all pyramidal cells and the majority of GABAergic interneurons in the neocortex (e.g., S1) contain GluR2. A similar pattern
was found in hippocampus, suggesting that the majority of the GABAergic interneurons in hippocampus are GluR2-positive, although
a subset of GABAergic neurons lacks any detectable GluR2 (52, 53), as in neocortex. These results are in excellent accord with the
GluR2 mRNA results obtained by single cell RT-PCR studies (45, 46), and suggest that a minority of the GABAergic interneurons lack
GluR2 mRNA/protein. Thus, the differences in calcium permeability between GABAergic interneurons and pyramidal cells could not
be the result of a widespread lack of GluR2 in GABAergic interneurons.

A double label GABA/GluR2 analysis that was extended to the ultrastructural level further clarified the issue of GluR2 representation
in GABAergic interneurons (54). It was hypothesized that if the difference in calcium permeability between pyramidal and GABAergic
neurons was related to differences in GluR2 expression, then it would likely be more apparent on the synaptic than the cellular level
(54). Ultrastructural analysis revealed that there is a consistently lower number of immunogold particles at the labeled asymmetric
synapses on GABAergic dendrites than those on pyramidal cell dendrites or spines, suggesting that a cell class-specific difference in
synaptic abundance of GluR2 is the substrate for the observed differences in calcium permeability across these two cell classes
revealed electrophysiologically (45, 46).

As demonstrated in the GluR2 studies discussed in the preceding, cellular colocalization may not adequately reflect the localization
patterns at the synaptic level. This was reinforced in studies of GluR2/NR1 colocalization in hippocampus and neocortex, designed
to delineate the degree of synaptic colocalization of NMDA and AMPA receptors in asymmetrical synapses (39, 40, 41, 42 and 43).
Although NR1 and GluR2 are broadly colocalized on a cellular level, extensive synaptic heterogeneity exists in their representation.
NR1 and GluR2 are often colocalized at the same synapse; however, there are also a large group of NR1-containing synapses that
lack GluR2 labeling (33% in [39 ]), many of which were on spines. These may be candidates for the “silent synapses” that have been
described electrophysiologically (55, 56) that might be activated by insertion of AMPA subunits (40, 42, 55, 56 and 57).

It has been generally recognized that AMPA receptors play a dominant role in mediating EPSCs. A physiologic role for KA receptors
has been elucidated only recently with the development of more selective agonists and antagonists. In the hippocampal slice in
which the AMPA, NMDA, and GABA receptors have been blocked pharmacologically, stimulation of the mossy fibers generates a slow
excitatory synaptic current system with the biophysical properties of the KA receptor (58). This current is absent in mice
homozygous for null mutation of the GluR6 subunit and less vulnerable to the epileptogenic effects of systemic KA (59). The
presynaptic inhibitory effect on GABA release in the CA 1 region of the hippocampus is mediated by the GluR5 subunit (60).

Although KA subunits have not been localized as extensively at the ultrastructural level as have AMPA or NMDA receptors,
immunocytochemical studies have demonstrated their broad distribution in the hippocampus and neocortex and broad colocalization
with AMPA and NMDA receptor subunits (38, 61) in both pyramidal and GABAergic interneurons (35, 36).

NMDA RECEPTORS
Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

The NMDA receptor, as its name indicates, was identified by the selective excitatory effects of the synthetic analogue of glutamate,
N-methyl-D-aspartic acid (1). A number of properties distinguishes the NMDA receptor from the non–NMDA iGluRs. First, its activity is
voltage dependent. At resting membrane potential, the channel is blocked by Mg2+, which is relieved by membrane depolarization.
Second, the receptor requires occupancy of another ligand binding site, the so-called glycine modulatory site, in order for glutamate
to gate channel opening. Recent evidence indicates that not only glycine but also D-serine, which is synthesized in astrocytes by
serine racemase, is a potent endogenous agonist at the glycine site (62). Third, the NMDA receptor possesses a number of
modulatory sites of physiologic significance. Zn2+ is a potent inhibitor of NMDA receptor conductance, especially those containing the
NR2A subunit (24). Zn2+ is concentrated in some glutamatergic
 P.74

terminals (e.g., the mossy fibers) and released with glutamate (63). A binding site for polyamines, when occupied, enhances
conductance in part through increasing the affinity of the glycine modulatory site on the NMDA receptor (64). Receptor function is
also modulated by redox status (65). Within the channel, there is a binding site for the dissociative anesthetics such as phencyclidine
(PCP), MK-801, and ketamine, which serve as noncompetitive inhibitors (66). The effects of the dissociative anesthetics occur only
with open channels, thereby causing a use-dependent inhibition. Finally, the NMDA channel provides ready passage of Ca2+, a cation
involved in a number of intracellular signaling processes.

Molecular cloning has disclosed at least six genes that comprise a family of polypeptides that form the various subtypes of the NMDA
receptor (1). NR1 was the first component cloned and, when expressed in Xenopus oocytes, was shown to possess the primary
electrophysiologic and pharmacologic features of the NMDA receptor–channel complex. Seven splice variants of NR1 have been
described, which reflect the exclusion or inclusion of three exons, two in the C terminal and one in the N terminal portion (1). These
splice variants significantly impact the biophysical characteristics of the receptor. The NR2 subunits, NR2A-D and the recently
identified NMDARL or NR3A, do not form channels (67); however, when coexpressed with NR1, the heteromeric channels exhibit a
markedly increased current as compared to the homomeric NR1 channels. Each of the NR2 subunits, when complexed with the NR1
subunit, exhibits different biophysical and pharmacologic properties. The NR2A and NR2B subunits, more highly expressed in adult
cortex in contrast to NR2C/D receptors, appear to be less sensitive to NMDA receptor antagonists, not as vulnerable to Mg2+ blockade,
and have lower Ca2+ conductance (1).

NR1 is expressed in most neurons, whereas the NR2 mRNAs exhibit different regional and developmental patterns of expression (68,
69). The NR2A subunit is highly expressed in the neocortex, hippocampus, cerebellum, and several thalamic nuclei. The NR2B
subunit is found in the neocortex, hippocampus, striatum, septum, and thalamic nuclei of the adult rat brain. The expression of the
NR2C subunit is much more restricted in the adult brain, being enriched in the olfactory bulb, thalamic nuclei, and cerebellum.
Finally, the NR2D subunit is enriched in brainstem nuclei, midline thalamic nuclei, and bipolar cells of the retina. The NR2B and
NR2D subunits appear early in brain development, followed by a decline in NR2D expression in the third week after birth of the rat,
whereas the acquisition of NR2A and NR2C subunits appears primarily postnatally in the rat (1).

With respect to NMDA receptor localization, as in the case of AMPA/kainate receptors, the early in situ hybridization studies
discussed in the preceding offered important information as to the regional distribution of NMDA receptors in the brain. These
studies have been followed with very extensive immunocytochemical analyses, particularly of the obligatory subunit NR1. With
respect to hippocampus and neocortex, NR1 is very broadly distributed and present in virtually all pyramidal neurons and
nonpyramidal GABAergic interneurons (29, 38), and in fact, appears to be present in over 90% of asymmetric synapses (30, 41). NR1
distribution has also been shown to be modifiable on both the cellular and synaptic level with respect to plasticity. For example,
deafferentation causes rearrangements of NR1 distribution at the cellular and synaptic level in a matter of days (70, 71). In addition,
although NMDA receptors have a very broad distribution on a cellular level, they can display a high degree of specificity on a
synaptic basis. The most dramatic example of this is in CA3 of the hippocampus, where NMDA receptors are present postsynaptically
in the distal dendrites (i.e., stratum moleculare), yet are absent in the stratum lucidum terminal zone, which is noted for LPT being
NMDA receptor-independent (37, 56, 72). This suggests that there are intracellular trafficking mechanisms or local synthesis that can
position NMDA receptors in a subset of synapses receiving a particular input to a given cell while not mediating other inputs.

The data on the distribution of other subunits are less well developed, and this is partly owing to the fact that it has been very
difficult to develop antibodies that differentiate NR2A from NR2B, the two dominant subunits in the NR2 group in the hippocampus
and neocortex. In general, it appears that NR2A and NR2B overlap in their distribution with NR1 to a large degree (73, 74 and 75);
however, there are regions such as CA3 where they differ in their distribution with NR1 (75, 76), but this has yet to be worked out at
the synaptic level. The detailed delineation of the synaptic distribution of NR2A and NR2B is an important task for the future given
that the presence of these subunits confer different functional attributes on the receptor, and the analysis of genetically
manipulated mice have suggested that up- or down-regulation of one of these subunits can profoundly impact their function. For
example, NR2B overexpression in mouse enhances learning and memory (77). Thus, as is the case for AMPA and kainate receptors,
delineating the subunit representation and potential stoichiometry at specific circuits and synapses for the NMDA receptor is of
paramount importance if this receptor is to be definitively linked to circuits that mediate specific behaviors and suffer under certain
pathologic conditions.

Several endogenous amino acids, aside from glutamate, are selective agonists at the NMDA receptor, including L-homocysteic acid,
L-aspartic acid, L-cysteine sulfate, L-serine-O-sulfate, L-cysteic acid, and quinolinic acid in order of decreasing potency (78). Given
the multiple modulatory sites and agonist binding sites for the NMDA receptor, it is not surprising that the antagonist pharmacology
for this receptor is complex. Several phosphonate analogues of glutamate, including D-aminophosphonovaleric acid (APV),
 P.75

D-aminophosphonoheptanoic (APH) acid, D-aminoadipic acid, and the cyclic analogue of AHP, (2-carboxypiperazin-4-yl)-propyl-1-
phosphonate (CPP), are competitive inhibitors to glutamate. As described, the NMDA receptor channel has a binding site for
dissociative anesthetics, which shares pharmacologic features with the σ receptor. The glycine modulatory site, which must be
occupied for glutamate gating of the ion channel, is subject to inhibition by the endogenous metabolite of tryptophan, kynurenic
acid as well as synthetic analogues such as 7-chlorokynurenate (79). For greater selectivity, attention is now directed at developing
subtype specific antagonists such as ifenprodil, which inhibits NMDA receptors bearing the NR2B subunit (80).

METABOTROPIC GLUTAMATE RECEPTORS

Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

It was generally believed that the neurophysiologic effects of glutamate were mediated exclusively by iGluR, until Sladeczek and
colleagues (81) reported that glutamate catalyzed phosphoinositide hydrolysis through a receptor coupled to a G-protein. Since then,
research has disclosed the existence of a family of glutamate receptors whose effects are largely mediated by G-proteins, the so-
called metabotropic glutamate receptors (mGluRs). The mGluRs appear to play an important role modulating both presynaptically
and postsynaptically the effects of glutamate at glutamatergic synapses where iGluRs are also engaged (82).

Cloning of mGluR 1 indicated that the predicted amino acid sequence shared negligible homology with any of the other G-protein
coupled receptor (GPCR) families except the parathyroid Ca2+-sensing receptor (83) and the GABA-B receptor (84). On the basis of
pharmacology, physiologic effects, and sequence homology, the eight mGluRs have been subdivided into three groups (85). Group I
includes mGluR 1 and mGluR 5, which act via phospholipase C; group II includes mGluR 2 and mGluR 3, which are negatively coupled
to adenylyl cyclase; and group III, which includes mGluR 4, 6, 7, and 8, and are also negatively coupled to adenylyl cyclase. Adding
to this complexity, mGluR 1 has four splice variants, whereas mGluR 4 and 5 each have two. The mGluRs within a group exhibit
greater than 70% sequence homology, whereas the homology falls to approximately 45% between groups (82).

Like other GPCRs, the mGluRs have seven putative transmembrane domains separated by short intracellular and extracellular loops
and an unusually large extracellular domain that contains nearly a score of cysteine residues (86). In further contradistinction to
other GPCRs, the agonist binding sites for the mGluRs are located in the extracellular domain. As a family, mGluRs are broadly
expressed in the nervous system, with group I having primarily a postsynaptic localization, whereas groups II and III primarily have a
presynaptic localization where they serve as autoreceptors and heteroreceptors that negatively regulate neurotransmission (82).
mGluR 1 is prominently expressed in the hippocampal granule and pyramidal cells, the Purkinje cells of the cerebellum, the
thalamus, and the lateral septum (87). mGluR 5 exhibits somewhat of a complementary distribution with high levels expressed in the
neocortex, the pyramidal cells in CA1 sector of the hippocampus, lateral septum, striatum, and nucleus accumbens (88). Although
studies in vivo suggest a low level of expression of mGluR 5 in glia, cultured astrocytes exhibit high expression (89). The expression
of group II receptors is more restricted than group I, with mGluR 2 found in the cerebellum, pyramidal cells in the entorhinal cortex
and the dentate gyrus, and presynaptically on corticostriatal afferents (90). The majority of the cerebellar Golgi cells have mGluR 2
but a minor subset express mGluR 5 in a complementary fashion (91). mGluR 3 is found in astrocytes throughout the brain and in
neurons in the neocortex, caudate putamen, thalamic reticular nucleus, and granule cells of the dentate gyrus (92). With regard to
group III, mGluR 4 is found in the thalamus, lateral septum, dentate gyrus, and cerebellar granule cells (92). mGluR 7 has a
widespread distribution with prominent representation in sensory afferent systems including the dorsal root ganglia and the
trigeminal nucleus in addition to the cerebral cortex, hippocampus, striatum, thalamus, and Purkinje cells (93). mGluR 6 is
restricted to on-bipolar cells in the inner nuclear layer of the retina (94), with a highly specific synaptic distribution (95). Finally,
mGluR8 is found in the mitral cells of the olfactory bulb, piriform cortex, and lateral thalamic reticular nucleus (96).

Ultrastructural studies have demonstrated that mGluRs differ in their predominant synaptic distribution from AMPA and NMDA
receptors. Although AMPA and NMDA receptors are localized predominantly in the postsynaptic density, mGluRs are principally
localized presynaptically and perisynaptically (97, 98 and 99). In addition, the mGluRs exhibit a high degree of specificity in this
regard with mGluR7 (100, 101) and mGluR4 (102) (which are primarily present presynaptically) and mGluRs 1 and 5 (which are
primarily present perisynaptically) (103, 104 and 105). These patterns suggest that synaptic localization is partially segregated by
mGluR group (99), with group I primarily perisynaptic, surrounding the postsynaptic density, group II primarily presynaptic or
extrasynaptic, and group III optimally situated as an autoreceptor on the presynaptic terminal. These synaptic distribution patterns
position mGluRs to play a critical role in modulating excitatory neurotransmission.

Group I mGluRs stimulate phospholipase C and the hydrolysis of phosphoinositide. These two receptors have been reported to
stimulate cAMP formation in different model systems (106). They also increase the excitability of neurons by reducing the K+
currents, through a mechanism that appears to be independent of G-protein action (107). Although the AMPA receptor agonist,
quisqualic acid, is the
 P.76

most potent agonist at the group I mGluRs, 3,5-dihydroxyphenylglycine (3,5-DHPG) is the most specific agonist. Group II mGluRs
inhibit adenylyl cyclase by coupling with the GI-protein. (See ref. 43 for a detailed review of mGluR pharmacology.) Group II
receptors also inhibit the N-type Ca2+ channels through GI-protein coupling. The group III mGluRs inhibit adenylyl cyclase via GI-
protein, although they may utilize other transduction mechanisms.

The most specific agonist at group I mGluRs appears to be 2R, 4R-4-aminio pyrrolidine-2, 4 dicarboxylate (APDC). LY354740 is an
exceptionally potent and selective agonist at group II mGluRs and is effective with systemic administration (108). Notably, N-
acetylaspartyl glutamate (NAAG), an endogenous neuropeptide, is a relatively potent agonist at mGluR 3, although it also serves as
an antagonist at the NMDA receptor (109). L-aminophosphonobutyric acid (L-AP4) is the most potent and selective agonist at the
group III mGluRs with the exception of mGluR 7, where L-serine-O-phosphate has greater potency. Presently, 2-carboxy-3-
phenylcyclopropyl glycine (CPCG) is the most potent antagonist against the group II mGluRs, and α-methyl-4-phosphonophenyl-
glycine appears to be an effective antagonist against the group III mGluRs, although the pharmacology of these receptors remains
less well developed than the group I mGluRs.

The heterogeneity of the mGluRs and their role in modulating glutamatergic neurotransmission make them attractive potential
therapeutic targets for drug development. Thus, activation of group II and group III mGluRs has been associated with protection
against excitotoxicity, whereas activation of group I mGluRs may actually enhance NMDA receptor-mediated neuronal degeneration
(110, 111). Recently it has been shown that inhibition of GCP II, the enzyme that degrades the selective mGluR 3 agonist NAAG,
provides potent protection against neuronal degeneration caused by transient occlusion of the middle cerebral artery (112). A
similar reciprocal relationship has been observed with regard to the effects on epilepsy with group I agonists exacerbating and group
II and III agonists attenuating seizures (113). Finally, metabotropic receptors, both in the dorsal root and thalamus, have been
implicated in modulating neuropathic pain (114).

GLUTAMATE TRANSPORTERS
Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

The demonstration of sodium-dependent high-affinity transport of glutamate in synaptosomal preparations was the first evidence
supporting the hypothesis that glutamate serves as a neurotransmitter (115). The presence of these transporters on excitatory nerve
terminals was exploited to label putative glutamatergic pathways such as the climbing fibers of the cerebellum through the
autoradiographic visualization of retrogradely transported radiolabel in axons and cell bodies. The transporters are capable of
maintaining extraordinary gradients with the extracellular concentration of glutamate in brain low μM (i.e., below the threshold for
iGluRs) in the face of a tissue concentration in the mM range (116). Energy deprivation not only collapses the sodium gradient across
the membrane that drives the transporter, thereby inhibiting glutamate uptake, but also results in reverse transport with massive
efflux of glutamate stores (117). Pharmacologic inhibition of glutamate transport in tissue culture models has been shown to
promote excitotoxic neurodegeneration (118).

Early pharmacologic studies pointed to the existence of subtypes of sodium-dependent glutamate transporters in brain with
cerebellum exhibiting a form that is much more sensitive to inhibition by L-α-aminoadipate and forebrain sensitive to dihydrokainate
(119). Both the pharmacologic heterogeneity as well as the diverse cellular distribution of the sodium-dependent glutamate
transporters have been illuminated recently by their cloning and molecular characterization. (See refs. 120 and 121 for review.)
Although acronyms have varied with the sequence of discovery, five excitatory acidic amino acid transporters (EAAT) that are
sodium dependent and chloride independent have been cloned. EAAT 1 or GLAST has its highest expression in brain but is also found
in peripheral tissue and placenta. The cerebellum appears to have the highest level within brain, depending on the species. EAAT 2
(GLT 1) is primarily expressed in brain, although low levels have been reported in pancreas and placenta; the highest expression
occurs in the forebrain, the lowest in the cerebellum. Its expression is predominantly if not exclusively astroglial in localization. The
predominant neuronal transporter is EAAT 3, which is also expressed in kidney and to a lesser extent in other peripheral tissues.
Consistent with the broad distribution of glutamatergic neuronal systems in brain, the levels of EAAT 3 are fairly uniform. EAAT 3 is
not consistently expressed in all glutamatergic systems, and some nonglutamatergic systems express it (122). Thus, EAAT 3 does not
appear to be a specific marker for glutamatergic neurons. Two additional minor forms have been identified: EAAT 4, which is
expressed in cerebellar Purkinje cells; and EAAT 5, which is limited to the retina.

Like the mGluRs, the neuronal transporter EAAT3 (i.e., EAAC1) has been shown to have a synaptic distribution different from the
AMPA or NMDA receptors in that it is primarily perisynaptic and presynaptic (44, 123). A double-label postembedding immunogold
study demonstrated the value of such ultrastructural data for revealing the importance of differential distribution of these proteins
with respect to the synapse (44). The double-label analysis targeted the AMPA subunit GluR2 and the glutamate transporter, EAAT3
(i.e., EAAC1), the neuron-specific glutamate transporter. This study revealed differential spatial distribution of these two proteins
very clearly. At the light microscopic level, as expected, GluR2 was broadly colocalized with EAAC1 in hippocampal projection
neurons, and both proteins
 P.77

had substantial cytoplasmic pools. However, the postembedding immunogold localization offered additional insights into the spatial
relationships between EAAT3 and GluR2 localization in and near the synapse, revealing morphologic and molecular constraints on
excitatory synaptic transmission. Specifically, synaptic GluR2 was present primarily in the postsynaptic specialization, appropriately
positioned to mediate the synaptic effects of glutamate. EAAT3 was not intermingled with GluR2 postsynaptically, but was generally
present perisynaptically, often immediately outside the synaptic specialization, with a small but significant presynaptic pool as well
(44). This arrangement positions EAAT3 to both confine glutamate to the synaptic site that contains the inotropic receptor molecules,
as well as to regulate its levels in immediately adjacent presynaptic and postsynaptic domains. This distribution is also interesting
with respect to the distribution of mGluRs, which are located perisynaptically and extrasynaptically (see the preceding). This
suggests that EAAT3 is also optimally positioned to regulate the exposure of perisynaptic and presynaptic mGluRs to glutamate (44).

The EAATs exhibit affinities for glutamate in the low μM range, two orders of magnitude more avid than the vesicular transporter for
glutamate, which is not sodium dependent. A generally accepted model for transport involves the binding of three Na+, one H+, and
glutamic acid, which is linked to the counter transport of one K+ (124). In addition, there is increasing evidence that individual EAAT
subtypes may also subserve signal transduction activity; thus, EAATs have been implicated in inhibition of adenylyl cyclase, altering
Ca2+ levels and Cl- flux by mechanisms independent of glutamate transport (121).

Pharmacologic inhibition of EAAT activity potentiates excitotoxic effects in tissue culture (118). Mice homozygous for the nul
mutation of EAAT 2 develop fatal epilepsy and exhibit increased vulnerability to excitotoxic insults (125), and mice homozygous for
the nul mutation for EAAT 1 exhibit cerebellar dysfunction (126). Thus, in contrast to the original hypothesis of the essential role of
neuronal glutamate transport in terminating excitatory neurotransmission, it is now apparent that the glial transporters play the
dominant role in neuroprotection from glutamate. In this regard, astroglial processes are tightly interdigitated with glutamatergic
and GABAergic synapses, where the transporters are expressed in high density, which accounts for the previous identification of the
“synaptosomal” localization of glutamate transport (127).

The activity of the glutamate transporters is regulate by transcriptional as well as posttranslational mechanisms. (See ref. 128 for
review.) GLT-1 expression, which is normally low in primary astrocyte cultures, is significantly increased in astrocytes by coculture
with neurons (129). Furthermore, subsequent destruction of the neurons results in down-regulation of GLT-1 but an up-regulation of
GLAST protein (130). Fornix transection results in down-regulation of both GLT-1 and GLAST in the innervation field (131). The
neuronal signal mediating this interaction has proved to be somewhat elusive but may in fact be glutamate itself acting at AMPA/KA
receptors on astrocytes. PKC has also been implicated in the regulation of glutamate transporter activity both directly and by
altering trafficking (136).

GLUTAMATE AND GLIA

Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

The neuroprotective action of glutamate transporters expressed by astroglia represents only one facet of the critical role astroglia
play in modulating glutamatergic neurotransmission. Astrocytes express a high-affinity Na+-dependent transporter for glycine, GlyT-1,
which maintains concentrations of glycine that are subsaturating for its modulatory site (Kd = 20 nM) on the NMDA receptor in spite
of μM levels in cerebrospinal fluid (CSF) (133). In addition, forebrain astroglia express serine racemase, which generates D-serine, a
potent agonist at the glycine modulatory site (31). The metabolism of tryptophan by a series of enzymes expressed in glia generates
both positive and negative modulators of the NMDA receptor (134). Of these, quinolinic acid is an agonist at NMDA receptors.
Although its affinity for the receptor is relatively low, lack of efficient clearance mechanisms renders it a pathophysiologically
significant NMDA receptor agonist. For example, the levels can achieve toxic concentrations in HIV encephalopathy with the
activation of microglia and infiltration of macrophages highly expressing quinoline (135). Kynurenic acid, another metabolite of
tryptophan, is a noncompetitive antagonist at the NMDA receptor acting at the glycine modulatory site. The synthesis of kynurenic
acid in brain takes place almost exclusively in astrocytes and is regulated by cellular energy status, ionic environment, local 2-oxo
acid concentration, and dopaminergic neurotransmission (136). Altered levels of kynurenic acid in disease states such as
Huntington’s disease (HD) and schizophrenia appear to correlate with NMDA receptor dysregulation (137).

N-acetylaspartyl glutamate is an abundant neuropeptide found in many but not all glutamatergic systems and several well-
characterized nonglutamatergic systems such as the locus ceruleus and motor neurons. Located in storage vessels and subject to
evoke release by a Ca2+-dependent process, NAAG is a noncompetitive antagonist at NMDA receptors (138) and a selective agonist at
the mGluR 3 (60). It is metabolized to N-acetylaspartate and glutamate by GCP II, which is selectively expressed by astrocytes (139).
Postmortem studies indicate reduced activity of GCP II in hippocampus, temporal cortex, and frontal cortex in schizophrenia, a
disorder potentially involving hypofunction of NMDA receptors (140), and increased activity in motor cortex and dorsal horn, which
would increase extracellular glutamate generated from NAAG in the neurodegenerative disorder ALS (141).

GLUTAMATE AND NEURODEGENERATION

Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "
 P.78

The term “excitotoxicity” was coined by Olney 30 years ago to designate a selective form of neurodegeneration caused by systemic
treatment of newborn rodents with monosodium glutamate (5). Degeneration of neurons with their perikarya in the arcuate nucleus
of the hypothalamus was apparent 90 minutes after injection; notably, axons passing through the lesion and glia were spared. As the
neurotoxic effects of glutamate analogues correlated with their excitatory potency, Olney hypothesized that the neuronal
degeneration resulted from excessive activation of glutamate receptors expressed on neurons. Through the use of potent excitatory
analogues of glutamate, the relevance of this phenomenon to neurodegenerative diseases became apparent. Thus, Coyle and
Schwarcz reported that intrastriatal injection of kainic acid in the rat replicated the neuropathologic and synaptic neurochemical
alterations that occur in HD (142). Systemic treatment with kainic acid produced a behavioral syndrome and histopathology
associated with temporal lobe epilepsy. GluR-mediated neurodegeneration now has been implicated in a broad range of
neurodegenerative conditions including stroke, ALS, cerebellar degeneration, and trauma (5, 143, 144).

Our knowledge of the role of iGluRs in neurodegenerative diseases has greatly increased since the realization that excitotoxicity is
linked to programmed cell death (PCD) in many neurodegenerative disorders. A seminal paper by Kerr and colleagues (145)
illustrated the morphological differences between apoptosis and necrosis. Subsequently, several genes that regulate cell death in C.
elegans were identified, leading to the discovery of the caspases and Bcl-2 and homologues (146). As a consequence, the idea that
cells could control their own death through the synthesis of new proteins was formulated. Subsequently, inhibitors of
macromolecular synthesis were proved to prevent the naturally occurring cell death in both sensory and motor neurons. Recently,
numerous molecular pathways and their components that activate or prevent neuronal cell death in response to iGluR activation
have been identified.

A major effort has been undertaken to identify the specific iGluRs and the downstream events following receptor stimulation that
mediate the death processes. Nevertheless, effective therapies to prevent or limit neuronal damage in neurodegenerative diseases
remain elusive, reflecting an incomplete understanding of the mechanisms of neuronal death in vivo. It has become apparent that
the boundary between apoptosis and necrosis is not well defined, leading to the realization that there exists a gradual shift from an
apoptotic to a necrotic cell death in many cases, referred to as the “apoptotic-necrotic” continuum (6). Weaker insults typically
promote apoptosis, whereas stronger ones favor necrosis. In other cases the apoptotic mechanism is activated along with the
necrotic one, hampering attempts to distinguish the two (147).

Apoptotic cell death is typically associated with caspase activation, chromatin condensation, DNA laddering, and cell membrane
blebbing that lead to cell shrinkage (6). Necrosis, on the other hand, is usually associated with the failure of ion pumps that causes
cells to swell and burst, and is identified in tissues by the presence of invading macrophages and disruption of plasma membrane
integrity, whereas in cell culture the absence of apoptotic markers and the rapid time course of death are the best indicators.
Proteolysis by calpain and the caspases is often an early event following iGluR activation (148). Calcium overload mediated by iGluRs
has a significant role in neurodegeneration (149). In addition, reactive oxygen species (ROS) play an important role in neuronal
death mediated by iGluRs (150).

Mitochondria are almost invariably involved in the pathways triggered by iGluR activation. There are several general mechanisms at
play, including release of proteins that activate caspases, disruption of electron transport, and alteration of cellular redox potential.
The discovery that Bcl-2 is localized to the mitochondria directed attention toward the role of this relationship in cell death (151).
Subsequently, it was found that Bcl-2 could prevent cytochrome c release, an inducer of apoptosis, from mitochondria. Bcl-2 also
blocks the onset of the mitochondrial permeability transition (MPT) (152). The MPT represents an increase in permeability of the
mitochondrial inner membrane to solutes of 1,500 daltons or less that results in membrane depolarization, uncoupling of oxidative
phosphorylation, ion release, and mitochondrial swelling (153). Many of the insults that lead to the opening of the MPT function in a
positive feedback manner (154). Glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule
cells, with mitochondrial function a critical factor that determines the mode of neuronal death (6).

Excitotoxicity is more commonly associated with necrosis because activation of iGluRs results in cation flux into the cell; however,
apoptosis can follow stimulation of both AMPA/KA and NMDA receptors. The iGluRs may activate apoptosis using existing cellular
components; for example, Simonian and colleagues demonstrated that kainate neurotoxicity in cerebellar granule cells was
apoptotic but independent of protein synthesis (155). Alternatively, cells may require a prior insult or the addition or withdrawal of
a trophic factor to become sensitive to iGluR activation that ordinarily would not be toxic. Cultured cerebellar granule cells remain
resistant to AMPA–receptor-mediated toxicity when maintained in medium containing serum or insulin-like growth factor I (IGF-I),
but become sensitive 4 to 5 days following the removal of trophic factors (156). In other cases, iGluR activation triggers PCD that
utilizes a signaling pathway. Jiang and associates reported that NMDA receptor-mediated influx of extracellular Ca2+ rapidly and
transiently activated ERK1/2, leading to apoptosis in cultured rat cortical neurons (157). Activation of the NMDA receptor
 P.79

up-regulated p53 expression in cultured cerebellar granule cells, whereas blockade of p53 induction by an antisense oligonucleotide
resulted in a complete inhibition of apoptosis (158). Similarly, systemic administration of kainate increased p53 mRNA levels in
neurons exhibiting morphological features of damage within kainate-vulnerable brain regions (159).

Apoptosis may be a favored route in PCD partly because the reactive microglia that usually accompany necrosis often stimulate
secondary cell death. Caspase-mediated degradation of AMPA receptor subunits occurs early during periods of cell stress in cultured
rat hippocampal neurons (160). This may favor apoptosis, because levels of the AMPA receptor subunits GluR1 and GluR4 are rapidly
decreased in neurons undergoing apoptosis in response to withdrawal of trophic support, whereas levels of NMDA receptor subunits
NR1, NR2A, and NR2B are unchanged. Activation of calpain I by NMDA in cultured hippocampal neurons prevented the entry of cells
into a caspase-dependent cell death program after the mitochondrial release of cytochrome c, possibly by inhibiting the processing
of procaspase-3 and -9 into their active subunits (161). Thus, moderate NMDA receptor activation can prevent apoptosis without
stimulating caspase-independent cell death, whereas a more severe stimulus favors apoptosis.

Necrotic cell death following iGluR activation is often attributed to alterations in receptor desensitization, subunit expression or
other regulatory mechanisms. Human NT2-N neurons, which express calcium-permeable AMPA receptors, become vulnerable to
excitotoxicity when desensitization is blocked with cyclothiazide (162). Necrosis is induced by insulin treatment within 48 hours in
cultured mouse cortical neurons (163). Insulin exposure increased the level of the NR2A subunit of the NMDA receptor without
altering NR1 or NR2B levels. Macromolecular synthesis inhibitors and NMDA antagonists blocked cell death, suggesting that an
activity-dependent emergence of excitotoxicity contributed to insulin neurotoxicity. Cultured rat hippocampal neurons pretreated
with BDNF exhibited increased levels of NR1 and NR2A, greater calcium responses to NMDA, and enhanced vulnerability to
excitotoxic necrosis and reduced vulnerability to apoptosis (164). Cultured cerebellar granule cells, which show primarily an
apoptotic death following KA treatment, undergo necrosis when L-type voltage-dependent calcium channels are blocked (147).

GLUTAMATE AND BRAIN DISORDERS

Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

Neurodegenerative Diseases
HD is an autosomal dominant, progressive neurodegenerative disease that typically has its symptomatic onset in midlife. Its
manifestations include chorea, dementia, and death 15 to 20 years after onset. Afflicted individuals have an expanded CAG repeat
in the gene encoding huntingtin on chromosome 4, resulting in an elongated series of glutamines. The number of CAG repeats is 10
to 34 in normal individuals and 37 to 100 in HD patients (165). The identification of the HD gene has enabled the production of
mouse models transgenic for huntingtin such as line R2/6, which has exon 1 with 92 repeats as well as transgenic cell lines that
reiterate cellular characteristics of the disease (see the following).

HD was the first neurodegenerative disease for which iGluR-mediated neurodegeneration was implicated. Intrastriatal injection of
kainate in the rat caused a striatal neuronal degeneration resembling HD (142); however, subsequent studies revealed that NMDA
receptor agonists replicated the selective neuronal vulnerability in HD much more faithfully (166). Chronic treatment of rats with
the mitochondrial toxin 3-nitropropionic acid elevated striatal lactate and selective striatal neuronal degeneration mediated by
NMDA receptors (167). In this regard, lactate is elevated in the cerebral cortex and basal ganglia of HD patients. There is also
reduced phosphocreatine/inorganic phosphate in resting muscle of HD patients, and mitochondrial electron transport enzymes are
reduced in HD postmortem tissue. (See ref. 168 for review.) Consistent with these observations, mitochondria from HD lymphoblasts
and fibroblasts display an increased tendency to depolarize (169). It may be that as a consequence of lowered energy levels, striatal
neurons in HD can not maintain the resting membrane potential (thereby relieving the Mg2+ block), leading to increased [Ca2+]i via
NMDA receptors and ultimately cell death. Noteworthy is a report by Ferrante and associates that dietary creatine supplementation
improved survival, slowed the development of striatal atrophy, and delayed the formation of huntingtin-positive aggregates in mice
transgenic for huntingtin exon 1 with the expanded CAG repeat (170). Creatine may exert neuroprotective effects by increasing
phosphocreatine levels or stabilizing the MPT, either of which could mitigate excitotoxicity mediated by GluRs.

Altered expression or composition of iGluR subunits may also contribute to neuronal death in HD. The editing of GluR2 mRNA is
compromised in a region-specific manner in HD as well as in schizophrenia and AD, although there is still a large excess of edited
GluR2 in each of these disorders (171). Chen and co-workers found that coexpression of huntingtin containing 138 repeats with NMDA
receptors resulted in an increased number of functional NR1/NR2B-type receptors at the cell surface as compared to cells with
normal huntingtin (172). Striatal spiny neurons are selectively vulnerable in HD and ischemia, whereas large aspiny (LA) cholinergic
interneurons of the striatum are spared in these pathologic conditions. Because NR1/NR2B is the predominant NMDA receptor
expressed in medium spiny neostriatal neurons, this may contribute to the selective vulnerability of these neurons in HD (172).
Calabresi and associates found that membrane depolarization and inward currents produced by AMPA, KA, and NMDA were
 P.80

much larger in spiny neurons than LA interneurons (173); moreover, concentrations of agonists producing reversible membrane
changes in LA interneurons caused irreversible depolarization in spiny cells. The striatal and cortical neurons of R6/2 mice and mice
with 94 CAG repeats displayed more rapid and increased swelling following NMDA treatment than controls, whereas AMPA and KA
treatments had no differential effects. These findings suggest that NMDA antagonists or compounds that alter sensitivity of NMDA
receptors may be useful in the treatment of HD (174).

The mGluRs may adversely affect iGluR function within the striatum in HD. The selective group I mGluR agonist 3,5-DHPG strongly
enhanced membrane depolarization and intracellular calcium accumulation induced by NMDA application in striatal spiny neurons
but not in LA interneurons, indicating a positive interaction between NMDA receptors and group I mGluRs, which are differently
expressed between these two neuronal subtypes (175). Cha and colleagues found that 12-week-old R6/2 mice displayed decreased
expression of AMPA- and KA- but not NMDA-type iGluR receptors compared to age-matched littermate controls. These mice also had
decreased expression of mGluR1-3 that preceded the appearance of motor symptoms; therefore, altered mGluR function may
contribute to subsequent pathology (176).

Approximately half of the variation in onset age for HD can be explained by the size of the repeat expansion. MacDonald and
associates examined a TAA repeat polymorphism, which is closely linked to the GluR6 gene, in 258 unrelated HD-affected persons
and found that younger onset age of HD was associated with linkage disequilibrium for this polymorphism (177). Rubinsztein and co-
workers found that 13% of the variance in the age of onset of HD that was not accounted for by the CAG repeat size could be
attributed to GluR6 genotype variation (178). These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD in
addition to NMDAR-mediated neurodegeneration.

Alzheimer’s disease (AD) is a progressive dementia characterized by a cortical neurodegeneration, particularly in the entorhinal
cortex, hippocampal CA1 region, and subiculum. The etiology of AD is complex, with age, trauma, health, and environmental and
genetic factors all playing a role (179). iGluR-mediated excitotoxicity is postulated to play a role in the neurodegeneration of AD (5).
Mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited autosomal dominant AD. Mice
transgenic for the PS1M146V gene are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the
hippocampus (180). Cultured hippocampal neurons from PS1M146V knock in mice display increased vulnerability to glutamate, which
is correlated with perturbed calcium homeostasis, increased oxidative stress, and mitochondrial dysfunction. Glutamate toxicity is
potentiated by ROS mediated inhibition of EAATs; two studies have shown that ROS generated by Aβ peptide inhibits astrocyte
glutamate uptake (181, 182).

Immunocytochemical studies indicate that virtually all projection neurons in the hippocampus express iGluR subunits from each
receptor class; however, regional differences in immunoreactivity were apparent in AD versus normal brain. In the vulnerable
regions (i.e., CA1), GluR1, GluR2(4), GluR5/6/7, and NR1 were reduced, presumably owing to cell loss (183). In contrast, GluR2(4)
immunolabeling appeared to be increased in the inner portion of the molecular layer of the dentate gyrus. Quantitative receptor
autoradiography was also used to measure the laminar distribution of NMDA and AMPA receptors in three areas of visual cortex in
control and AD postmortem human brains. The hierarchical pattern of the laminar loss of NMDA receptor binding correlated with the
increasing complexity of associational visual cortices and increasing numbers of neurofibrillary tangles; however, AMPA receptor
losses did not directly correlate with the pathology (184). Hyman and colleagues found no difference for the pattern of
immunostaining between control and AD in either hippocampi or adjacent temporal cortices for GluR1, GluR2/3, and GluR4 (185);
however, age-related loss of GluR2/3 immunoreactivity prior to degeneration has been reported in nucleus basalia of Meynert (186)
and entorhinal cortex (187), suggesting that an increase in calcium permeability of AMPA receptors may leave these neurons
vulnerable to degeneration in AD. Western blot analysis revealed average reductions of approximately 40% for GluR1 and GluR2/3 in
the entorhinal cortex of patients with AD pathology versus age-matched controls, but neither GluR1 nor GluR2/3 protein
concentration correlated significantly with tangle density (188). Thus, the relationship between excitotoxicity and neuronal loss in
AD is complex and requires additional investigation.

Amyotrophic lateral sclerosis is a disorder characterized by a selective and progressive degeneration of motor neurons in the spinal
cord and pyramidal neurons in the motor cortex, with onset in midlife (189). Death results from complications of the progressive
paralysis. The two forms of ALS, sporadic and familial (FALS), have similar clinical symptoms and neuropathology, although the
latter only accounts for 10% of the cases. Rosen and associates first reported a tight genetic linkage between FALS and the gene
encoding Cu/Zn superoxide dismutase (SOD1), and identified 11 different SOD1 missense mutations in 13 different FALS families
(190). Expression of high levels of a mutant form of human SOD1 for which the glycine at position 93 was replaced with an alanine
(G93ASOD1; a change that has little effect on enzyme activity) caused a progressive motor neuron disease resulting in death by 6
months in transgenic mice (191). Because the mouse gene for SOD1 is unaffected in the transgenic mice, the results indicate that
these mutations in SOD1 cause a gain-of-function that results in motor neuron death.
 P.81

The reason for the selective vulnerability of motor neurons in ALS is unknown. Various molecular and neurochemical features of
human motor neurons may render this cell group differentially vulnerable to such insults. Motor neurons are large cells with long
axonal processes that require a high level of mitochondrial activity and have greater neurofilament content than other neuronal
groups. Motor neurons have a very high expression of the cytosolic free radical scavenging enzyme Cu/ZnSOD1, which may render
this cell group more vulnerable to genetic or posttranslational alterations interfering with the function of this protein. The low
expression of calcium binding proteins and GluR2 AMPA receptor subunit by vulnerable motor neuron groups may render them unduly
susceptible to calcium-mediated toxic events following GluR activation (192).

High levels of mRNA for GluR1, GluR3, and GluR4 are expressed in normal human spinal motor neurons; however, GluR2 subunit
mRNA was not detectable in this cell group, predicting that normal human spinal motor neurons express calcium-permeable AMPA
receptors unlike most neuronal groups in the human CNS (193); however, this has not been borne out in studies of mouse spinal cord
in the context of the mouse models of ALS, where GluR2 is well represented in spinal cord motor neurons (194). AMPA or kainate
exposure triggers substantial mitochondrial calcium loading in motor neurons, but causes little mitochondrial accumulation in
forebrain GABAergic interneurons, neurons that express large numbers of calcium-permeable AMPA/kainate channels but do not
degenerate in ALS. Brief exposure to either AMPA or kainate caused mitochondrial depolarization in motor neurons, whereas these
effects were only observed in the GABAergic neurons after exposure to the nondesensitizing AMPA receptor agonist kainate. Finally,
blocking mitochondrial calcium uptake attenuated AMPA/kainate receptor-mediated motor neuron injury. Thus, mitochondrial
calcium uptake and consequent ROS generation may be central to the injury process (195). Quantification of mRNA expression in
spinal cord showed a significant widespread loss of NR2A from both dorsal and ventral horns with losses of 55% and 78%, respectively,
in ALS as compared to control. These results were substantiated by analysis of spinal cord homogenates, which showed a significant
total decrease of 50% in NR2A message for ALS as compared to control (196).

Riluzole, which attenuates the glutamate neurotransmitter system, has been shown to prolong survival in patients with ALS (197).
Riluzole affects neurons using three mechanisms: by inhibiting excitatory amino acid release, inhibiting events following stimulation
of iGluRs, and stabilizing the inactivated state of voltage-dependent sodium channels. Mennini and associates studied inotropic
glutamate receptor subtypes and the effect of chronic treatment with NBQX in the spinal cord of motor neuron disease (mnd) mice.
NBQX significantly improved the behavioral scores in mnd mice. These findings suggest that selective antagonism of inotropic non–
NMDA receptors may be of value in the treatment of motor neuron disease (198). Further research may allow the development of
therapies that target specific glutamate receptor subunits and modulate “downstream” events within motor neurons, aimed at
protecting vulnerable molecular targets in specific populations of ALS patients.

SCHIZOPHRENIA
Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "

Kim and associates reported diminished concentrations of glutamate in CSF of patients with schizophrenia and first proposed that
hypofunction of glutamatergic systems might cause the disorder (199). This finding has been replicated by some studies but not by
several others (200, 201, 202 and 203). In a postmortem study, Tsai and associates (140) studied eight brain regions and found
decreased concentrations of glutamate and aspartate in the frontal cortex and decreased concentration of glutamate in the
hippocampus of patients with schizophrenia as compared to controls. Furthermore, the concentration of NAAG was increased in the
hippocampus and the activity of GCPII was selectively reduced in the frontal cortex, temporal cortex, and hippocampus of people
with schizophrenia. Subsequent studies with magnetic resonance spectroscopy have revealed significant reductions in the level of N-
acetylaspartate (NAA), the product of NAAG by GCPII, in the very same regions—frontal cortex, temporal cortex, and hippocampus
(204).

Initial ligand binding studies in postmortem schizophrenic brain have revealed increases in the non–NMDA iGLURs in the prefrontal
cortex (205, 206) and decreases in the hippocampus (207, 208). Strychnine-insensitive binding, which labels the glycine modulatory
site on the NMDA receptor, is increased throughout the primary sensory cortex and related associational fields in schizophrenia (209).
Molecular approaches have shown a reduction in mRNA encoding luR2 in the hippocampus and parahippocampus of people with
schizophrenia, and reduced editing of GluR2 in the prefrontal cortex (210, 211). Although the density of NMDA receptors in the
prefrontal cortex of people with schizophrenia was normal, the relative subunit composition differed significantly from controls with
a large increase observed for NR2D (212).

A convincing link between glutamatergic dysfunction and schizophrenia came from anecdotal and subsequent controlled studies of
the neuropsychologic effects of dissociative anesthetics, which are noncompetitive antagonists of the NMDA receptor (213). When
chronically abused, PCP produces a syndrome in normal individuals that closely resembles schizophrenia and exacerbates symptoms
in patients with chronic schizophrenia. Subanesthetic doses of ketamine administered to normal subjects produces positive
symptoms, including delusions and thought disorder, negative symptoms, and frontal lobe cognitive impairments characteristic of
schizophrenia (214). When administered
 P.82

to schizophrenic subjects, subanesthetic doses of ketamine exacerbate delusion, hallucinations, and thought disorders that are
consistent with the patient’s typical pattern of psychotic relapse (215, 216). These effects are attenuated by the atypical
antipsychotic clozapine but not haloperidol.

Although acute administration of ketamine to normal subjects causes increased (217) prefrontal cortical perfusion, chronic exposure
to PCP is associated with the classical “hypofrontality” of schizophrenia (218, 219). Chronic PCP treatment produced more
perseveration and fewer nonspecific cognitive deficits in monkeys that persisted after discontinuation. Notably, these memory
deficits were prevented by clozapine treatment (220).

Administration of NMDA receptor antagonists markedly increased the release of dopamine and glutamate in prefrontal cortex and
subcortical structures in rats (221, 222), which was associated with impaired performance on a memory task sensitive to prefrontal
cortical function (217); these alterations could be ameliorated by treatment with an AMPA/KA receptor antagonist. Furthermore,
administration of a group I mGluR agonist blocked PCP-induced glutamate release without affecting dopamine release (223). These
effects of NMDA receptor antagonism observed in the rodent have been shown to be comparable to humans in a positron emission
tomographic study in which [11C]-raclopride binding in striatum was used to measure dopamine release; subanesthetic doses of
ketamine cause increased dopamine release in human subjects (224).

If the symptoms of schizophrenia result from hypofunction of NMDA receptors, then agents that enhance NMDA receptor function
would be predicted to reduce symptoms. Because full agonists could be excitotoxic, studies have focused primarily on agents that
act via the glycine modulatory site (225). Electrophysiologic studies in the hippocampal slice indicate that the glycine modulatory
site is not fully occupied because of efficient transport of glycine by the GLYT-1 transporter on astroglia so that the modulatory site
is subject to pharmacologic manipulation (133). In most of the studies, the drugs were added to typical antipsychotics in stable
patients with prominent negative symptoms. Javitt and colleagues have performed a series of placebo-controlled crossover trials in
which high doses of glycine (30 to 60 g per day) were added to antipsychotic drugs. They demonstrated improvement in negative
symptoms and cognitive function without effects on psychotic symptoms or extrapyramidal side effects (226, 227 and 228). Tsai and
associates added D-serine at a dose of 30 mg per kg to typical antipsychotic drugs for 8 weeks and found significant improvements in
negative symptoms, cognitive function (as measured by the Wisconsin Card Sorting test), and psychosis (229). The more robust
effect of D-serine may reflect the fact that it has better penetrance of the blood–brain barrier, is a full agonist and has a higher
affinity than glycine.

Another drug that has been extensively studied is the antitubercular agent, D-cycloserine, which is a partial agonist at the glycine
modulatory site with 60% efficacy and readily crosses the blood–brain barrier (230). A blinded dose finding study in patients receiving
typical antipsychotics and exhibiting prominent negative symptoms revealed a U-shaped dose–response curve with significant
reductions in negative symptoms and improvement in cognitive function at 50 mg per day (231). van Berckel and associates observed
improvement in negative symptoms at a D-cycloserine dose of 100 mg per day in a small open trial with medication-free
schizophrenics (232). D-cycloserine at 50 mg per day significantly improved negative symptoms when added to conventional
antipsychotics in an 8-week fixed dose placebo-controlled parallel group trial with patients meeting criteria for deficit syndrome of
schizophrenia (233); however, performance on a cognitive battery did not change. Notably, full response for negative symptoms was
not achieved until after 4 to 6 weeks of treatment.

It was of interest to determine whether the addition of D-cycloserine would have further ameliorative effects in clozapine
responders because clozapine has substantial effects on negative symptoms in many patients who respond poorly to typical
antipsychotics. To the contrary, two separate trials of D-cycloserine at 50 mg per day added to clozapine resulted in worsening of
negative symptoms (234, 235). In contrast, trials in which the full agonists, glycine or D-serine, were added to clozapine yielded no
additional change in negative symptoms or cognitive function (236, 237). A plausible explanation for these findings is that clozapine
may exert its effects on negative symptoms and cognitive functions in part by increasing occupancy of the glycine modulatory site
on the NMDA receptor, thereby transforming the partial agonist D-cycloserine into an antagonist. Support for this inference comes
from electrophysiologic studies in the hippocampal slice where clozapine enhances NMDA receptor currents (238).

As hippocampal interneurons appear more sensitive to NMDA receptor antagonists owing to the presence of NR2C (239),
hypofunction of these NMDA receptors because of an excess of endogenous antagonists such as NAAG or kynurenic acid could account
for many features of schizophrenia. The disinhibition of cortico-hippocampal efferents appears to increase subcortical dopamine
release associated with positive symptoms (240). This would also interfere with the precision of cortical/hippocampal activations
consistent with schizophrenic subjects’ inability to increase hippocampal neuronal activity in a memory task because of a ceiling
effect (241). The effects of glycine modulatory site activation, particularly on negative symptoms and cognitive impairment, are
consistent with this model. Finally, the fact that ketamine reproduces the eye tracking impairments found in schizophrenics and
some of their first-degree relatives suggest that NMDA receptor hypofunction could be part of the endophenotype (242).
 P.83

Age-Associated Memory Impairment

Glutamate receptors have also been implicated in the functional decline seen in normal aging in the absence of neurodegeneration.
Spatial memory is particularly vulnerable to aging (243), and is also disrupted by pharmacologic blockade of NMDA receptor function
(244) or hippocampal knockout of NR1 (245). Electrophysiologic investigations of aging in rat hippocampus have revealed that
certain aspects of excitatory synaptic transmission are unaffected or even compensatory, whereas others are compromised (246).
One component of synaptic transmission that is compromised is maintenance and induction of long-term potentiation (LTP) that
could be related to impaired NMDA receptor-mediated processes and the decreased stability of spatial information coding by “place
cell” in aged rats (247). At the regional level, receptor binding studies have reported decreases in NMDA binding in hippocampus
(248) with confirmatory declines in mRNA expression (249); however, other studies suggested that there is no change in NMDA
receptors with aging (250).
In studying age-related changes in receptors, it is particularly important to be able to take the analysis from the regional level to
that of cell classes, circuits, individual neuronal compartments, and synapses, because the changes are very likely to be cell-,
circuit-, and synapse-specific and therefore difficult to resolve at the regional level; for example, age-related shifts in NR1, have
been reported in the molecular layer of the dentate gyrus (251). The projection from the entorhinal cortex (ERC) to the DG is
strictly confined to the outer molecular layer (OML), that is, the distal dendrites of granule cells; whereas other excitatory inputs
terminate in a nonoverlapping fashion in the inner molecular layer (IML), the proximal dendrites. Aged monkeys, compared to young
adult monkeys, exhibit a decrease in the fluorescence intensity for NR1 in the OML of the DG as compared to the IML. Given the
tight laminar organization of these circuits, this pattern means that decreased NR1 levels primarily affect the input from the ERC,
pointing to the ERC input to the hippocampus as a key element in age-related changes, and suggests that the intradendritic
distribution of a neurotransmitter receptor is modified in an age-related and circuit-specific manner (251).
Although these results suggest that age-related circuit-specific shifts in NMDA receptors might underlie memory defects, they were
not done in behaviorally characterized animals, and need to be followed up in the context of behavior. In addition, the data on NR1
changes were limited to the dendrite and did not directly address the GluRs at the synapse. Thus, these data need to be extended,
particularly in the nonhuman primate model. Species issues may be particularly relevant because in rat hippocampus decreases in
presynaptic markers such as synaptophysin correlate with age-associated memory impairment more directly than do any age-related
shifts in GluRs (252, 253).

CONCLUSION
Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "
In closing, glutamate sits at the epicenter of signal transduction in brain, not only mediating excitatory neurotransmission, but also
modulating neuroplasticity at the genetic, synaptic, and structural levels. Furthermore, insufficient glutamatergic signaling causes
the degeneration of immature neurons through apoptosis (254), whereas excessive activation of iGluRs kills neurons through necrosis
and/or apoptosis. Dysregulation of glutamatergic neurotransmission has been implicated in an expanding number of neuropsychiatric
disorders. Although the clinical pharmacology of glutamate is currently embryonic, the remarkable advances in the molecular
characterization of this system hold promise for the development of a rich array of specific drugs in the future.

ACKNOWLEDGMENT
Part of "6 - The Diverse Roles of L-Glutamic Acid in Brain Signal Transduction "
Dr. Coyle serves as a consultant for Jansson, Abbott, and Prestwick and owns stock in Merck, Celera, Genzyme, and PESystems.

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7
Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role
in Central Nervous System Disorders
Errol B. De Souza
Dimitri E. Grigoriadis
Errol B. De Souza: Aventis Pharmaceuticals, Inc., Bridgewater, New Jersey.
Dimitri E. Grigoriadis: Neurocrine Biosciences, Inc., San Diego, California.

 HISTORICAL PERSPECTIVES
 CHARACTERISTICS OF THE CRF PEPTIDE AND GENE SEQUENCES
 ANATOMY OF CRF
 UROCORTIN: A NOVEL MAMMALIAN CRF-RELATED PEPTIDE
 CRF RECEPTORS
 CRF-BINDING PROTEIN
 CRF REGULATION OF NEUROENDOCRINE FUNCTION
 CRF REGULATION OF CNS ACTIVITY
 ROLE FOR CRF IN NEUROPSYCHIATRIC DISORDERS AND NEURODEGENERATIVE DISEASES
 POTENTIAL THERAPEUTIC STRATEGIES DESIGNED FOR THE CRF SYSTEM
 CONCLUSION
 ACKNOWLEDGMENT

HISTORICAL PERSPECTIVES
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "
In 1948 Sir Geoffrey Harris first proposed the concept that the hypothalamus plays a primary role in the regulation of the pituitary-
adrenocortical axis. Subsequently, during the 1950s, Guillemin and Rosenberg, and Saffran and Schally independently observed the
presence of a factor in hypothalamic extracts (termed corticotropin-releasing factor, CRF) that could stimulate the release of
adrenocorticotropic hormone (ACTH, corticotropin) from anterior pituitary cells in vitro. Although CRF was the first hypothalamic
hypophysiotropic factor to be recognized, its chemical identity remained elusive until 1981, when Wylie Vale and colleagues at the
Salk Institute reported the isolation, characterization, synthesis, and in vitro and in vivo biological activities of a 41-amino acid
hypothalamic ovine CRF (1 ). Just over a decade later, Vale and colleagues were the first to report the cloning of the human CRF1
receptor from a single human Cushing’s corticotropic adenoma using an expression cloning techniques (2 ). This initial discovery led
to the identification of a second receptor subtype (termed CRF2), which has now been localized and characterized in a variety of
species (see the following).
This chapter provides an overview of the CRF system and its related receptor targets. More detailed and comprehensive information
on CRF is available in recent reviews (3 ,4 ) and books (5 ,6 ) on the topic.

CHARACTERISTICS OF THE CRF PEPTIDE AND GENE SEQUENCES

Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Amino Acid Sequence and Structure of CRF

The sequence of CRF has been determined in a variety of species including sheep, humans, rats, pigs, goats, and cows. In all species,
CRF is a 41-amino acid residue single chain polypeptide (Fig. 7.1 ). Rat and human CRFs are identical to one another and differ from
ovine CRF by seven amino acid residues. All three CRFs have close amino acid homology and share some biological properties with
sauvagine, a 40-amino acid peptide that exists in frog skin, and urotensin I, a 41-amino acid peptide derived from fish urophysis.
Caprine and ovine CRF are identical and differ from bovine CRF by one amino acid. Porcine CRF more closely resembles rat and
human CRF. CRF and related peptides are amidated at their carboxy terminal; CRF COOH-terminal-free acid has less than 0.1% of
the potency of native CRF, suggesting the importance of amidation to biological activity of the peptide. Studies to determine the
solution structure of CRF using proton nuclear magnetic resonance suggest that human CRF comprises an extended N-terminal
tetrapeptide connected to a well-defined α-helix between residues 6 and 36 (7 ). An α-helical oCRF(9-41) has been demonstrated to
be an antagonist of CRF (8 ), which underscores the necessity of the α-helical conformation for receptor binding and biological
activity.

FIGURE 7.1. Amino acid sequences of ovine and rat/human

corticotropin-releasing factor (CRF) and related peptides
including human and rat urocortin. Rat CRF and human CRF
are identical and differ from ovine CRF by seven amino
acid residues, which are denoted by the shading. All
peptides are amidated at the carboxy terminus; the
amidation is essential for biological activity. See color
version of figure.

Organization of the CRF Gene and Protein Precursor

The nucleotide sequences encoding ovine and rat CRF cDNA precursors as well as human, rat, and ovine CRF
 P.92

genes have been determined (9 ,10 ). The locus of the CRF gene is on chromosome 8q13 in the human. The CRF genes are quite
similar to one another, containing two exons separated by intervening intron 686 to 800 base pairs long. The first exon encodes most
of the 5′-untranslated region of the mRNA and the second encodes the entire prepro-CRF precursor polypeptide, which is 187 to 196
amino acids long; the carboxy end of the precursor contains the 41-amino acid peptide sequence. The high incidence of homology
among species suggests that the gene has been highly conserved through evolution.

As previously demonstrated for other systems, the 5′-flanking DNA sequences are most likely to contain the DNA sequence elements
responsible for glucocorticoid, cAMP and phorbol ester regulation, tissue-specific expression, and enhancer activity. Although a
consensus cAMP response element has been identified, located 200 base pairs upstream from the major transcription initiation site,
no obvious glucocorticoid response elements or activation protein (AP) 1-binding elements are present. A potential AP-2 binding site,
which may mediate the responses to protein kinase A and C, is present 150 base pairs upstream from the major start site.

ANATOMY OF CRF
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Distribution of CRF in the CNS

The distribution and localization of CRF mRNA in the central nervous system (CNS) have been evaluated using Northern blot analysis
and in situ hybridization histochemistry, respectively. Radioimmunoassay and immunohistochemical studies have been critical in the
determination of the neuroanatomic organization of CRF immunoreactive cells and fibers in the CNS. Overall, there is good
concordance between studies demonstrating a widespread distribution of CRF cell bodies and fibers in the CNS. Detailed descriptions
of the organization of CRF immunoreactive cells and fibers in rat brain have been published (11 ,12 and 13 ).

Morphologic data clearly indicate that the paraventricular nucleus of the hypothalamus (PVN) is the major site of CRF-containing cell
bodies that influence anterior pituitary hormone secretion. These neurons originate in the parvocellular portion of the PVN and send
axon terminals to the capillaries of the median eminence. CRF is also present in a small group of PVN neurons that project to the
lower brainstem and spinal cord; this group of neurons may be involved in regulating autonomic nervous system function. Other
hypothalamic nuclei that contain CRF cell bodies include the medial preoptic area, dorsomedial nucleus, arcuate nucleus, posterior
hypothalamus, and mammillary nuclei.

The neocortex contains primarily CRF interneurons with bipolar, vertically oriented cell bodies predominantly localized to the
second and third layers of the cortex and projections to layers I and IV. In addition, scattered cells are present in the deeper layers
that appear to be pyramidal cells. Although CRF-containing neurons are found throughout the neocortex, they are found in higher
densities in the prefrontal, insular, and cingulate areas. CRF neurons in the cerebral cortex appear to be important in several
behavioral actions of the peptide, including effects on cognitive processing; furthermore, dysfunction of these neurons may
contribute to many CNS disorders (see the following).

Large and discrete populations of CRF perikarya are present in the central nucleus of the amygdala, bed nucleus of the stria
terminalis, and substantia innominata. CRF neurons in the central nucleus of the amygdala project to the parvocellular regions of
the PVN, the parabrachial nucleus of the brainstem, and thus may influence both neuroendocrine and autonomic function in addition
to behavioral activity. CRF neurons originating in the bed nucleus of the stria terminalis send terminals to brainstem areas such as
the parabrachial nuclei and dorsal vagal complex, which coordinate autonomic activity. CRF fibers also interconnect the amygdala
with the bed nucleus of the stria terminalis and hypothalamus. Scattered CRF cells with a few fibers are also present in
telencephalic areas such as regions of the amygdala in addition to the central nucleus, septum, diagonal band of Broca, olfactory
bulb, and all aspects of hippocampal formation, including the pyramidal cells, dentate gyrus, and subiculum.

Several groups of CRF cell bodies are present throughout the brainstem. In the midbrain, CRF perikarya are present in the
periaqueductal gray, Edinger-Westphal nucleus, dorsal raphe nucleus, and ventral tegmental nucleus. Projections from the dorsal-
lateral tegmental nucleus to a variety of anterior brain areas such as the medial frontal cortex, septum, and thalamus have also
been described. In the pons, CRF cell bodies are localized in the locus ceruleus, parabrachial nucleus, medial vestibular nucleus,
paragigantocellular
 P.93

nucleus, and periaqueductal gray. CRF neurons originating in the parabrachial nucleus project to the medial preoptic nucleus of the
hypothalamus. In the medulla, the large groups of cell bodies are present in the nucleus of the solitary tract and dorsal vagal
complex with ascending projections to the parabrachial nucleus. Scattered groups of cell bodies are also present in the medullary
reticular formation, spinal trigeminal nucleus, external cuneate nucleus, and inferior olive. The inferior olive gives rise to a well-
defined olivocerebellar CRF pathway with projections to the Purkinje cells of the cerebellum. No CRF cell bodies are present in the
cerebellar formation.

Within the spinal cord, CRF cell bodies are present in laminae V to VII and X and in the intermediolateral column of the thoracic and
lumbar cord. CRF fibers originating in the spinal cord form an ascending system terminating in the reticular formation, vestibular
complex, central gray, and thalamus. This ascending CRF system may play an important role in modulating sensory input. In addition,
spinal cord CRF neurons may represent preganglionic neurons that modulate sympathetic outflow.

Distribution of CRF in Peripheral Tissues

In addition to its CNS distribution, CRF has been localized in a variety of peripheral tissues (14 ). CRF-like immunoreactive fibers are
present in the intermediate lobe of the pituitary; these fibers originate in the hypothalamus. A physiologic role has been proposed
for CRF in regulating pro-opiomelanocortin (POMC)-derived peptide secretion from the intermediate pituitary. CRF has also been
localized in the adrenal medulla of a variety of species and is increased following stimulation of the splanchnic nerve stimulation
and hemorrhagic stress. CRF-like immunoreactivity and CRF mRNA have been detected in lymphocytes, where they may play a role
in regulating immune function. Other tissues in which CRF has been localized include the testis (Leydig cells and advanced germ
cells), pancreas, stomach, and small intestine. Although CRF is not detected in the circulation under normal circumstances, very
high levels have been measured in the plasma of pregnant women; the source of CRF in pregnancy appears to be the placenta. (See
CRF-Binding Protein .)

UROCORTIN: A NOVEL MAMMALIAN CRF-RELATED PEPTIDE

Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Urocortin is the newest member of the CRF peptide family and has been demonstrated to possess many of the intrinsic properties of
CRF itself as well as some unique properties of its own. Originally, the nonmammalian CRF-related analogues urotensin I (teleost fish)
(15 ) and sauvagine (frog) (16 ) were thought to subserve the functions of CRF in the respective species; however, the discovery of
peptides even closer to the structure of CRF in those species (17 ,18 ) led to the suggestion that other forms of CRF may exist in
mammals. Furthermore, with the cloning of the CRF2 receptor subtype, it became apparent that because sauvagine and urotensin
had even higher affinity and activity at this subtype than CRF itself (19 ), a mammalian form of these peptides may exist that would
serve as the endogenous ligand for the CRF2 subtype. Indeed, a mammalian form of urotensin was recently discovered termed
“urocortin” and the cDNA cloned from the rat (20 ) and human (21 ), respectively.

Amino Acid Sequence and Structure of Urocortin

The sequence of urocortin has been determined in both rats and humans. In rat, urocortin was identified using a library derived from
rat midbrain and a carp urotensin cDNA probe. A full-length cDNA was described and encoded a putative 40-amino acid peptide that
was related to CRF (20 ). The human form was subsequently identified using a cDNA probe encoding the peptide region of rat
urocortin and screening a human genomic library (21 ). The resulting putative peptide demonstrated 88% identity to rat at the
nucleotide level and 95% identity at the amino acid level (Fig. 7.1 ). In both species, urocortin is a 40-amino acid residue single-
chain polypeptide; the two forms differ by only two amino acids at positions 2 (Asn to Asp) and 4 (Ser to Pro) (21 ). In addition to the
homology between the species, the deduced amino acid sequence of rat and human urocortin exhibits sequence identity with
urotensin I (63%) and human CRF (45%) (20 ). Consistent with the other CRF-related peptides, urocortin is also amidated at its
carboxy terminal, again suggesting the importance of amidation to this family of peptides (Fig. 7.1 ).

Anatomic Distribution of Urocortin

The distribution of urocortin in rat tissues was elucidated first by examining the cellular localization of urotensin-like
immunoreactivity and correlating that distribution with the in situ hybridization of urocortin mRNA. The highest areas of correlation
and overlapping localization were the Edinger-Westphal nucleus and the lateral superior olive (20 ). In addition, cellular staining was
observed in the external plexiform layer of the rat olfactory bulb and lateral hypothalamus. Interestingly, terminal projection fields
in the lateral septum also demonstrated distinct localization where CRF2 receptors have been uniquely localized (see the following).
Although the localization of urocortin appears to be in very discrete brain regions, these regions demonstrate no CRF mRNA,
suggesting that urocortin subserves some unique functionality within the CRF system. As described in the following, its affinity and
functional activity at the
 P.94

CRF2 receptor subtype suggest that this may be one endogenous ligand for this subtype.

In Vitro and In Vivo Pharmacologic Effects

Urocortin binds with high affinity to all the known effectors of CRF function, including CRF1, CRF2α, CRF2B receptors, and CRF-binding
protein (described in the sections that follow). This profile makes urocortin unique in the CRF system because endogenous r/hCRF
has been shown to have relatively low affinity for the CRF2 receptor subtypes and oCRF, which also has lower affinity for the CRF2
subtype, also has very low affinity at the CRF-binding protein. Urocortin binds to cells stably transfected with the CRF1, CRF2α, or
CRF2B receptors with affinities in the 100 to 500 pM range and has 100 pM affinity for the CRF-BP (20 ). In in vitro studies, urocortin
stimulates cAMP accumulation in cells transfected with either CRF receptor subtype, and is extremely potent in stimulating ACTH
release from cultured anterior pituitary cells (20 ). The effects on the CRF1 receptor subtype are comparable to the effects of CRF
itself; however, the activities observed at both CRF2 receptor isoforms are approximately tenfold more potent than CRF itself
(20 ,21 ). Furthermore, as has been shown for CRF, the presence of CRF-BP can decrease the ability of urocortin to stimulate ACTH
release in vitro. Moreover, specific CRF-BP inhibitors such as r/hCRF(9-33) can restore the ability of urocortin to stimulate ACTH,
further confirming the functional activity of urocortin at the CRF-BP (21 ).

In unanesthetized freely moving rats, urocortin administered IV was fivefold more potent than CRF in increasing plasma ACTH
concentrations and demonstrated a longer duration of action. Similarly, urocortin reduced mean arterial pressure more potently and
for a longer period of time than CRF or urotensin I (20 ). Thus, although capable of interacting with the CRF1 receptor with
equivalent potency and activity, the anatomic distribution, localization, and potency at the CRF2 subtypes support the notion that
urocortin is likely one endogenous ligand for this receptor subtype. Clearly, further study is required to determine the specific role
that this novel endogenous peptide plays in the regulation of the CRF system.

CRF RECEPTORS
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Cloning of CRF Receptor Subtypes

Molecular cloning studies have enabled the elucidation of receptor subtypes for the CRF system. Structurally, the CRF receptor
subtypes all contain seven putative transmembrane domains and share considerable sequence homology with one another. These
receptors are members of the family of “brain-gut” neuropeptide receptors, which includes receptors for calcitonin, vasoactive
intestinal peptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, growth hormone-releasing factor, glucagon,
and secretin. In addition, the known members of this neuropeptide receptor family also belong to the superfamily of G-protein
coupled receptors; thus far, all have been shown to stimulate adenylate cyclase in response to their respective agonist activation.

The CRF1 receptor was cloned first from a human Cushing’s corticotropic adenoma using an expression cloning technique and
characterized as a 415-amino acid protein with potential N-linked glycosylation sites, protein kinase C phosphorylation sites in the
first and second intracellular loops and in the C-terminal tail, as well as casein kinase II and protein kinase A phosphorylation sites in
the third intracellular loop (2 ). Independently, this receptor subtype was also identified in mouse (22 ) and rat (23 ,24 ). In all three
species, CRF1 receptor mRNAs encode proteins of 415 amino acids, which are 98% identical to one another. The potential N-linked
glycosylation sites on the N-terminal extracellular domain are characteristic of most G-protein coupled receptors and confirm the
glycosylation profiles determined by chemical affinity cross-linking studies (25 ). In those studies, although the molecular weights of
the proteins labeled from brain or pituitary appeared different when labeled with [125I]oCRF, the deglycosylation and peptide
mapping studies suggested that the protein itself was identical and that the differences were owing to posttranslational modification
(25 ). Indeed, the molecular weight predicted from the deglycosylated forms of the CRF1 receptor was virtually identical to that
obtained from the cloned amino acid sequence. These data taken together established the fact that the CRF1 receptor subtype is the
dominant form in both the brain and pituitary.

CRF Receptors
2

Following the cloning of the CRF1 subtype, two forms of a second family member were discovered in the rat and termed CRF2α and
CRF2B. The rat CRF2α receptor (19 ) is a 411-amino acid protein with approximately 71% identity to the CRF1 receptor. The CRF2B
receptor has been cloned from both rat (19 ) and mouse (24 ,26 ), and is a 431-amino acid protein that differs from the CRF2α
subtype in that the first 34 amino acids in the N-terminal extracellular domain are replaced by 54 different amino acids. The
genomic structure and corresponding cDNA of the human CRF2α receptor subtype was cloned and characterized. The cDNA sequence
in the protein-coding region had 94% identity with the previously reported rat CRF2α receptor (27 ). In addition, the human CRF2α
receptor protein was found to be a 411-amino acid protein that had an overall 70% identity with the human CRF1 receptor sequence
(less in the N-terminal extracellular domain; 47%). In stably transfected cells, the human CRF2α receptor had the same pharmacologic
characteristics as those demonstrated for the
 P.95

rat and increased intracellular cAMP levels in response to either sauvagine or CRF (see the following for details). Very recently, the
human form of the CRF2β receptor was cloned from human amygdala and demonstrated 94% identity to human CRF2α receptors at the
protein level. Preliminary characterization of this novel human isoform indicated that this form also had higher affinity for sauvagine
and urotensin than for r/hCRF (28 ). The CRF2γ receptor was the most recently identified isoform and has thus far only been found in
human brain. This splice variant uses yet a different 5′ alternative exon for its amino terminus and replaces the first 34-amino acid
sequence of the CRF2α receptor with a unique 20 amino acid sequence. Thus, although the CRF2 receptor exists as three isoforms,
CRF2α, CRF2β, and CRF2γ, there are at present no known functional splice variants for the CRF1 receptor. Figure 7.2 illustrates the
differences among human CRF2α, CRF2β, and CRF2γ in the N-terminal extracellular domain. Between the CRF1 and CRF2 receptors,
there exist very large regions of amino acid identity, particularly between transmembrane domains five and six. This similarity
strongly argues for conservation of biochemical function because this region is thought to be the primary site of G-protein coupling
and signal transduction. All three CRF2 receptor subtypes contain five potential N-glycosylation sites, which are analogous to those
found on the CRF1 receptor subtype. The genomic structure of the human CRF2 receptor gene is similar to that of the mouse CRF1
receptor described in the preceding and has 12 introns, the last ten of which interrupt the coding region in identical positions. These
gene sequences, however, diverge significantly at the 5′ end, and the chromosomal mapping of the human CRF2 gene has been
localized to chromosome 7 p21-p15.

FIGURE 7.2. Amino acid sequences of the human

CRF2α, CRF2β, and CRF2γ receptor subtypes. The CRF2
receptor isoforms are demonstrated with the seven
putative transmembrane domains. The arrows
indicate where the different CRF2 receptor isoforms
diverge at the N-terminus. See color version of
figure.

Pharmacologic Characteristics
The literature is replete with information on the pharmacologic and biochemical characterization of CRF receptors in a variety of
tissues and animal species. (See refs. 29 and 30 ,31 ,32 and 33 for reviews.) The radioligand binding characteristics of CRF
receptors that have been performed thus far in brain, endocrine, and immune tissues have used the available radioligands at the
time, which were [125I]-Tyr0 oCRF, [125I]-Tyr0 r/hCRF, and [125I]-Nle21-Tyr32 r/hCRF. These ligands have all demonstrated high affinity for
the CRF1 receptor subtype and lower affinity for the CRF2 subtypes (as described in the following). Thus, the discovery of the CRF2
receptor subtype and its isoforms has not confused the earlier literature owing to the apparent “selectivity” of the r/hCRF and oCRF
analogue radioligands for the CRF1 receptor. Recently, [125I]-Tyr0 sauvagine, a novel radioligand for the CRF2 receptor, has been
described that binds to both receptor subtypes with equal affinity and has become a useful tool in the study of CRF2 receptors (34 ).

CRF receptors fulfill all of the criteria for bona fide receptors. The kinetic and pharmacologic characteristics of CRF1 receptors are
comparable in brain, pituitary, and spleen. The binding of [125I]oCRF in a variety of tissue homogenates as well as in CRF1 receptor-
expressing cell lines is dependent on time, temperature, and tissue concentration, and is saturable, reversible, and of high affinity
with Kd values of 200 to 400 pM. The pharmacologic rank order profile of these receptors from various tissues has been compared
using closely related analogues of CRF. Bioactive analogues of CRF have high affinity for [125I]oCRF binding sites, whereas biologically
inactive fragments of the peptide and unrelated peptides are all without inhibitory binding activity in brain, endocrine, and immune
tissues.

CRF1 receptors exhibit the typical properties of neurotransmitter receptor systems linked to the adenylate cyclase system through a
guanine nucleotide binding protein. In in vitro radioligand binding studies, divalent cations (e.g., magnesium ions) have been shown
to enhance agonist binding to receptors coupled to guanine nucleotide binding proteins by stabilizing the high-affinity form of the
receptor–effector complex. In contrast, guanine nucleotides have the ability to selectively decrease the affinity of agonists for their
receptors by promoting the dissociation of the agonist high-affinity form of the receptor. Consistent with CRF receptors being
coupled to a guanine nucleotide regulatory protein, the binding of [125I]oCRF to pituitary, brain, and spleen homogenates is
reciprocally increased by divalent cations such as Mg2+ and decreased by guanine nucleotides. Furthermore, in expressed cell lines
using a β-galactosidase reporter system, CRF and related analogues could stimulate the production of β-galactosidase in whole cells
with the same pharmacologic rank order of potencies as those in a variety of tissues from different species (35 ).

The cloning of the CRF2 receptor subtype gave the first indication that other family members of this receptor system exist and have
unique properties that could subserve functions that were previously undefined. As mentioned, a fundamental element in the
characterization of any receptor system is the availability of high-affinity and selective ligands that can be used to label the proteins
in a reversible manner. The initial observations clearly demonstrated that the CRF2 receptor subtype recognized the nonmammalian
analogues of CRF with high affinity (similar in profile to the CRF1 subtype) but unlike the CRF1 receptor, had low affinity for the
endogenous CRF ligands (r/hCRF and its analogues) (19 ). Thus, the available radioligands used in the initial studies of CRF receptors
were not useful in providing information about this subtype.

The development of a high-affinity radioligand suitable for the characterization of the CRF2 receptor subtype was recently described
(34 ). Using one of the high-affinity nonmammalian analogues of CRF (sauvagine), a radiolabel was developed, and its binding
specificity and selectivity determined. [125I]Tyr0-sauvagine was found to bind reversibly, saturably, and with high affinity to both the
human CRF1
P.96
 P.97

and CRFsα receptor subtypes expressed in mammalian cell lines. The specific signal for the labeling of the human CRFaα receptors was
greater than 85% over the entire concentration range of the radioligand, which suggested very low nonspecific binding in the
expressed cell line. The radioligand bound in a reversible, time- and protein-dependent manner, reaching equilibrium within 60
minutes with the binding being stable for at least 4 hours at 22°C. Scatchard analyses demonstrated an affinity of about 200 pM for
the CRF2 receptor subtype and a maximum receptor density in the expressing cells of about 180 fmol/mg protein (34 ).
The pharmacologic rank order of potencies for the CRF2 receptor labeled with [125I]sauvagine was essentially identical to the in vitro
effects of the same unlabeled peptides in the production of cAMP in cells expressing the receptor as described. That is, the
nonmammalian analogues sauvagine and urotensin I that were more potent in stimulation of cAMP production were also more potent
at inhibiting the binding of [125I]sauvagine than oCRF or r/hCRF. Interestingly, the putative antagonists for CRF receptors, D-
PheCRF(12-41) and α-helical CRF(9-41) exhibited approximately equal affinity for the two receptor subtypes either in inhibiting
[125I]sauvagine binding or inhibiting sauvagine-stimulated cAMP production (34 ). These data clearly indicated that although distinct
pharmacologic differences exist between the two receptor subtypes of the same family (in terms of their rank order profile), they
still must share some structural similarities. Further study is required to determine the precise common structural features of these
two family members.
In addition to the pharmacologic rank order profile, [125I]sauvagine binding to the CRF2 receptor was guanine nucleotide-sensitive,
confirming the agonist activity of this peptide for the receptor. Although there is as yet no direct evidence, this modulation of the
binding of [125I]sauvagine to the human CRF2α receptor by guanine nucleotides suggests that this receptor exists in two affinity states
for agonists coupled through a guanine nucleotide binding protein to its second messenger system. Unfortunately to date, the only
ligands available for the biochemical study of these receptors have been agonists, making it very difficult to examine the
proportions and affinities of high- and low-affinity states of these receptors. Further study is required, possibly using labeled
antagonists as tools in order to characterize the affinity states of these receptors.
The high affinity of the nonmammalian CRF analogues for this subtype has raised the possibility that other endogenous mammalian
ligands exist that have high affinity and selectivity for this receptor subtype. As described, the recent discovery of urocortin (36 ),
although not selective for the CRF2 subtype, has provided the first evidence for one such endogenous molecule that has high affinity
for the CRF2 receptor. With the increase in the complexity of the CRF system recently elucidated, it is highly likely that more
members, from both the receptor and ligand families, remain to be discovered that will lead to a much more comprehensive
understanding of this system and its role in both normal and pathologic physiology.

Autoradiographic Localization of CRF Receptor Subtypes

Many studies to date have described the distribution of CRF receptors in various tissues, including the pituitary, brain, and spleen
(29 ,31 ,32 and 33 ). The autoradiographic localization of CRF1 receptors in the anterior pituitary demonstrates a clustering of
binding sites that corresponds to the distribution of corticotrophs. The intermediate lobe shows a more uniform distribution of
binding sites characteristic of the homogeneous population of POMC-producing cells in this lobe. Overall, the distribution pattern of
CRF1 receptors within the pituitary supports the functional role of CRF as the primary physiological regulator of POMC-derived
peptide secretion from the anterior and intermediate lobes of the pituitary.
Receptor autoradiography and binding studies in discrete areas of rat and primate CNS demonstrate that, in general, the highest
concentration of CRF binding sites are distributed in brain regions involved in cognitive function (cerebral cortex), limbic areas
involved in emotion and stress responses (amygdala, nucleus accumbens, and hippocampus), brainstem regions regulating autonomic
function (locus ceruleus and nucleus of the solitary tract), and olfactory bulb. In addition, there is a high density of CRF1 receptor
sites in the molecular layer of the cerebellar cortex and the spinal cord where the highest concentrations are present in the dorsal
horn.
CRF receptors in spleen are primarily localized to the red pulp and marginal zones. The localization of [125I]oCRF binding sites in
mouse spleen to regions known to have a high concentration of macrophages suggests that CRF receptors are present on resident
splenic macrophages. The absence of specific [125I]oCRF-binding sites in the periarteriole and peripheral follicular white pulp regions
of the spleen suggests that neither T nor B lymphocytes have specific high-affinity CRF receptors comparable to those localized in
the marginal zone and red pulp areas of the spleen or in the pituitary and brain.
The availability of nucleotide sequences for CRF1 and CRF2 receptors has allowed a detailed examination of the regional and cellular
distribution of CRF receptor subtype mRNA expression utilizing both RNAse protection assays and in situ hybridization histochemistry.
A comparison of the distribution of CRF1 and CRF2 mRNA and receptor protein defined by ligand autoradiography is demonstrated in
adjacent horizontal sections of rat brain (Fig. 7.3 ).

FIGURE 7.3. Digitized, color-coded images of CRF1 (Panel A) and CRF2

(Panel B) receptor mRNA expression and receptor autoradiography in
adjacent horizontal sections of rat brain. The highest levels of mRNA
expression are coded in red, whereas the lowest concentrations are coded
in blue. Similarly, the highest densities of receptors labeled with either
[125I]oCRF (CRF1 only; Panel C) or [125I]sauvagine (CRF1 and CRF2; Panel D) are
coded in red. There was a good correspondence between the message for a
particular receptor subtype and its protein localization; the pharmacologic
selectivity was retained for the two radioligands. (Compare Panels C and
D.) See color version of figure.

The distribution of CRF2 message clearly differs from that of the CRF1 and exhibits a distinct subcortical pattern. In the rat brain, the
CRF1 mRNA was most abundant in neocortical, cerebellar, and sensory relay structures and generally
 P.98

corresponded to the previously reported distribution of [125I]oCRF binding sites (Fig. 7.3 ). On the other hand, the CRF2 receptor
mRNA was localized primarily in subcortical regions such as the lateral septal nuclei, hypothalamic nuclei, bed nucleus of the stria
terminalis, and amygdaloid nuclei. Using the radioligand [125I]sauvagine described, CRF2 receptors could be localized to areas of high
CRF2 message. In addition, because [125I]sauvagine has equal affinity for both receptor subtypes (34 ), the autoradiography revealed
the localization of both the CRF1 and CRF2 receptor subtypes, demonstrating the utility of this novel radioligand. (See ref. 37 for a
complete and detailed account of the mRNA distribution patterns of CRF1 and CRF2 receptors.)

The heterogeneous distribution of CRF1 and CRF2 receptor mRNA and protein suggests distinctive functional roles for each receptor
within the CRF system. For example, the lateral septum, by virtue of widespread reciprocal connections throughout the brain, is
implicated in a variety of physiologic processes. These range from higher cognitive functions such as learning and memory to
autonomic regulation, including food and water intake (38 ). In addition, the septum plays a central role in classical limbic circuitry
and thus is important in a variety of emotional conditions, including fear and aggression. Thus, the lack of CRF1 receptor expression
in these nuclei suggests that CRF2 receptors may solely mediate the postsynaptic actions of CRF inputs to this region and strongly
suggests a role for CRF2 receptors in modulating limbic circuitry at the level of septal activity. In addition, the selective expression
of CRF2 receptor mRNA within hypothalamic nuclei indicates that the anxiogenic and anorexic actions of CRF in these nuclei may
likely be CRF2 receptor-mediated. In contrast, within the pituitary, there is a predominance of CRF1 receptor expression with little
or no CRF2 expression in either the intermediate and anterior lobes, indicating that it is the CRF1 receptor that is primarily
responsible for CRF regulation of the HPA axis.

In addition to the differences in distribution between the CRF1 and CRF2 receptor subtypes, there exists a distinct pattern of
distribution between the CRF2 isoforms (CRF2α and CRF2B) as well. The CRF2α isoform is primarily expressed within the CNS, whereas
the CRF2B form is found both centrally and peripherally. Within the brain, the CRF2α form is the predominant one, whereas the CRF2B
form is localized primarily to non-neuronal structures, the choroid plexus of the ventricular system, and cerebral arterioles (37 ,39 ).
The identification of the CRF2B form in cerebral arterioles suggests a mechanism through which CRF may directly modulate cerebral
blood flow. Peripherally, the highest detectable levels of mRNA were found in heart and skeletal muscle with lower levels detected
in lung and intestine (24 ,39 ). Taken together, the results of these studies demonstrating a distinct heterogeneous distribution
pattern of CRF receptor subtypes in brain and peripheral tissues, strongly suggest that these receptor subtypes subserve very
specific physiological roles in CRF related function both centrally and peripherally.

Second Messengers Coupled to CRF Receptors

Radioligand binding studies have demonstrated that CRF receptors in the brain-endocrine-immune axis are coupled to a guanine
nucleotide regulatory protein. In all of these tissues, the primary second messenger system involved in transducing the actions of
CRF is stimulation of cAMP
 P.99

production (29 ,31 ,32 and 33 ,40 ). CRF initiates a cascade of enzymatic reactions in the pituitary gland beginning with the
receptor-mediated stimulation of adenylate cyclase, which ultimately regulates POMC-peptide secretion and possibly synthesis.
POMC-derived peptide secretion mediated by the activation of adenylate cyclase in the anterior and neurointermediate lobes of the
pituitary is dose-related and exhibits appropriate pharmacology. Similarly in the brain and spleen, the pharmacologic rank order
profile of CRF-related peptides for stimulation of adenylate cyclase is analogous to the profile seen in pituitary and in keeping with
the affinities of these compounds for receptor binding. In addition, the putative CRF receptor antagonist α-hel ovine CRF(9-41)
inhibits CRF-stimulated adenylate cyclase in brain and spleen homogenates.

In addition to the adenylate cyclase system, other signal transduction mechanisms may be involved in the actions of CRF. For
example, CRF has been shown to increase protein carboxyl methylation, and phospholipid methylation in AtT-20 cells (41 ).
Preliminary evidence suggests that CRF may regulate cellular responses through products of arachidonic acid metabolism (42 ).
Furthermore, although the evidence in anterior pituitary cells suggests that CRF does not directly regulate phosphatidylinositol
turnover or protein kinase C activity (42 ), stimulation of protein kinase C either directly or by specific ligands (vasopressin or
angiotensin II), enhances CRF-stimulated adenylate cyclase activity, ACTH release, and inhibits phosphodiesterase activity (42 ).
Thus, the effects of CRF on anterior pituitary cells and possibly in neurons and other cell types expressing CRF receptors are likely to
involve complex interactions among several intracellular second messenger systems.

CRF-BINDING PROTEIN
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

CRF and Its Binding Protein in Human Plasma

Under normal conditions, the plasma levels of CRF remain low; however, CRF levels are markedly elevated in plasma during the late
gestational stages of pregnancy (43 ,44 and 45 ). The source of the pregnancy-associated CRF is most likely the placenta because
previous studies have demonstrated that the human placenta synthesizes CRF (46 ). The CRF in the maternal plasma is bioactive in
releasing ACTH from cultured pituitary cells (44 ). In spite of the high levels of CRF in the maternal plasma, there is no evidence of
markedly increased ACTH secretion or hypercortisolism in pregnant women (43 ). A plausible explanation for this paradoxic situation
could be the presence of a binding protein in the plasma of pregnant women that could specifically inhibit the biological actions of
CRF (44 ,45 ). This hypothesis was validated by the isolation of a CRF-binding protein (CRF-BP) from human plasma and its
subsequent cloning and expression (see the following).

cDNA and Amino Acid Sequences

The CRF-BP was first isolated and purified to near homogeneity for sequencing and generation of oligonucleotide probes (47 ).
Screening a human liver cDNA library using probes generated from the original amino acid sequence revealed a full-length cDNA
containing a 1.8-kb insert that coded for a novel protein of 322 amino acids (48 ). A single putative N-linked glycosylation site was
found at amino acid 203, which agrees with the previous observation of the presence of asparagine-linked sugar moieties on the
native protein (49 ). Subsequent screening of a rat cerebral cortical cDNA library, revealed the presence of a single clone containing
a 1.85-kb insert predicting a protein of 322 amino acids, which was 85% identical to the human CRF-BP. The putative glycosylation
site on the rat protein seems to be conserved between the rat and human sequences (48 ). The pharmacology of these proteins
appears to be similar with both the rat and human binding proteins having high affinity for the rat/human CRF (Kd ˜ 0.2 nM) and very
low affinity for the ovine form of CRF (Kd ˜ 250 nM). Although there may be some similarities in the binding domains of the binding
protein and the CRF receptor (as evidenced by the equal affinity of r/hCRF), these are distinct proteins, each with unique
characteristics and distributions.

Distribution in Brain and Pituitary

Although the human and rat forms of the CRF-BP are homologous (as indicated), there is a somewhat different anatomic distribution
pattern in the two species. The human form of the binding protein has been found abundantly in areas including liver, placenta, and
brain, whereas in the rat levels of mRNA for the binding protein have only been localized in the brain and pituitary (48 ). Peripheral
expression of the binding protein may have its greatest utility in the modulation and control of the elevated levels of CRF in
circulating plasma induced by various normal physiologic conditions (see the preceding). In addition, expression of this binding
protein in the brain and pituitary offers additional mechanisms by which CRF-related neuronal or neuroendocrine actions may be
modulated.

CRF-binding protein has been localized to a variety of brain regions including neocortex, hippocampus (primarily in the dentate
gyrus), and olfactory bulb. In the basal forebrain, mRNA is localized to the amygdaloid complex with a distinct lack of
immunostained cells in the medial nucleus. CRF-binding protein immunoreactivity is also present in the brainstem particularly in the
auditory, vestibular, and trigeminal systems, raphe nuclei of the midbrain and pons, and reticular formation (50 ). In addition, high
expression levels of binding protein mRNA are seen in the anterior pituitary, predominantly restricted to the corticotrope cells.
Expression of this protein in the corticotropes strongly suggests that the CRF-BP is involved in the regulation of neuroendocrine
 P.100

functions of CRF by limiting and/or affecting the interactions of CRF with its receptor, which is also known to reside on
corticotropes; however, the detailed role of the binding protein in regulating pituitary–adrenal function remains to be elucidated.

CRF REGULATION OF NEUROENDOCRINE FUNCTION

Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Regulation of Pituitary Hormone Secretion

CRF is the major physiologic regulator of the basal and stress-induced release of ACTH, B-endorphin, and other POMC-derived
peptides from the anterior pituitary. (See refs. 3 , 4 , and 51 for reviews.) CRF stimulates the release of POMC-derived peptides in
anterior pituitary cells in culture and in vivo; these actions of CRF can be antagonized by the CRF receptor antagonist α-helical
ovine CRF(9-41) or by immunoneutralization with an anti-CRF antibody. Several other lines of evidence support a critical role for
endogenous CRF in regulating ACTH secretion. For example, increases in CRF in the hypophysial portal blood are observed following
application of stress. Administration of CRF antisera or the CRF receptor antagonist results in attenuation of stress- or
adrenalectomy-induced ACTH secretion further substantiating a role for CRF in regulating ACTH secretion from the anterior pituitary.
In addition to effects in the anterior pituitary, CRF also has been reported to stimulate POMC-derived peptide secretion from the
intermediate lobe of the pituitary gland.

Central administration of CRF inhibits the secretion of luteinizing hormone (LH) and growth hormone without any major effects on
follicle-stimulating hormone, thyroid stimulating hormone, or prolactin secretion (3 ,4 ). The effects of CRF to inhibit LH secretion
appear to be mediated at the hypothalamic level through effects of CRF to inhibit gonadotropin releasing hormone secretion. CRF-
induced inhibition of LH secretion may also involve endogenous opioids since the effects are attenuated by administration of
naloxone or antiserum to B-endorphin (3 ,4 ).

Regulation of Hypothalamic CRF Release

Plotsky and associates (52 ) and Owens and Nemeroff (4 ) provide a comprehensive review of the neurotransmitter regulation of
hypothalamic CRF release. Most studies demonstrate stimulatory effects of cholinergic and serotonergic neurons on CRF release. The
muscarinic and/or nicotinic cholinergic receptor subtypes involved in the stimulatory effects of acetylcholine on CRF secretion
remain to be precisely elucidated. The effects of serotonin to stimulate CRF release appear to be mediated by a variety of receptor
subtypes, including 5-HT2, 5-HT1A, and 5-HT1C receptors. The effects of catecholamines and opioids on hypothalamic CRF release are
less well defined. Norepinephrine has been reported to have both stimulatory and inhibitory effects on CRF release that may be a
consequence of the dose administered as well as the receptor subtype involved. For example, in studies sampling hypophysial portal
concentrations of CRF, Plotsky and colleagues (52 ) noted that low doses of norepinephrine stimulated CRF release in vivo via α-
adrenergic receptors and inhibited CRF release at high doses via B-adrenergic receptors. Similarly, opioids have been reported to
either inhibit or stimulate CRF release depending on the nature of the opioid tested, dose utilized, and receptor specificity (μ versus
κ) involved. Drugs acting at GABA–benzodiazepine–chloride ionophore complex are potent inhibitors of CRF secretion.

Stress is a potent general activator of CRF release from the hypothalamus. The extent and time course of changes in CRF in the
paraventricular nucleus and median eminence of the hypothalamus following application of stress are highly dependent on the
nature of the stressor as well as the state of the animal. The effects of stress to increase the release and synthesis of CRF are
mediated by many of neurotransmitter systems described in the preceding.

Glucocorticoids, which are involved in the negative feedback regulation the hypothalamic–pituitary–adrenocortical axis, are potent
inhibitors of CRF release. Conversely, the absence of glucocorticoids following adrenalectomy results in marked elevations in the
synthesis and release of CRF. The actions of glucocorticoids to inhibit CRF release are mediated directly at the level of the
paraventricular nucleus of the hypothalamus as well as indirectly through actions on receptors in the hippocampus.

Modulation of Pituitary CRF Receptors

Stress (29 ,32 ,33 ,53 ) or adrenalectomy (29 ,32 ,33 ) result in hypersecretion of CRF and a consequent down-regulation of receptors
in the anterior pituitary. The adrenalectomy-induced decreases in anterior pituitary receptors can be prevented by glucocorticoid
replacement with corticosterone or dexamethasone (29 ,32 ,33 ). In addition, chronic administration of corticosterone has been
reported to cause dose-dependent decreases in anterior pituitary CRF receptor number (29 ,32 ,33 ). An age-related decline in
anterior pituitary CRF receptors has also been reported (54 ). In contrast, lesions of the paraventricular nucleus that result in
dramatic reductions in hypothalamic CRF secretion have been reported to increase the density of pituitary CRF receptors (4 ). Thus,
CRF receptors in the anterior pituitary appear to be reciprocally regulated by hypothalamic CRF release.

CRF REGULATION OF CNS ACTIVITY

Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Electrophysiologic Effects of CRF

CRF stimulates the electrical activity of neurons in various brain regions that contain CRF and CRF receptors, including
 P.101

locus ceruleus (55 ), hippocampus (56 ), cerebral cortex, and hypothalamus as well as in lumbar spinal cord motor neurons (3 ,4 ). In
contrast, CRF has inhibitory actions in the lateral septum, thalamus, and hypothalamic PVN (3 ,4 ). The electrophysiologic effects of
CRF on spontaneous and sensory-evoked activity of locus ceruleus neurons are well documented (55 ). Activation of the locus
ceruleus, a brainstem nucleus comprising of noradrenergic cells, results in arousal and increased vigilance. Furthermore, dysfunction
of this nucleus has been implicated in the pathophysiology of depression and anxiety. Centrally administered CRF increases the
spontaneous discharge rate of the locus ceruleus in both anesthetized and unanesthetized rats, while decreasing evoked activity in
the nucleus (55 ). Thus, the overall effect of CRF in the locus ceruleus is to decrease the signal to noise ratio between evoked and
spontaneous discharge rates.

The effects of CRF on EEG activity have been reviewed in detail (3 ,4 ,57 ). CRF causes a generalized increase in EEG activity
associated with increased vigilance and decreased sleep time. At CRF doses below those affecting locomotor activity or pituitary–
adrenal function, rats remain awake, vigilant and display decreases in slow-wave sleep compared to saline-injected controls (57 ).
Higher doses of the peptide, on the other hand, cause seizure activity that is indistinguishable from seizures produced by electrical
kindling of the amygdala, further confirming the role of CRF in brain activation.

Autonomic Effects of CRF

A great deal of anatomic, pharmacologic, and physiologic data support the concept that CRF acts within the CNS to modulate the
autonomic nervous system (3 ,4 ,58 ,59 ). For example, central administration of CRF results in activation of the sympathetic
nervous system resulting in stimulation of epinephrine secretion from the adrenal medulla and noradrenergic outflow to the heart,
kidney, and vascular beds. Other consequences of central administration of CRF include increases in the mean arterial pressure and
heart rate. These cardiovascular effects of CRF can be blocked by the ganglionic blocker chlorisondamine underscoring the
sympathetic actions of the peptide. In contrast, CRF acts in brain to inhibit cardiac parasympathetic nervous activity. (See ref. 58
for review.) Peripheral administration of CRF causes vasodilation and hypotension in a variety of species including humans
(3 ,4 ,58 ,59 ). The physiologic role of CRF in regulating the autonomic nervous system is supported by data demonstrating central
effects of the CRF receptor antagonist, α-helical ovine CRF(9-41) to attenuate adrenal epinephrine secretion resulting from stressors
such as insulin-induced hypoglycemia, hemorrhage, and exposure to ether vapor (59 ). Overall, these data substantiate a major role
for CRF in coordinating the autonomic responses to stress.

Gastrointestinal Effects of CRF

Studies examining the gastrointestinal effects of CRF have determined that CRF modulates gastrointestinal activity by acting at
central and possibly peripheral sites, and that these effects are qualitatively similar to those observed following exposure to various
stressors. (See refs. 3 , 4 , and 60 for reviews.) CRF inhibits gastric acid secretion, gastric emptying, and intestinal transit while
stimulating colonic transit and fecal excretion in a dose-dependent manner when administered centrally or systemically to dogs or
rats. CRF is equipotent in inhibiting gastric emptying in both species following both central and peripheral routes of administration.
The central effects of CRF on gastric acid secretion do not appear to result from leakage of the peptide into peripheral blood
because measurable quantities of CRF are not present in the circulation following injection of CRF into the third ventricle of the dog.
Furthermore, an intravenous injection of anti-CRF serum completely abolishes the peripheral but not the central effect of CRF on
gastric acid secretion. These data strongly implicate CRF in the mechanisms through which various stressors alter gastrointestinal
function and are consistent with its proposed role in integrating the autonomic nervous system’s response to stress.

Behavioral Effects of CRF

The behavioral effects of CRF in the CNS have been reviewed extensively (3 ,4 ,61 ). The effects of CRF on behavior are dependent
on both the dose of peptide administered and the specific conditions under which the tests are performed. In a familiar or “home”
environment, central administration of CRF produces a profound increase in locomotor activity. Although very low doses of CRF
produce locomotor activation when tested in an open field test, higher doses produce a dramatic decrease in locomotor activity.
CRF administered intracerebrally also produces additional behavioral effects including increases in sniffing, grooming, and rearing in
a familiar environment, increased “emotionality” and assumption of a freeze posture in a foreign environment, decreased feeding
and sexual behavior, and increased conflict behavior. The behavioral effects of CRF are not an indirect consequence of actions of
the peptide to activate pituitary–adrenocortical hormone secretion because they are not seen following peripheral administration of
CRF or following pretreatment with doses of dexamethasone that adequately block pituitary–adrenal activation. Of critical
importance is the observation that these effects of CRF can all be blocked by administration of the peptide antagonist α-helical
ovine CRF(9-41), strongly supporting a specific CRF receptor-mediated event in these behaviors. Furthermore, the CRF receptor
antagonist by itself attenuates many of the behavioral consequences of stress underscoring the role of endogenous peptide in
mediating many of the stress-related behaviors.

ROLE FOR CRF IN NEUROPSYCHIATRIC DISORDERS AND

NEURODEGENERATIVE DISEASES
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "
 P.102

Major Depression and Anxiety Disorders

Many patients with major depression are hypercortisolemic and exhibit an abnormal dexamethasone suppression test. Given the
primary role of CRF in stimulating pituitary–adrenocortical secretion, the hypothesis has been put forth that hypersecretion or
hyperactivity of CRF in brain might underlie the hypercortisolemia and symptomatology seen in major depression. (See refs. 62 and
63 for review.) The concentration of CRF is significantly increased in the cerebrospinal fluid (CSF) of drug-free individuals
(4 ,64 ,65 ), and a significant positive correlation is observed between CRF concentrations in the CSF and the degree of
postdexamethasone suppression of plasma cortisol (66 ). Furthermore, the observation of a decrease in CRF binding sites in the
frontal cerebral cortex of suicide victims compared to controls is consistent with the hypothesis that CRF is hypersecreted in major
depression (67 ). The elevated CSF concentrations of CRF seen in depressed individuals are decreased following treatment with
electroconvulsive therapy (68 ). In addition, a blunted ACTH response to intravenously administered ovine or human CRF is observed
in depressed patients when compared to normal controls (69 ). The blunted ACTH response to exogenous CRF seen in depressed
patients may be caused by the intact negative feedback of cortisol on the corticotrophs, a compensatory decrease in CRF receptors
subsequent to chronic hypersecretion of the peptide, and/or desensitization of the pituitary corticotrophs to respond to CRF.

A number of studies suggest that anxiety-related disorders (e.g., panic disorder and generalized anxiety disorder) and depression are
independent syndromes that share both clinical and biological characteristics. The role that has been proposed for CRF in major
depressive disorders along with preclinical data in rats demonstrating effects of CRF administration to produce several behavioral
effects characteristic of anxiogenic compounds (61 ) have led to the suggestion that CRF may also be involved in anxiety-related
disorders. A role for CRF in panic disorder has been suggested by observations of blunted ACTH responses to intravenously
administered CRF in panic disorder patients when compared to controls (70 ). The blunted ACTH response to CRF in panic disorder
patients most likely reflects a process occurring at or above the hypothalamus, resulting in excess secretion of endogenous CRF.

Anorexia Nervosa
Anorexia nervosa is an eating disorder characterized by tremendous weight loss in the pursuit of thinness. There is similar
pathophysiology in anorexia nervosa and depression, including the manifestation of hypercortisolism, hypothalamic hypogonadism,
and anorexia. Furthermore, the incidence of depression in anorexia nervosa patients is high. Like depressed patients, anorexics show
a markedly attenuated ACTH response to intravenously administered CRF (4 ,64 ,65 ). When the underweight anorexic subjects are
studied after their body weight had been restored to normal, their basal hypercortisolism, increased levels of CRF in the CSF, and
diminished ACTH response to exogenous CRF all return to normal at varying periods during the recovery phase (4 ,64 ,65 ). CRF can
potently inhibit food consumption in rats, which further suggests that the hypersecretion of CRF may be responsible for the weight
loss observed in anorexics. In addition, the observation that central administration of CRF diminishes a variety of reproductive
functions (4 ,65 ) lends relevance to the clinical observations of hypogonadism in anorexics.

Alzheimer’s Disease
Several studies have provided evidence in support of alterations in CRF in Alzheimer’s disease (AD) (4 ,65 ,71 ,72 ,73 and 74 ).
There are decreases in CRF content and reciprocal increases in CRF receptors in cerebral cortical areas affected in AD such as the
temporal, parietal, and occipital cortex. The reductions in CRF and increases in CRF receptors are all greater than 50% of the
corresponding control values. The up-regulation in cerebral cortical CRF receptors in AD under conditions in which the endogenous
peptide is reduced suggests that CRF-receptive cells may be preserved in the cortex in AD. Chemical cross-linking studies have
demonstrated a normal pattern of labeling of cerebral cortical CRF receptors in AD when compared to age-matched controls (75 ).
Although these decreases in CRF content have a modulatory action on the receptors (up-regulation), there appears to be no effect
on the concentration or levels of CRF-binding protein in cerebral cortical areas affected in AD (76 ). The reduction in cortical CRF
content may be owing to selective degeneration of CRF neurons intrinsic to the cerebral cortex or dysfunction of CRF neurons
innervating the cortex from other brain areas. Additional evidence for a role for CRF in AD is provided by observations of decreases
in CRF in other brain areas including the caudate (71 ) and decreased concentrations of CRF in the CSF (77 ,78 ). Furthermore, a
significant correlation is evident between CSF CRF and the global neuropsychological impairment ratings, suggesting that greater
cognitive impairment is associated with lower CSF concentrations of CRF (79 ).

Immunocytochemical observations demonstrating morphologic alterations in CRF neurons in AD complement the studies described in
the preceding. In AD, swollen, tortuous CRF-immunostained axons, termed fiber abnormalities, are clearly distinguishable from the
surrounding normal neurons and are also seen in conjunction with amyloid deposits associated with senile plaques (80 ).
Furthermore, the total
 P.103

number of CRF-immunostained axons is reduced in the amygdala of Alzheimer’s patients (80 ). Interestingly, the expression of CRF
antigen in neurons is not globally reduced in Alzheimer’s patients. CRF immunostaining of perikarya and axons located in the
hypothalamic paraventricular nucleus is much more intense in AD patients than controls (80 ). Increased immunostaining of the
paraventricular neurons in AD, if truly representative of increased content of CRF, could be related to increased amounts of CRF
mRNA in these cells or increased translation of available mRNA. The increased expression and/or release of CRF from the
paraventricular nucleus of the hypothalamus would provide a reasonable explanation for the hypercortisolemia often seen in
Alzheimer’s patients.

At present, the cerebral cortical cholinergic deficiency seems to be the most severe and consistent deficit associated with AD.
Reductions in cerebral cortical CRF correlate with decreases in choline acetyl transferase (ChAT) activity (72 ). In Alzheimer’s, there
are significant positive correlations between ChAT activity and reduced CRF in the frontal, temporal, and occipital lobes. Similarly,
significant negative correlations exist between decreased ChAT activity and increased number of CRF receptors in the three cortices.
These data suggest that the reported reciprocal changes in presynaptic and postsynaptic markers in CRF in cerebral cortex of
patients with AD may be, in part, a consequence of deficits in the cholinergic projections to the cerebral cortex. Additional studies
are necessary to determine the functional significance of the interaction between CRF and cholinergic systems.

Other Neurological Disorders

Alternations in brain concentrations of CRF have been reported in other neurological diseases. For example, in cases of Parkinson’s
disease (PD) with dementia that also show pathologic features of AD, CRF content is decreased and shows a pattern similar to those
cases exhibiting the pathology of AD alone (74 ,81 ). Specimens from patients with PD who did not have the histopathology
characteristic of AD also demonstrate reductions of CRF content, although the reductions are less marked than in cases of combined
AD and PD. Normal levels of CRF have been reported in the hypothalamus in PD (82 ), suggesting that the loss of CRF in the cerebral
cortex is not generalized. CRF is deceased to approximately 50% of the control values in the frontal, temporal, and occipital lobes of
patients with progressive supranuclear palsy (74 ,81 ), a rare neurodegenerative disorder that shares certain clinical and pathologic
features with AD.

The similarity of the changes in CRF found in the context of the three neurological diseases associated with Alzheimer-type
pathology raised the possibility that cerebral cortical reduction is nothing more than a nonspecific sequela of the disease process. In
Huntington’s disease (HD), a neurological disorder in which minimal cerebral cortical pathology is present, the CRF content in the
frontal, temporal, parietal, occipital, and cingulate cortices and in the globus pallidus is not significantly different from that seen in
neurologically normal controls (73 ). However, the CRF content in the caudate nucleus and putamen of the basal ganglia (a brain
area that is severely affected in the disease) is less than 40% of the CRF concentrations seen in controls (73 ). The localization of the
CRF changes to only affected brain regions in the four neurodegenerative disorders described suggest that CRF has an important role
in the pathology of these dementias.

POTENTIAL THERAPEUTIC STRATEGIES DESIGNED FOR THE CRF

SYSTEM
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "

Nonpeptide CRF-Binding Protein Ligand Inhibitors

As mentioned, the CRF-binding protein has the capacity to bind and functionally inactivate CRF. Peptide CRF-BP ligand inhibitors
have been shown in vitro, to release CRF from the binding protein making it available for binding to its receptor and have been
theorized to have efficacy in diseases that are associated with low levels of CRF such as Alzheimer’s disease (76 ). Interestingly,
unlike the direct i.c.v. administration of CRF, inhibition of the CRF-BP by ligand inhibitors that release functional CRF does not
cause anxiogenic-like activity in animal models, validating the approach for diseases that require an increase in CRF function (76 ).
Thus, compounds that act to dissociate CRF from its binding protein complex will act to selectively increase synaptic concentrations
of CRF in discrete brain regions and may provide a novel treatment opportunity for disorders associated with low levels of CRF. To
date, however, no nonpeptide CRF-BP ligand inhibitors have been discovered to test this hypothesis. On the other hand, small
molecule nonpeptide CRF receptor antagonists have shown encouraging preliminary results in this complex system.

Nonpeptide CRF Receptor Antagonists

As indicated in this and previous reviews of the CRF system, there are a number of neuropsychiatric indications where clinical and
preclinical data have shown that the CRF system is hyperactivated as evidenced by abnormally high levels of CRF within the CNS. For
example, in clinical studies of major depression, increased CRF concentrations in the cerebrospinal fluid, increased HPA activity,
blunted ACTH responsiveness to exogenously administered CRF, and pituitary and adrenal hypertrophy have all indicated
hypersecretion of CRF associated with this disorder. (See refs. 4 , 5 , 32 , 65 , and 83 for review.) It follows that for these
indications the therapeutic strategy would involve functional blockade of the actions of CRF. This can be achieved
 P.104

through inhibition of CRF synthesis and secretion, inactivation of CRF (either by antibody neutralization or increased metabolism), or
direct antagonism by specific receptor blockade. Although anti-CRF antibodies have demonstrated antiinflammatory effects (84 ),
these types of therapies are limited to peripheral use and could not be readily formulated for oral administration for central activity.
Nevertheless, these therapeutics could be useful for the treatment of disorders with peripheral elevations of CRF such as
rheumatoid arthritis.

The best strategy for the blockade of elevated CRF levels is to design specific and selective nonpeptide receptor antagonists.
Particularly for use in the brain, these molecules can be designed to have receptor subtype specificity, good oral bioavailability, and
rapid penetration across the blood–brain barrier; characteristics that are difficult to optimize with peptide therapies. The recent
surge in combinatorial chemistry techniques, coupled with recent technological advances in robotic high-throughput screening and
data management of large libraries of molecules have enabled the field of small molecule drug discovery. These advancements have
led to the identification of several patented structural series of molecules known to antagonize the effects of CRF at the CRF1
receptor subtype. (See ref. 85 for a complete review.) Several of these small molecules have recently appeared in the literature
and reported to have good CRF1 receptor antagonistic activity. Compounds such as CP 154,526 (86 ), NBI 27914 (87 ), Antalarmin
(88 ), and most recently DMP 696 (89 ), all demonstrate a good in vitro profile, showing selectivity for the CRF1 receptor subtype.
Systemic administration of these compounds have been found to attenuate stress-induced elevations in plasma ACTH levels in rats,
demonstrating that CRF1 receptors can be blocked in the periphery. Furthermore, nonpeptide CRF1 antagonists administered
peripherally have also been demonstrated to inhibit CRF-induced seizure activity (90 ). Until recently, however, these compounds
have suffered from poor solubility and pharmacokinetics, thus limiting their utility in in vivo characterization of the individual
compounds as well as the overall proof of concept for the mechanism of CRF receptor efficacy. One compound has very recently
been described as a water-soluble nonpeptide CRF1 receptor antagonist (NBI 30775, also referred to as R121919) that demonstrates
high affinity, and has a superior in vitro and in vivo profile compared to other nonpeptide CRF receptor antagonists (91 ).

NBI 30775, is a pyrazolopyrimidine with high affinity for the CRF1 receptor and over 1,000-fold weaker activity at the CRF2 receptor
subtype. This compound does not interact at all with the CRF binding protein and was shown to be as potent as the peptide
antagonist D-Phe CRF(12-41) at inhibiting the CRF-stimulated cAMP accumulation from cells that express the human CRF1 receptor
and CRF-stimulated ACTH release from cultured rat anterior pituitary cells in vitro. In vivo, this molecule potently attenuated the
plasma elevations of ACTH observed following a stressful stimulus in the rat, and demonstrated both dose- and time-dependent CRF1
receptor occupancy concomitant with the levels of drug measured in whole brain (91 ). Owing to the promising preclinical profile of
this compound this particular compound was assessed in full Phase I clinical trials and an initial open label Phase IIa study where the
compound was assessed in patients with major depressive disorder.

First Clinical Experience with CRF Receptor Antagonists

1

Preclinically, studies have demonstrated that attenuation of the CRF system, either by decreasing synthesis and release or by
selective blockade of the CRF receptor, results in decreased anxiety and behavioral activation in stressed animals; however,
clinically it will probably not be beneficial to the overall outcome of the patient if the stress axis is maximally compromised. The
preclinical studies described, prompted the development of NBI 30775 in Phase I safety studies in humans and in an open-label
clinical trial in patients with major depressive disorder (92 ). In this latter study severely depressed patients were given NBI 30775
orally once daily in a dose-escalating manner and their hypothalamic-pituitary adrenal (HPA) function assessed. In addition, the
patients’ level of depression or anxiety was measured using the Hamilton depression (HAM-D) and anxiety (HAM-A) scales. The
results demonstrated that this compound was safe and well tolerated under the conditions of this study. Moreover, the data
suggested that blockade of the CRF1 receptor in these patients did not result in an impairment of the HPA axis either at baseline or
following an exogenous CRF challenge (92 ). This demonstration was critical in setting this potential therapy apart from existing
therapies that blunt basal functioning of multiple neurotransmitter systems. Furthermore, although under the limited conditions of
an open-label trial, there was a statistically significant dose-dependent reduction in the depression and anxiety scores using both
clinician and patient ratings, suggesting that this mechanism may provide an exciting novel therapy in patients suffering with major
depressive disorder. Although it is of great importance at this stage to develop these compounds as tools in the ultimate
understanding of the CRF system and the role it plays in neuropsychiatric disorders, evidence is now beginning to emerge that
compounds of this class, and more importantly of this mechanism, will prove beneficial in neuropsychiatric disorders such as
depression or anxiety. Should compounds such as this continue to demonstrate efficacy in these disorders without severely
compromising the stress axis as a whole, they would validate the CRF hypothesis for depression and anxiety and provide an entirely
novel treatment for these devastating diseases.

CONCLUSION
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "
 P.105

Corticotropin-releasing factor is the key regulator of the organism’s overall response to stress. CRF has hormone-like effects at the
pituitary level to regulate ACTH secretion that, in turn coordinates the synthesis and secretion of glucocorticoids from the adrenal
cortex. CRF also functions as a bona fide neurotransmitter in the CNS. CRF neurons and receptors are widely distributed in the CNS
and play a critical role in coordinating the autonomic, electrophysiologic, and behavioral responses to stress.
Clinical data have implicated CRF in the etiology and pathophysiology of various endocrine, psychiatric, and neurological disorders.
Hypersecretion of CRF in brain may contribute to the symptomatology seen in neuropsychiatric disorders such as depression,
anxiety-related disorders, and anorexia nervosa. In contrast, deficits in brain CRF are apparent in neurodegenerative disorders such
as AD, PD, and HD as they relate to dysfunction of CRF neurons in brain areas affected in the particular disorder. The recent
discovery of novel receptor family members as well as novel alternative ligands for these subtypes serve not only to increase our
understanding of the system but provide a basis for selective and rational drug design for the treatment of disorders that are
associated with aberrant levels of CRF. Strategies directed at developing specific and selective CRF agents have yielded many
nonpeptide small molecule CRF1 receptor antagonists and a preliminary proof-of-concept has encouraged the further development of
such agents. Compounds such as those described in this chapter may hold promise for novel therapies for the treatment of these
various neuropsychiatric disorders without severely compromising this highly complex hormonal system. Clearly with the recent
advances made within a very short period of time, it now seems possible to begin a full understanding of this increasingly complex
neurohormone system.

ACKNOWLEDGMENT
Part of "7 - Corticotropin-Releasing Factor: Physiology, Pharmacology, and Role in Central Nervous System Disorders "
Dr. De Souza is a stockholder in Neurocrine Biosciences, Inc.

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8
Neurogenesis in Adult Brain

Fred H. Gage

Henriette Van Praag

Fred H. Gage and Henriette van Praag: The Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California.

 HISTORIC PERSPECTIVE
 CHARACTERIZATION OF CELL GENESIS IN VIVO
 PROPERTIES OF STEM CELLS IN VITRO
 REGULATION OF PROLIFERATION AND DIFFERENTIATION IN VIVO
 FUNCTIONAL SIGNIFICANCE OF NEUROGENESIS
 POTENTIAL THERAPEUTIC IMPLICATIONS
 ACKNOWLEDGMENTS

HISTORIC PERSPECTIVE
Part of "8 - Neurogenesis in Adult Brain "

The idea that the adult brain retains the capacity to generate new neurons has been proposed several times over the last 40 years,
and in each case both conceptual and technical constraints have led to resistance. Joseph Altman first reported that some dividing
cells in the adult brain survived and differentiated into cells with morphology similar to neurons using tritiated thymidine
autoradiography (1 ). Over the subsequent years he and his colleagues confirmed these initial observations and focused on the few
areas where neurogenesis was apparent in the adult, at the light microscopic level, while systematically documenting the birth
dates of neurons throughout the brain during development (2 ). When contrasting adult neurogenesis with the extensive
neurogenesis in development, adult neurogenesis seemed almost like an epiphenomenon. The continued skepticism surrounding
adult neurogenesis and the absence of definitive phenotypic markers limited the development of the field. In the mid-1970s and
early 1980s Michael Kaplan reexamined the initial observations using the electron microscope and added substantial confidence that
not only could neurogenesis occur in the adult brain, but also that the cells appear ultrastructurally, similar to sister cells in the
dentate gyrus of the hippocampus, one of the structures shown to be neurogenic (3 ). In the mid-1980s, Fernando Nottebohm and his
student Steve Goldman further stimulated this fledgling field by showing that songbirds experience a seasonal cell death and
neurogenesis in a region of the brain important for song production (4 ). They have continued to reveal more about the environment
and molecular regulators of this process in the adult avian brain (5 ). Despite these observations of neurogenesis in the adult brain,
confusion over the mechanism origin of cell genesis in the adult brain persisted. In the early 1990s a series of papers in the adult
mouse and rat revealed that cells with stem cell properties could be isolated and expanded in culture. Under a variety of culture
conditions with different factors, these isolated cells can be induced to differentiate into glia and neurons (6 ,7 ,8 and 9 ). This
later observation provided a mechanism for the neurogenesis in the adult. Mature committed neurons were not dividing, but rather
a population of immature stem-like cells exists in the brain and it is likely that it is the proliferation and differentiation of this
population that is resulting in neurogenesis. With this conceptual framework the original statement by Cajal that, “Once
development was ended, the fonts of growth and regeneration of the axons and dendrites dried up irrevocably. In adult center, the
nerve paths are something fixed and immutable: everything may die, nothing may be regenerated,” still holds true for most areas of
the adult brain. The new lesson that we have learned is that development never ended in some areas of the brain.

CHARACTERIZATION OF CELL GENESIS IN VIVO

Part of "8 - Neurogenesis in Adult Brain "

Areas of Neurogenesis
Neurogenesis is a process that includes cell division, migration, and differentiation. There appear, at present, to be only two areas
of the brain where stems cells initially reside and proliferate prior to migration and differentiation. Those areas are the lateral
subventricular and subgranular zones of the dentate gyrus. The exact cell that corresponds to the initiating stem cell in the lateral
ventricle is a point of contention. One view is that this cell is the ependymal cell facing the ventricle (10 ), whereas an alternative
view is that a glial population one cell layer in from the ependyma are the stem cells (11 ). In any case, after cell division one of the
stem cells begins to differentiate and migrate in what Alvarez-Buylla has described as “chain migration” along the rostral migratory
stream toward the olfactory bulb where they differentiate into interneurons in the bulb (12 ). This process continues throughout life;
the functional importance and consequences of this process are not understood. The stem cell in the adult mammalian subgranular
zone of the dentate gyrus is likely an ectopically displaced cell
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originating from the developing ventricular zone. It remains formally possible that a more primitive cell exists elsewhere in the
adult brain in a quiescent state, and migrates to the dentate gyrus where the cells begin to divide. However, from the subgranular
zone, one of the progeny migrates into the granule cell layer once the cells divide; there the majority become neurons with axons
extending to the CA3 pyramidal neurons and receiving synaptic connections. The function of these newly born cells is being
investigated.

Although these are the two principal areas where neurogenesis occurs in the mammalian brain, cell genesis occurs throughout the
adult brain including cortex, optic nerve, spinal cord, and many brainstem and forebrain structures. To date the function of this cell
genesis in the normal intact brain and spinal cord is not known, but some of these new cells can become glial cells (13 ). A clear
challenge for the future is to document all the areas of the adult brain where cell genesis continues, and to understand the normal
function as well as the factors that regulate this process.

Mammalian Species in Which Adult Neurogenesis Is Documented

The first studies demonstrating adult neurogenesis were in the rat. Subsequently, rabbit and cat were shown to exhibit similar
characteristics, although little additional work has been done in those animals since the original publications (14 ,15 ). It was not
until 1997 that Kempermann and associates showed the mice retain neurogenesis and that significant genetic variability exists
among mouse strains (16 ). These findings, together with the important role for environmental stimulation as a regulator of
neurogenesis, have placed adult neurogenesis as paradigm for examining the interactions of nature and nurture (17 ,18 ). Although
there was debate in the mid-1980s as to whether nonhuman primates retained adult neurogenesis, a series of papers by Fuchs and
associates beginning with tree shrew followed by marmosets and finally with Rhesus monkeys demonstrated and confirmed that
neurogenesis in occurs in adult nonhuman primates (19 ,20 ). Recently, Gould presented data suggesting that neurogenesis occurs in
the adult primate frontal cortex and concluded that the cells are derived from the subventricular zone where they migrate to
specific cortical regions of the adult primate brain. This observation awaits confirmation (21 ). One of the markers for determining
cell division is bromo-deoxyuridine (BrdU); a traceable analogy of uridine, which in incorporated into the genome of, cells
undergoing cell division. Administering BrdU and then examining cell proliferation in tumor biopsies is occasionally used to monitor
tumor progression in patients with cancer. Because BrdU is a small soluble molecule, it is distributed throughout the body including
the brain, thus can be a marker for cell and neurogenesis in humans. In 1998 Eriksson and colleagues (22 ) reported that in five of
the cancer patients they examined who received BrdU at between 15 days and over 2 years early, all of them showed neurogenesis
as revealed by colabeling of BrdU with markers of mature neurons in the dentate gyrus. Together these studies clearly demonstrate
that neurogenesis, at least in the dentate gyrus, is a process that persists throughout life and in all mammalian species (Fig. 8.1 ).

The extent to which or whether neurogenesis can occur in other brain areas remains an area of intense investigation.

FIGURE 8.1. Birth of new neurons in the adult hippocampus has been documented in a variety of species, including rodents
and humans. See color version of figure.

PROPERTIES OF STEM CELLS IN VITRO

Part of "8 - Neurogenesis in Adult Brain "

CNS Areas from Which Stem Cells Can Be Isolated

Neural stem cells can be derived from the adult brain and propagated in vitro (6 ,7 and 8 ,10 ,11 ,23 ,24 ,25 and 26 ). Ongoing
adult neurogenesis, however, has only been documented convincingly in two brain areas, the subventricular zone/olfactory system
and the dentate gyrus of the hippocampus. Interestingly, stem cells are found not only in these regions, but also have been isolated
from areas that are non-neurogenic such as the septum, striatum (27 ), spinal cord (28 ), cerebral cortex, corpus callosum, and optic
nerve (29 ) and eye (30 ). In culture, these cells are multipotent and can give rise to neurons and glia. However, cells isolated from
areas outside of the hippocampus and subventricular zone require high levels of FGF-2 in order to give rise to neurons, rather than
only glial cells (29 ). These findings suggest that either different
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populations of stem cells exist in the nervous system or that they require unique culture conditions to become multipotent.
Alternatively, the cells isolated from different CNS regions may already be committed toward a specific lineage. Indeed, there are
no antigenic markers that allow unambiguous identification of stem cells in the nervous system. In the subventricular zone, stem
cells are suggested to divide slowly, whereas and their offspring, progenitor cells, may divide more frequently (31 ). Stem cells in
this area have been suggested to ependymal cells (10 ) or a subclass of glial cells in the subependymal zone (11 ). The location and
identity of the hippocampal stem cell remains to be determined.

Factors That Affect Proliferation and Differentiation of Stem Cells In Vitro

A variety of cytokines, neurotrophins, and conditioned media are used to culture neural progenitor cells (32 ,33 and 34 ). The two
major factors are EGF and FGF. Progenitor cells responsive to EGF have been isolated and cultured from adult mouse subventricular
zone (6 ,7 ,31 ). FGF-2 has been found to be mitogenic for adult neural progenitors from brain and spinal cord (9 ,27 ,28 ). FGF-2,
however, is member of a family of 10 related, but genetically and functionally distinct polypeptides. Among those, only FGF-2 and
FGF-4 are mitogens for neural progenitor cells. Moreover, a comparison of amino acid sequences between the FGFs revealed a
striking similarity between a 10-amino acid sequence of FGF-2 and FGF-4. This 10-amino acid sequence was been shown to elicit the
mitogenic effects of FGF-2 and FGF-4 on neural progenitor cells, whereas similar regions in FGF-1 and -5 were found to be inactive
(35 ).
Several factors have been found to be important for neuronal differentiation in cultured progenitor cells. In particular, retinoic acid
and cAMP increase neuronal differentiation (27 ,36 ,37 ). In addition, neurotrophins such as NGF, BDNF, and NT-3 have been found
to influence neuronal differentiation and transmitter phenotype (34 ,37 ,38 ), whereas CNTF can regulate glial differentiation of
precursor cells (39 ).

Transplanted Cells and Responses to Local Cues

Progenitor cells may play an important role in brain or spinal cord repair. In particular, grafting of progenitor cells into degenerated
or injured areas may be used to replace cells that are no longer functional. The phenotype and function that these cells acquire
appears to be very much dependent on the specific environment into which they are transplanted. Thus, cultured hippocampal
progenitors become granule cell neurons when grafted into the dentate gyrus, tyrosine hydroxylase, and calbindin positive neurons
in the olfactory bulb after grafting into the rostral migratory stream (40 ). In addition, after implantation into the developing retina
these cells showed properties of several types of retinal neurons (37 ). Moreover, progenitors isolated from a non-neurogenic area
such as the spinal cord, acquired the morphologic characteristics of granule cell neurons when grafted into the dentate gyrus, and
had a glial phenotype when grafted back in to the spinal cord (41 ). These studies suggest that neural stem cells derived from the
adult mammalian brain retain multipotentiality. Recent research suggests that neuronal stem cells are multipotent outside the CNS
as well. It was reported that neural progenitor cells repopulate experimentally depleted bone marrow and reconstitute the
hematopoietic system (42 ). It remains to be determined what the local cues are, that are driving the neuronal precursor cells to
acquire such specific fates when transplanted in vivo.

REGULATION OF PROLIFERATION AND DIFFERENTIATION IN VIVO

Part of "8 - Neurogenesis in Adult Brain "
The mechanisms that generate new granule cells in the dentate gyrus are poorly understood. A variety of environmental, behavioral,
genetic, neuroendocrine, and neurochemical factors can regulate adult neurogenesis. Two critical processes that lead to
neurogenesis, cell proliferation and the subsequent differentiation and survival of newborn neurons, can undergo differential
regulation by these factors (Table 8.1 ).

TABLE 8.1. REGULATION OF CELL

PROLIFERATION AND
NEUROGENESIS IN THE DENTATE
GYRUS IN VIVO

Genetics
In 1997 Kempermann and colleagues found that strains of mice differ with respect to rate of cell division and amount of cell survival
and neurogenesis. Comparisons were made among C57BL/6, BalB/c, CD1, and 129/SVJ strains. Proliferation was found to be highest
in C57BL/6 mice; however, net neurogenesis was highest in the CD1 strain. 129/SVJ produced relatively more astrocytes and fewer
neurons than other strains (16 ). The degree to which environmental, behavioral, and biochemical factors can affect cell
proliferation and neurogenesis may also differ depending on the species or strain of animal involved. Indeed, exposure to an
enriched environment (18 ) had different effects on two of these strains of mice, C57BL/6 and 129/SVJ, respectively. In C57BL/6
mice enrichment promoted the survival of progenitor cells but did not affect proliferation, whereas the net increase in neurogenesis
in 129/SVJ mice was accompanied by a twofold increase in proliferation (18 ). Thus, strain differences not only influence the
baseline rate of adult hippocampal neurogenesis, but also influence how adult hippocampal neurogenesis is regulated in response to
environmental stimulation. Indeed, proliferation, survival and differentiation of progenitor cells and their progeny are each
separately influenced by inheritable traits and are not uniformly
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up-regulated in response to environmental stimulation.

Growth Factors
During development, growth factors provide important extracellular signals for regulating the proliferation and fate determination
of stem and progenitor cells in the CNS (43 ). Several studies have been carried out to investigate progenitors in the adult brain
respond to such growth factors. Intracerebroventricular infusion of EGF and FGF-2 in adult rats increased proliferation in the
subventricular zone (44 ). Neither EGF nor FGF enhanced proliferation in the subgranular zone of the dentate gyrus. With regard to
differentiation, EGF promoted glial differentiation, whereas FGF-2 did not influence phenotype distribution (44 ). In another series
of experiments, FGF was administered systemically during the first postnatal weeks and in the adult rat. Cell proliferation was
increased the dentate gyrus of infant rats but not in the adult hippocampus (45 ,46 ). Recent research has shown that intracerebral
infusion of IGF increases both cell proliferation and neurogenesis in hypophysectomized rats (47 ). In songbirds, seasonal regulation
of adult neurogenesis depends on testosterone levels that mediate their effect through BDNF (48 ). In addition, IGF-1, FGF mRNA,
and BDNF mRNA are elevated in rodents by exercise (49 ,50 ,51 and 52 ). Expression of BDNF (53 ) and GDNF is increased by
exposure to an enriched environment (54 ). Both running and enrichment increase net neurogenesis (17 ,55 ,56 ). The effects of
intracerebral administration of trophins such as BDNF, NT-3, and GDNF remain to be determined; however, it appears that growth
factors do play a role in in vivo regulation of proliferation and neurogenesis in the adult hippocampus. Better understanding of their
mechanisms of action may lead to therapeutic application of these factors after brain injury or disease.

Neuroendocrine Factors and Stress

McEwen and Gould at Rockefeller University first investigated the effects of glucocorticoids or stress on adult hippocampal
neurogenesis. The initial study reported that adrenalectomy, which leads to a reduction in serum glucocorticoid levels, elicits cell
division in the dentate gyrus. This effect could be reversed by corticosterone replacement (57 ). Conversely, stress or increased
levels of glucocorticoid hormones inhibit proliferative activity in the dentate gyrus (58 ,59 ). For example, administration of high
levels of corticosterone diminishes cell division in the adult rat hippocampus (59 ). In addition, exposure of a rat to the odor of a
natural predator (fox), causing stress and elevated corticosterone levels, transiently suppressed cell proliferation in the
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adult rat dentate gyrus (60 ). Furthermore, exposure of marmoset monkeys to a resident intruder causes stress and results in a
decrease in cell proliferation (61 ). In a recent study, rats that are highly reactive to novelty and exhibit a prolonged corticosterone
secretion in response to novelty and stress were found have reduced dentate gyrus cell proliferation (62 ). Aging is accompanied by
a reduction in neurogenesis (63 ), which may be caused in part by elevated glucocorticoid levels. Adrenalectomy in aged rodents has
been shown to increase cell proliferation and neurogenesis (64 ). The effects of glucocorticoids on cell genesis appear to be
mediated via a downstream effect on NMDA glutamate receptors (65 ). Thus, glucocorticoids and stress associated with increased
corticosterone secretion inhibit cell genesis in the hippocampus. Enhanced stress or glucocorticoid levels therefore may impair
hippocampal function, and lead to deficits in learning and memory. In contrast to the glucocorticoids, other steroid hormones, such
as testosterone, enhance neurogenesis in birds (66 ), whereas estrogen results in a transient increase in proliferation in rats (67 ).
Thyroid hormone can affect neuronal differentiation of hippocampal progenitor cells in vitro (27 ). In vivo, hypothyroidism
interferes with cell migration (68 ), but does not affect postnatal cell proliferation (69 ).

Neurotransmitters
Neurotransmitters have also been suggested to play a role in adult dentate gyrus neurogenesis. Systemic injection of glutamate
analogs inhibits birth of new cells, whereas an antagonist, such as MK801, enhances cell division (65 ,70 ). Recently, another class of
neurotransmitters, the monoamines, has been suggested to be important as well. Prolonged administration of fluoxetine, as well as
therapeutic agents acting on norepinephrine and dopamine receptors, and electroconvulsive shock enhance the number of BrdU-
positive cells in rats (71 ,72 and 73 ). Acute administration of fluoxetine did not affect cell genesis (73 ). Grafting of fetal raphe
neurons also stimulated granule proliferation in the hippocampus, whereas embryonic spinal tissue had no effect (74 ). Furthermore,
depletion of serotonin reduces stem cell proliferation in the dentate gyrus (75 ). It is possible that these effects are mediated by the
1A receptor, because administration over 4 days of a specific 1A receptor antagonist (WAY) reduced basal rate of cell proliferation
(Jacobs et al., unpublished observations). Taken together, these findings suggest that induction of cell proliferation is dependent on
chronic administration of monoamines, consistent with the therapeutic time course for antidepressant treatments. Indeed, these
studies have led to the hypothesis that therapeutic interventions that increase serotonergic transmission may act in part by
augmenting dentate neurogenesis, promoting recovery from depression (76 ,77 ). It is of interest to note in this context that
voluntary exercise increases cell proliferation (55 ), enhances monoamine levels and has an antidepressant effect (78 ). Thus,
monoamines can affect cell genesis in the dentate gyrus. The receptors and mechanisms by which they exert their effects as well as
possible interactions with other classes of neurotransmitters and/or growth factors remain to be determined.

Experience
As mentioned, stress (19 ) and depression may reduce the birth of new neurons. In addition, the aging process is accompanied by a
decrease in neurogenesis (63 ); however, there are several environmental and behavioral interventions that can enhance
neurogenesis (Fig. 8.2 ). In 1997 Kempermann and colleagues carried out the first of these studies comparing mice living under
standard conditions with those housed in an enriched environment (17 ). Exposure to an enriched environment, consisting of larger
housing; toys; and more opportunity for social stimulation, physical activity, and learning than standard laboratory conditions (79 ),
resulted in a significant increase in neurogenesis, without affecting cell proliferation in mice and rats (17 ,80 ). Subsequent studies
showed that the age-related decline in neurogenesis could be attenuated by enrichment (81 ). In addition, it was shown that
enrichment inhibits cell death by apoptosis and prevents seizures (54 ). Moreover, it was determined that the most important
components of enrichment are increased physical activity and possibly learning. Similar to enrichment, voluntary exercise in a
running wheel increases net neurogenesis (55 ). In addition, running increases
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cell proliferation in the dentate gyrus (55 ). It is interesting to note that enrichment and running had the same net effect on
neurogenesis, but that running increased proliferation, whereas enrichment did not. Thus, not only the genetic factors mentioned,
but also different environmental and behavioral factors can have differential effects on cell proliferation and neurogenesis. Others
reported that hippocampus-dependent tasks, such as spatial learning in the Morris water maze (82 ), increases the number of
surviving BrdU-positive cells (83 ,84 ); however, in our laboratory there was no effect of learning on proliferation or survival of
newborn hippocampal cells (55 ).

FIGURE 8.2. Proliferation and neurogenesis in the dentate gyrus. Photomicrographs of BrdU-positive cells 1 day (a–c) and 4
weeks (d–f) after the last injection in control (a,d), running (b,e), and enriched (c,f) mice. Confocal images of BrdU positive
cells in control (g), running (h), and enriched (i) mice, 4 weeks after the last injection. Sections were immunofluorescent
triple labeled for BrdU (red), NeuN indicating neuronal phenotype (green), and s100B selective for glial phenotype (blue).
Orange (arrow, newborn neuron) is red + green. Scale bar is 100 μm. See color version of figure.

Apart from these rather innocuous manipulations, there are several pathologic events that can affect granule cell number. Damage
to the hippocampus by kindling (85 ,86 ), seizures (87 ,88 and 89 ), ischemia (90 ,91 ), or mechanical lesions (92 ) enhances
proliferation. Thus, both normal and pathologic circumstances can affect cell genesis. Whether increased proliferation is beneficial
for function or may represent compensation for lost cells and/or function remains to be determined.

FUNCTIONAL SIGNIFICANCE OF NEUROGENESIS

Part of "8 - Neurogenesis in Adult Brain "

Adult neurogenesis has been reported to exist over more than three decades, and to occur in a variety of species, including humans;
however, the functional role of these new cells has yet to be determined. Given that the hippocampus is important for some forms
of learning and memory and related mechanisms of neural plasticity such as long-term potentiation (LTP), much of the research has
focused on finding a relationship between neurogenesis and memory function.

Behavior
It was found in several studies that animals living in an enriched environment not only had more new neurons, but also performed
better on a spatial learning task (17 ,18 ,80 ). In addition, mice that were housed with a running wheel had more new neurons than
their sedentary counterparts (55 ) and performed better on the water maze task (56 ). As mentioned, neurogenesis declines with
aging (63 ); however, exposure to an enriched environment can restore some of the neurogenesis and improve performance on the
water maze task in aging mice (81 ). Whether voluntary exercise in a running wheel would yield similar results in aged animals
remains to be determined.

Electrophysiology
Although we can identify new granule cells using histologic and morphologic techniques, the question remains whether these cells
are physiologic functional and if so, how similar or different are they from existing granule cells (Fig. 8.3 ). Moreover, what could be
the functional significance of their (possible) neuronal activity? It has been shown that new hippocampal granule cells send axons
along the mossy fiber tract to CA3 as do all other granule cells (93 ,94 and 95 ) and that they receive synaptic contacts (93 ). A
recent study compared LTP, a physiologic model of certain forms of learning and memory (96 ) in the dentate gyrus, and CA1 in
hippocampal slices from running and control mice. LTP amplitude was selectively enhanced in the dentate gyrus of running mice
(56 ). It is possible that the newborn granule cell neurons play a role in increased dentate gyrus LTP because running increases
learning and neurogenesis. Although the new cells are a small percentage of the granule cell layer, the possibility exists that they
have greater plasticity than do mature cells. Indeed, dentate gyrus LTP last longer in immature rats than adults (97 ); however, in
order to test this hypothesis it is necessary to record activity from individual new granule cells. In a recent study granule cells from
the inner and outer layer of the dentate gyrus were compared. The inner layer cells were considered to be “young” cells and the
outer layer “old” cells. The researchers found that the putative
 P.115
young cells had a lower threshold for LTP and were unaffected by GABA-A inhibition, suggesting enhanced plasticity in the “young” cells (98 ); however, the problem with this study is
that the definition of “young” cells is ambiguous. In our studies we have found newly generated cells throughout the granule cell layer. Moreover, the only way to be certain that a cell
is a newborn neuron is by labeling the cell when it divides with a mitogenic marker. Ideally, recordings would be made from such a labeled cell and compared with preexisting neurons.

FIGURE 8.3. The three major areas of the hippocampus are the dentate gyrus, CA3, and CA1. The
perforant pathway (1) from the subiculum forms excitatory connections with granule cells of the
dentate gyrus. Both newborn and existing granule cells give rise to axons that form the mossy fiber
pathway (2). This pathway projects to area CA3 pyramidal cells. The CA3 cells project to CA1 pyramidal
cells by Schaffer collaterals. Within the hippocampal system, only the dentate gyrus gives rise to new
neurons in the adult brain. These cells proliferate, migrate, and differentiate into mature neurons in
the dentate gyrus (see box). See color version of figure.

In summary, adult hippocampal neurogenesis appears to be associated with memory function. Indeed, performance on a learning task is better in animals in which neurogenesis is
stimulated, such as by running or enrichment, than in controls. This link may between neurogenesis and behavior may be causal rather than correlational, given that
electrophysiologically measurable changes occur in the brain region where adult neurogenesis occurs.

POTENTIAL THERAPEUTIC IMPLICATIONS

Part of "8 - Neurogenesis in Adult Brain "

Several areas of interest emerge when considering the therapeutic potential for neurogenesis. First, a strategy that takes advantage of the ability of stem cells from the adult brain to
be isolated and induced to divide in culture opens the opportunity for cellular transplantation to replace cells that have died because of injury or disease. Although evidence supports
that ability of adult stem cells to survive grafting to the adult brain, the fate of the grafted cells appears to be dictated by the local environment. Thus, in order to accurately replace
cells in damaged areas of the brain significant new information about the cellular and molecular mechanisms that control fate decisions is required in order to “train” the immature
cells in culture to respond to the unique features of each of the environment to which they are grafted.

A second strategy emerges as a direct result of the fact that the adult brain retains stem cells in situ throughout life. By understanding the internal factors, molecules and mechanisms
as well as the external stimuli and influences that control and regulate each of the steps in neurogenesis in vivo, eventually the potential of the endogenous cells could be harnessed.
To this end, cell number could be amplified, direction of migration could be targeted, and finally terminal fate could be specified to the extent that a form of “self-repair” could be
induced or orchestrated in the adult damaged brain. In all cases a more complete understanding the cellular and molecular events directing neurogenesis is a prerequisite for the use
of this process in rational strategies for therapy.

ACKNOWLEDGMENTS
Part of "8 - Neurogenesis in Adult Brain "

This work was supported by NIA, NINDS, the Lookout Fund, Pasarow Foundation, Holfelder Foundation, and APA. We thank M.L. Gage for comments on the manuscript.

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9
Dopamine

Anthony A. Grace

Anthony A. Grace: Departments of Neuroscience and Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania.

Studies into the regulation of the dopamine (DA) system and its postsynaptic actions are often stymied by the myriad of actions that
this neurotransmitter can produce. Thus, DA has been found to exert actions on the neurons it innervates both directly and via G-
protein–coupled receptors. Moreover, this transmitter can modulate afferent input within these target regions, as well as alter
intercellular communication via its actions on gap junctions. Finally, DA can potently modulate its own dynamics, acting via
autoreceptors on DA nerve terminals and on DA neuron somata. In fact, the DA system is under potent dynamic regulation in the
short term by a multitude of feedback systems, and in response to prolonged alterations is subject to powerful homeostatic
mechanisms that can compensate for dramatic changes in DA system function. Such homeostatic alterations can be compensatory in
nature, such as those that occur in response to a partial DA system lesion, or pathologic, such as the sensitization that can occur
with repeated psychostimulant administration. Nonetheless, the importance of this neurotransmitter system in a broad array of
human disorders ranging from Parkinson’s disease to schizophrenia has driven an intensive array of investigations oriented toward
increasing our understanding of this complex system in normal conditions as well as disease states. This chapter attempts to
summarize some of the major research findings that have occurred within the last 5 years, and place them into a functional
framework. This is not meant to be inclusive: A search of Medline indicated that there were over 16,000 papers published on DA
during the past 5 years! Because of the exceedingly broad range that this topic encompasses, the focus is primarily on a subset of
the numerous improvements that are most related to advancing our understanding of psychiatric disorders in particular. Topics
related to specific disorders, such as drug abuse, schizophrenia, and so on, are deferred to the appropriate chapters in this volume.

 DA NEURON ANATOMY AND PHYSIOLOGY

 REGULATION OF DA RELEASE
 POSTSYNAPTIC EFFECTS OF DA
 BEHAVIORAL CORRELATES OF DA SYSTEM FUNCTION
 SUMMARY OF DA ACTIONS
 ACKNOWLEDGMENTS

DA NEURON ANATOMY AND PHYSIOLOGY

Part of "9 - Dopamine "

Both in vivo and in vitro studies have demonstrated that DA-containing neurons in the midbrain exhibit spontaneous spike firing that
is driven by an endogenous pacemaker conductance (1 ,2 and 3 ), with their activity modulated by afferent inputs. One of the
prominent regulators of DA neuron activity is the DA autoreceptor. It has been known for some time that DA neurons are very
sensitive to DA agonists, which inhibit spike firing as well as cause a presynaptic inhibition of DA synthesis and release. Studies
indicate that DA neuron somatodendritic autoreceptors are stimulated by an extracellular pool of DA released from the dendrites of
neighboring DA neurons rather than exclusively by autoinhibition back onto the releasing neuron. This is supported by data showing
that partial lesions of the DA system result in DA autoreceptor supersensitivity in the remaining neurons, which would only occur if
the remaining neurons were responding to the decrease in DA caused by the loss of neighboring neurons (4 ). The autoreceptors are
believed to exert a tonic down-regulation of DA neuron activity, maintaining their firing within a stable range of activity (4 ,5 ).
These autoreceptors appear to be primarily of the D2 type, because D2-deficient mice do not show autoreceptor-mediated inhibition
of firing (6 ). Moreover, inhibition of monoamine oxidase potentiates this inhibition (7 ,8 ), whereas inhibition of catechol-o-methyl
transferase does not alter this response (9 ).

Exogenous transmitters also potently regulate dopamine neurons. Thus, GABA afferents both from striatonigral neurons as well as
from local circuit neurons in the midbrain cause inhibition of DA neuron activity (10 ) by both a GABA-A– and GABA-B–mediated
action (11 ,12 and 13 ). Glutamate has also been shown to exert multiple actions on DA neuron activity. Glutamate applied in vivo
increases burst firing (14 ). N-methyl-D-aspartate (NMDA) receptor activation mediates a slow excitatory postsynaptic potential
(EPSP) in these neurons (8 ), whereas metabotropic glutamate agonists are reported to depress both excitatory and inhibitory
afferent input to these neurons (15 ). This latter effect is apparently shared by muscarinic receptors, which also depress both
excitatory and inhibitory afferents, presumably via a presynaptic action (16 ,17 ).
 P.120

Burst Firing
Studies have shown that DA neuron discharge is an essential component of the DA release process (18 ). The firing pattern of DA
neurons also is effective in modulating release, with burst firing in particular being an important regulator of DA transmission. Thus,
studies have shown that burst firing in DA neurons is associated with induction of c-fos and NG1-A in postsynaptic sites (19 ,20 ), and
this response demonstrates a spatial and temporal specificity with respect to brain region, genes activated, and cell phenotype. One
factor that is thought to regulate burst firing is the glutamatergic system. Several studies have shown that iontophoresis of
glutamate onto DA neurons in vivo lead to burst firing (14 ), as do stimulation of glutamatergic afferents to DA neurons (21 ,22 );
however, the evidence for glutamate acting alone to induce burst firing in vitro is equivocal (23 ). In contrast, evidence shows that
burst firing can be induced in vitro by blockade of apamin-sensitive potassium channels that modulate a nifedipine-sensitive calcium
conductance (24 ). One source of glutamatergic input to ventral tegmental area (VTA) DA neurons is proposed to arise from the
prefrontal cortex (PFC) (25 ); however, recent studies (26,27) show that the PFC input to the VTA innervates only the small
proportion of VTA DA neurons that project to the PFC, providing a direct feedback loop, whereas the VTA-accumbens neurons
innervated from the PFC are exclusively GABAergic neurons; therefore, it is unlikely that activation of the PFC can induce increased
DA levels in the accumbens by a direct projection to these neurons (28 ). On the other hand, there is evidence that activation of the
subiculum by excitatory amino acids increases accumbens DA (29 ,30 ) via activation of DA neurons that involves a pathway through
the nucleus accumbens (31 ).

Afferent Input
The feedback systems between DA neurons and their postsynaptic targets appear to be quite complex, particularly in the primate.
By analyzing a large number of retrograde and anterograde tracings, Haber and associates (32 ) found that different striatal
subdivisions are linked by overlapping feedback to DA neurons, in a manner that suggests an ascending spiral of regulation extending
from the shell to the core to the central striatum and finally to the dorsolateral striatum (Fig. 9.1 ). As pointed out by Haber, such
an anatomic arrangement could account for the parallel psychomotor, affective, and cognitive disturbances seen in a variety of
psychiatric disorders.

FIGURE 9.1. Studies using retrograde and anterograde tracers reveal that the feedback system between the midbrain DA
neurons and their striatal targets contains both reciprocal and feed-forward components. The shell of the accumbens (left)
receives inputs from hippocampus, amygdala, and limbic cortex, and projects to both the ventral tegmental area (VTA) and
dorsomedial substantia nigra DA neurons. Projections from the VTA back to the shell form the “closed” portion of this loop.
The core receives afferent input from the orbital and medial prefrontal cortex (OMPFC); the afferent projection to the core
from the medial substantia nigra (SN) then forms the first part of the spiral. The core in turn projects to more ventrodorsal SN
regions; therefore, ventral striatal regions can modulate the dopaminergic influence over more dorsal striatal regions via the
spiraling midbrain-striatal-midbrain connections. The magnified insert shows a model of the reciprocal versus feed-forward
loops. The reciprocal component is proposed to (a) directly inhibit the DA neuron, whereas the feed-forward, nonreciprocal
component terminates on a GABAergic interneuron; or (b) indirectly excite the DA neuron by disinhibition. DL-PFC, dorsolateral
prefrontal cortex; IC, internal capsule; S, shell; Snc, substantia nigra, zona compacta; Snr, substantia nigra, zona reticulata;
VTA, ventral tegmental area. (From Haber et al., 2000; used with permission.)

The striatum provides a powerful feedback regulation of DA neuron firing. Thus, alterations in striatal activity potently affect DA
cell activity states. Striatal neuron activation is known to cause an activation of DA neuron firing (10 ,33 ). Moreover, single-pulse
stimulation of the striatum directly, or indirectly via activation of the PFC in rats, causes an inhibition/excitation response pattern
(10 ,25 ). This relationship can be altered by manipulation of second messenger systems in the striatum. One system in particular
that seems to affect striatal activation, leading to an alteration
 P.121

in DA neuron activity, is the nitric oxide (NO) system. Increasing NO in the striatum by infusion of the substrate for the synthetic
enzyme nitric oxide synthetase (NOS), coupled with striatal or cortical stimulation, was found to increase the firing rate of striatal
neuron DA neurons. This effect was mimicked by infusion of the nitric oxide generator hydroxylamine. In contrast, NOS inhibitors
failed to affect baseline DA cell firing but did increase their response to stimulation (34 ); therefore, NO signaling in the striatum
facilitates DA neurotransmission by modulation of corticostriatal and striatonigral pathways. NO also appears to have a role in
regulating terminal DA release (see the following).

Stress and DA Neuron Activity

Stress appears to affect many monoamine systems. Stress plays a role both in acute behavioral responses and adaptations to chronic
stressful conditions. Although the noradrenergic system has played a major role in these processes, recent evidence supports a role
for the DA system as well. Studies have shown that, on presentation of stress, there are differential increases in DA dynamics
depending on the brain regions involved. Thus, stressful stimuli tend to cause the largest increase in DA levels in the PFC region,
with markedly smaller changes in the limbic and dorsal striatal regions (35 ); however, this relationship is altered by lesions of
different nuclei. Thus, stress causes release of DA in the amygdala (36 ), and lesions of the amygdala tend to block stress-induced
increases in PFC DA levels (37 ). Lesions of the PFC also affect this response. Studies in which the PFC DA innervation is lesioned
show that subsequent stressors cause a much larger increase in DA levels within the nucleus accumbens, particularly with respect to
the duration of the response (38 ). This suggests that PFC DA released in response to stress actually blunts the responsiveness of the
subcortical limbic DA system. In contrast, 6-OHDA lesions of PFC DA levels were found to decrease the basal electrophysiologic
activity of VTA DA neurons (39 ). Given that basal DA levels in the accumbens are normal, one interpretation is that the DA release
system has adapted to the diminished DA neuron drive, allowing normal levels of DA transmission to occur. However, if a stimulus
then causes an increase in DA neuron firing, the compensated release mechanism would produce an augmented response. Thus, the
magnitude of increase in action potential-dependent DA release into the accumbens that occurs in response to a challenge may be
augmented when the PFC DA response is attenuated (39 ).

Repeated stress also has important clinical implications with regard to the DA system and exacerbation of schizophrenia. A recent
study examined how chronic stress in the form of cold exposure affects the discharge of VTA DA neurons. Thus, after exposing rats
to cold, there was a 64% decrease in the number of spontaneously active DA neurons, with no significant alteration in their average
firing rate. Nonetheless, there was a subpopulation of neurons that exhibited excessive burst activity in the exposed rats (40 ).
Therefore, unlike acute exposure to stressful or noxious stimuli, chronic stress actually attenuates DA neuron baseline activity. Such
a decrease in baseline activity could enable the system to show a magnified response to activating stimuli, thereby producing a
sensitized DA response.

REGULATION OF DA RELEASE
Part of "9 - Dopamine "

DA appears to be released by multiple factors within its postsynaptic target; moreover, once it is released, there are several
mechanisms that can modulate its site of action. In general, the majority of evidence suggests that DA is released primarily in a
spike-dependent manner, because inactivation of DA neuron firing virtually eliminates DA release within the striatum (18 ). Carbon
fiber recordings, which allow rapid measurement of DA overflow, show that stimulation of DA axons causes rapid release of
transmitter. Moreover, the release varies with tissue content, with PFC showing much lower levels of release compared to
accumbens at a given stimulus frequency (41 ). DA released by impulse flow is then rapidly removed via the DA transporter, because
mice with knockouts of this transporter exhibit 300 times longer clearance half-life compared to controls (42 ). The amount of DA
released by impulses appears to depend on several factors. Previous volumes in the Generations of Progress series have detailed
how DA release can be modulated by both synthesis- and release-modulating autoreceptors on DA terminals. It is becoming more
evident that heteroceptors also play a significant role in modulating DA release (43 ).

DA release appears to occur via two functionally distinct components. One is the DA that is released in a high-amplitude, brief
pulsatile manner by means of action potentials, and then is rapidly removed from the synaptic cleft via reuptake. This has been
termed the phasic component of DA release (44 ), and is believed to underlie most of the behavioral indices of this transmitter. The
other is the level of DA present in the extrasynaptic space. This tonic DA exists in very low concentrations; too low to stimulate
intrasynaptic DA receptors, but of sufficient level to activate extrasynaptic receptors, including DA terminal autoreceptors (thereby
causing feedback-inhibition of phasic DA release) and other extrasynaptic receptor sites. It is this tonic DA compartment that is
sampled by slower measures of DA dynamics, such as microdialysis. Recently, evidence has been advanced to define what factors
may contribute to the regulation of this tonic DA compartment.

Although studies suggest that neuronal impulse flow is necessary for DA overflow in the striatum, there is substantial evidence that
the released DA can be controlled locally by a number of factors. For example, stimulation of cortical inputs increases DA release
within the striatum, and evidence suggests that this can occur via afferents to DA cell bodies or presynaptically onto DA terminals,
depending on the preparation and site of stimulation. Thus, infusion of excitatory amino acids into the hippocampus subiculum
increases
 P.122

DA neuronal activity (31 ) and DA levels in the striatum in a manner that is dependent on DA neuron impulse flow (29 ). It is
proposed that this subicular-driven DA release may be involved in the modulation of investigatory response to novel and conditioned
stimuli (45 ). Stimulation of the PFC also appears to result in impulse-dependent DA release in the striatum (28 ). On the other hand,
there is evidence suggesting that DA can be released in a manner not dependent on DA neuron firing via stimulation of the
hippocampal afferents (46 ), or amygdala afferents (47 ) to the accumbens, all of which use glutamate as a transmitter. This
purported presynaptic action on DA terminals appears to occur via activation of either NMDA receptors on DA terminals (48 ) or by
metabotropic glutamate receptors (49 ,50 and 51 ). There is also evidence that glutamate can release acetylcholine or serotonin in
the striatum, which in turn can trigger DA release (43 ). Glutamate may also stimulate DA release via an action on other local
systems, such as those producing NO. NO is known to be released from striatal interneurons containing the enzyme NOS, and exert
actions on neuronal elements in the vicinity of the release site. Infusion of NOS substrates or NO generator compounds was found to
facilitate the release of both glutamate and DA within the striatum in a calcium-dependent manner, and is dependent on vesicular
stores (52 ,53 ). Moreover, the NO-induced efflux of striatal glutamate was found to indirectly enhance extracellular DA levels in the
striatum in a manner dependent on NMDA and AMPA receptors (53 ,54 ). Therefore, it is likely that excitatory amino acids and NO
interact with DA neuron firing to regulate DA release from presynaptic sites within the striatum.

The ability of cortical glutamate to release tonic DA in the striatum is supported by studies showing that lesions of the cortical input
to the striatum cause a decrease in extracellular DA and glutamate within the striatum (55 ), which would thereby increase in the
behavioral response to amphetamine (56 ). Thus, evidence indicates that alterations in tonic DA levels produced by cortical
afferents can potently alter spike-dependent DA release, and thereby modulate DA-dependent behaviors (43 ,44 ,57 ). Such tonic
down-modulation of spike-dependent DA release could play a particular role when the uptake system is inactivated by
psychostimulants. Thus, although the DA transporter is normally highly effective at removing DA from the synaptic cleft before it
can escape into the extracellular space, blockade of the DA transporter would allow substantially higher levels of DA to escape the
cleft and contribute to the tonic extracellular DA pool (57 ). Such a condition is thought to underlie some of the therapeutic actions
of psychostimulants in attention deficit/hyperactivity disorder (ADHD) (58 ).

One problem in attempting to examine the relationship between DA neuron firing rate and DA overflow is the potential disruption in
the system caused by probe implantation. This was found to be a significant issue when testing the effects of chronic antipsychotic
drug treatment-induced DA neuron depolarization block (59 ) on DA levels in the striatum. Thus, implantation of a microdialysis
probe was found to disrupt DA neuron depolarization block when DA cell activity was assessed 24 hours following probe implantation.
However, if the probe was inserted via a preimplanted guide cannula, depolarization block was maintained, and the DA levels were
found to be approximately 50% less than in control conditions. Moreover, the relationship between DA neuron firing and release was
altered. Thus, although there was no significant correlation between DA cell population burst firing and DA release in control rats,
there was a significant correlation between burst firing in the remaining cells and DA levels following administration of chronic
antipsychotic drug (60 ). Thus, correlations between cell firing patterns and DA levels postsynaptically appear to depend on the
state of the system.

It is also possible that there may be local fluctuations in tonic DA stimulation that may be a consequence of increases in DA neuron
firing. Indeed, studies using voltametric measures have shown that brief elevations in extracellular DA may occur as a consequence
of rapid burst firing, overwhelming the DA uptake process (61 ). This relationship is particularly important during administrations of
drugs that interfere with the uptake process, such as cocaine or amphetamine (57 ,58 ). Such drugs would cause phasic DA release to
rapidly augment tonic DA levels, leading to high extracellular DA and abnormal levels of down-regulation of spike-dependent DA
release. In a similar nature, in mice lacking the DA transporter, the extracellular DA is already elevated fivefold over control (62 );
therefore, there appears to be a tight dynamic interdependence on DA neuron activity levels and DA uptake that determines the
contribution of phasic and tonic DA to activity within this system. This tonic/phasic balance has been proposed to underlie normal
and dysfunctional DA regulation as it relates to the pathophysiology of schizophrenia, drug abuse, and the treatment of ADHD
(44 ,57 ,58 ).

Given the importance of tonic DA system regulation, a literature has emerged regarding the functional relevance of extrasynaptic DA
receptors. Indeed, studies have shown that in the PFC, the DA terminals located in the deep layers of cortex do not contain DA
transporters (63 ). As a consequence, the DA released from these sites would be free to diffuse to a much greater extent than in
areas such as the striatum and accumbens. This is further substantiated by evidence that a substantial portion of the DA that is
released in the PFC is actually taken up and deaminated in norepinephrine (NE) terminals (64 ). This arrangement would have
substantial functional implications. First, it would provide a mechanism for stimulation of the numerous extrasynaptically located D1
terminals on pyramidal neurons (65 ), which have been proposed to regulate information flow between compartments on pyramidal
neurons (66 ,67 ,68 and 69 ). Moreover, such a condition could imply that NE uptake
 P.123

blockers could serve to increase the functional actions of DA in the PFC by preventing its removal via NE terminals. This may also
have implications regarding the clinical actions of NE-selective antidepressant drugs within this brain region.

POSTSYNAPTIC EFFECTS OF DA
Part of "9 - Dopamine "

DA exerts a myriad of actions on postsynaptic systems. These actions can occur at the level of individual cells in terms of direct
postsynaptic actions, as well as altering cellular interactions (via presynaptic effects and network modulation). Moreover, the nature
of these effects can vary depending on both the specific region examined and the time course of DA agonist administration.

Striatum
D1 stimulation decreases excitability of dorsal striatal and accumbens neurons (67 ,68 and 69 ), although others have reported
excitation by this agonist (70 ). Within the dorsal striatum, D1 receptor stimulation decreases current-evoked action potential
discharge in hyperpolarized neurons, although an enhancement in excitability can be obtained with longer duration or higher
frequency current pulses (71 ). The decrease in spiking is believed to be owing to a reduction in the peak amplitude of the fast
sodium conductance (72 ,73 ), and occurs through activation of protein kinase-A (PKA) (74 ). Studies show that the D1-mediated
inhibition can act synergistically with D2 stimulation-induced inhibition when the agonists are applied simultaneously. However, the
D1-mediated decrease in excitability can be reversed to facilitation if the D2 agonist is administered subsequently (75 ). This
temporal dependence of D1 and D2 activation may have functional implications with regard to the tonic/phasic model of DA system
regulation (44 ). For example, if the DA system exhibits sustained activation such as during a reward process, the large phasic DA
release that results should stimulate both D1 and D2 receptors located within synapses. In addition, the large DA level released
should be sufficient to escape the synaptic cleft, with the resultant elevated tonic DA levels stimulating the extrasynaptic D1
receptors (76 ,77 ). According to our data, this should produce synergistic inhibition. On the other hand, if the activity is maintained,
there would be tonic stimulation of the extrasynaptic D1 receptors. Under this condition, subsequent stimulation of the D2 receptors
preferentially located in the synaptic cleft (78 ) would be attenuated (75 ). Thus, the system appears to be oriented to provide a
maximal initial response, whereas continuous activation would cause an attenuation of subsequent responses.

In addition to effects on sodium conductances, D1 stimulation also affects high voltage-activated calcium conductances. Thus, both
D1 agonists and cAMP analogues reduce both N- and P-type calcium currents via a PKA-mediated process; however, these
manipulations also enhance L-type calcium currents (79 ). In contrast, D2-receptor stimulation has been shown to modulate voltage-
dependent potassium conductances in the striatum (80 ).

Evidence shows that a large part of the response to D1 stimulation requires the participation of a messenger cascade involving the
phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) (81 ). In particular, this phosphoprotein is a required
component in the cascade mediating D1 function (Fig. 9.2 ). Moreover, mice with knockouts of DARPP-32 have been shown to lack
D1 modulation of glutamate function, as well as other biochemical processes and behavioral responses known to involve D1
receptors (82 ). Recent studies have shown that DARPP-32 is also present in other, non–D1-containing neurons as well, including the
enkephalin-containing striatal neurons (83 ). In this case, D2-receptor stimulation has been shown to cause a dephosphorylation of
DARPP-32 via calcineurin activation by calcium influx. DARPP-32 is also present in striatal efferent projection areas, including the
globus pallidus, entopeduncular nucleus, and substantia nigra (SN) (83 ). Thus, DARPP-32 is positioned to exert modulatory
influences on DA function by affecting striatal outflow.

FIGURE 9.2. The DARPP-32 signaling pathway has a

central role in mediating signal transduction within
medium spiny neurons in the striatum. A variety of
neurotransmitters act on systems regulating the
phosphorylation of DARPP-32, which in turn modulates
the activity of protein phosphatase-1 (PP-1). DA
stimulation of D1 receptors acts via cAMP and PKA to
phosphorylate DARPP-32, which in turn inhibits PP-1; this
works in synergy with different protein kinases to
increase the level of protein phosphorylation of their
targets. In contrast, stimulation of D2 receptors
attenuates D1 activation of adenylate cyclase as well as
leading to calcium stimulation of protein phosphatase 2B;
together, this decreases the phosphorylation state of
DARPP-32. (From Greengard et al., 1999; used with
permission.)

Modulation of Intercellular Coupling

In addition to its effects on single neurons, DA also is capable of affecting neuronal interactions on a network level. In
 P.124

particular, substantial evidence has shown DA to have a potent effect over interactions among neighboring neurons in a region via its
modulation of gap junction conductance. The DA system appears to regulate this coupling in two ways: (a) acutely, presumably by
opening gap junctions that are already present between neurons in its target structures, and (b) as a compensatory change in
response to a chronic compromise of the DA system.

Studies have shown that neurons within the dorsal and ventral striatum exhibit dye coupling, which is the morphologic correlate of
gap junctions between neurons. In striatal slices recorded in vitro, application of the D2 agonist quinpirole causes a substantial
increase in coupling, from nearly undetectable levels in the basal state to approximately 80% coupling after the agonist. D1 agonists,
in contrast, do not affect coupling in a measurable way; however, in brain slices derived from a DARPP-32 knockout rat, the basal
level of coupling is significantly higher than in control, and furthermore, the D2 agonist fails to increase coupling above this
elevated baseline (75 ). These data suggest that coupling is normally suppressed by an action of DARPP-32, and that this suppression
can be overcome by D2 agonist administration.

Dye coupling is also affected by maintained changes in DA system function. Changes in coupling are observed following lesions of the
DA system with the neurotoxin 6-hydroxydopamine. Only the rats that exhibit severe loss of the DA innervation (i.e., >95%) also
show a substantial increase in the level of dye coupling among striatal neurons (84 ). In all cases, the coupling was present only
between cells of the same morphologic class; that is, between medium spiny neurons or between aspiny neurons. In addition,
withdrawal from repeated drug treatment such as amphetamine (Fig. 9.3 ) (85 ) or antipsychotic drugs (86 ,87 ) cause a regionally
selective increase in dye coupling. Amphetamine and antipsychotic drugs increase coupling in limbic striatum, whereas classic
antipsychotic drugs also cause an increase in coupling in the motor-related dorsal striatum. These effects are only observed
following withdrawal from the drug. Given that changes in gap junction composition are observed during repeated cocaine
administration (88 ), it is possible that the system compensates for the presence of the drug by altering gap junctions to allow
coupling to be maintained at its basal state. Under these conditions, the alteration is only observed when the adapted state is
altered by withdrawal of the drug. Indeed, the observation that coupling is maintained for weeks following drug withdrawal suggests
that the system may have reached a new stable steady state that could leave it more susceptible to destabilizing influences (85 ).

FIGURE 9.3. Long-term alterations in DA transmission lead to changes in dye coupling within the striatal complex. Medium
spiny neurons in the nucleus accumbens were injected during in vivo intracellular recording with Lucifer yellow, which was
then converted into a dense stain using antibodies. In a control rat, injection of Lucifer yellow typically labels only a single
neuron (left); overall, less than 15% of accumbens neurons injected in control rats exhibit labeling of more than a single
neuron. In contrast, in rats that had been administered amphetamine for 2 to 4 weeks and then withdrawn for at least 7 days,
the majority of injected neurons exhibited dye coupling (>60% of cells injected). In this case, four neurons were labeled after
injecting a single neuron with Lucifer yellow. This increase in coupling persisted for at least 28 days following amphetamine
withdrawal, but was not present if the rats were tested during the treatment phase. (From Onn and Grace, 2000; used with
permission.)

Interactions with Other Neurotransmitters

DA has also been shown to affect the response of striatal neurons to other neurotransmitters. Thus, DA was found to
 P.125

modulate the response of striatal neurons to glutamatergic excitation (89 ). Specifically, D1-receptor stimulation enhances NMDA-
mediated currents (90 ), which may occur via a combination of two effects: (a) a facilitation of L-type calcium conductances on
dendrites (90 ), and (b) activation of cAMP-PKA cascade (91 ). A similar D1-mediated cascade also attenuates responses to GABA in
the striatum (92 ,93 ). In contrast, D2 stimulation appears to preferentially attenuate non–NMDA-mediated responses (89 ). There is
also evidence that the activation of DA neuron firing by stimulation of DA axons (70 ,94 ) occurs via a D1-mediated facilitation of
glutamate transmission (94 ). This response, which occurs in parallel with a D1-mediated increase in c-fos in striatonigral neurons
(20 ), is more potent when the DA axons are stimulated in a burst-firing pattern (70 ). This suggests that, under physiologic
conditions, D1-induced facilitation of glutamate transmission in the striatum is mediated by burst-firing–dependent phasic DA
release (44 ).

In addition to its ability to modulate neurotransmitter actions on postsynaptic neurons in the striatum, DA also plays a significant
modulatory role in the presynaptic regulation of neurotransmitter release. D2 stimulation is reported to presynaptically decrease
GABA release from intrinsic neurons (95 ) and glutamate release from corticostriatal terminals. Several studies report that D2
agonists cause a down-regulation of glutamate-mediated EPSPs on neurons in the nucleus accumbens (96 ,97 ,98 and 99 ). This is
consistent with biochemical studies showing D2-mediated down-regulation of stimulated glutamate release in striatal tissue
(100 ,101 ) and the presence of D2 receptors on presynaptic terminals making asymmetric synapses in the striatum (78 ), which are
presumed to be the glutamatergic corticostriatal afferents. Interestingly, D2 stimulation does not inhibit all corticostriatal EPSPs in
normal preparations (97 ,99 ); however, after acute depletion of endogenous DA, all corticoaccumbens EPSPs are sensitive to DA
(99 ). This suggests that under normal circumstances, the presynaptic DA receptors may already be saturated with DA, as suggested
by the observation that sulpiride increase EPSP amplitude in a majority of cases when administered alone (99 ). This unusual
pharmacology may reflect a contribution of presynaptic D4 receptors on the corticoaccumbens terminals to this response (102 ).
Although another group has reported a D1-mediated presynaptic action EPSPs evoked by intrastriatal stimulation in slices, which was
interpreted as a presynaptic effect on corticostriatal terminals (103 ), this study employed exceedingly high doses of the D1 agonist
to achieve these effects (i.e., 100 μM, which is approximately two orders of magnitude higher than should be required for a
selective D1 action). Moreover, anatomic studies have shown that D1 immunoreactive axons are exceedingly rare in the striatum
(77 ). In contrast, recent studies suggest that DA acting on postsynaptic D1 receptors may actually cause a transsynaptic feed-
forward inhibition of glutamate release. Both NMDA antagonists and adenosine antagonists can block this effect. These data
suggested that dopamine depresses the excitatory postsynaptic conductance (EPSC) by causing an NMDA receptor-dependent
increase in extracellular adenosine, which acts presynaptically to depress glutamate release (104 ). The D1–NMDA-R interaction
appears to be postsynaptic and acts via PKC activation (105 ). It is of interest to note that there is other evidence of
interdependence between DA and adenosine. Thus, a recent study by Ginés and colleagues (106 ) have shown that D1 and adenosine
A1 receptors have the capacity to form heteromeric complexes, which appear to play a role in receptor desensitization and
trafficking.

Consequently, there appears to be a complex, dynamic equilibrium between dopamine and glutamate transmission within the
striatal complex, with glutamate contributing to DA release and DA causing a two-pronged inhibition of glutamate release, both
directly via D2 presynaptic receptors and indirectly using adenosine as an intermediary. Finally, glutamate-released NO also appears
to play a significant role in modulating DA systems and striatal neuron responsivity. The tight interdependence and coregulation
between DA and glutamate suggest that the system is designed to maintain stable levels of transmission to the striatal neurons over
the long term, whereas short-term changes in activity in either system in response to a signal are amplified by their coordinated
effects on each of these interdependent processes.

LTP and LTD

DA also appears to have a role in short- and long-term synaptic plasticity within the striatum. Specifically, DA was found to influence
two opposite types of synaptic plasticity within the striatum that depend on the history of synaptic input to this structure. In cases
in which striatal excitatory amino acid afferents arising from the cortex are stimulated with high frequencies in the absence of
magnesium (to enhance NMDA conductances), a long-term facilitation in synaptic transmission is induced, known as long-term
potentiation. In contrast, if the stimulation is carried out at a low frequency, the opposite type of plasticity is induced; that is, long-
term depression (LTD) (107 ). These forms of synaptic plasticity have been proposed to play a major role in learning and memory
formation in other structures, such as the hippocampus. Such plasticity within the striatum may be involved in such phenomena as
the acquisition of complex motor skills. Repetitive stimulation of corticostriatal fibers to release glutamate is required for the
induction of LTP and LTD, which only occurs in the presence of DA afferent input (108 ). Thus, D1 and/or D2 antagonist
pretreatment prevents the induction of LTD (107 ), suggesting that a synergistic interaction between these receptor subtypes is
required for this process to occur. In contrast, cortical stimulation-induced LTP is blocked selectively by D1 antagonists, but is
actually enhanced by D2 antagonists or in D2 receptor knockout mice (109 ).
 P.126

Prefrontal Cortex
The effects of DA within the PFC have been controversial, in that several groups have failed to produce consistent results. Thus,
although studies done in vivo have consistently shown that direct DA application inhibits PFC neuron firing, studies using in vitro
slice preparations have found a DA-mediated increase (110 ,111 ) and a decrease (112 ,113 ) in neuronal excitability in this region.
D1 stimulation has been shown to affect sodium conductances by increasing the sodium plateau potential and shifting the activation
of sodium currents to more negative potentials (114 ). This increase in excitability was augmented by a D1-induced decrease in slow
potassium conductances (110 ). D1 stimulation may also activate L-type calcium conductances located in proximal dendrites of
pyramidal neurons to further increase excitability in these neurons (66 ). Such an interaction has been postulated to differentially
modulate afferent input to these neurons (Fig. 9.4 ). Indeed, the highly organized DAergic input onto virtually every dendrite of PFC
pyramidal neurons in the primate provides a means for this neurotransmitter to regulate nearly the entire complement of
glutamatergic afferents to this cell type (115 ). In contrast, at least part of the inhibitory action of DA on PFC pyramidal neurons
may occur by DA-induced excitation of GABAergic interneurons (116 ), which also receive a direct DA innervation (115 ,117 ).

FIGURE 9.4. A simplified diagram illustrating the basic processing

units within the prefrontal cortex. Each unit consists of a deep layer
pyramidal neuron that projects to the nucleus accumbens or the
ventral tegmental area (VTA), and a GABAergic interneuron. The
apical and basal dendrites of the pyramidal neuron receive
functionally segregated inputs from various cortical and subcortical
regions, whereas the GABAergic interneuron, which is also
modulated by DA, exerts inhibitory influences over both the apical
dendrite and soma of the pyramidal neuron. By acting on both the
interneuron and pyramidal neuron dendrites, the DA input has the
capacity to modulate the integration of the functionally diverse
array of inputs to this neuron. DA acting on D1/D5 receptors to
modulate calcium channel subtypes on the apical dendrite are
proposed to “sharpen” or, with greater stimulation, attenuate
afferent signals arising from these distal regions. DA activation of
the GABAergic interneuron can also serve to suppress information
input from the apical dendrites. In contrast, DA modulation of
conductances at the somatodendritic region amplifies low-level
afferent inputs from neighboring pyramidal neurons. In this way, the
DAergic input is proposed to change the pyramidal neuron from
responding primarily to long-loop afferents to a state in which it
responds primarily to local circuit interactions that may subserve
working memory functions. (From Yang et al., 1999, with
permission.)

It is known that PFC neurons in vivo exhibit bistable membrane potentials, which alternate between a hyperpolarized, nonfiring
condition and a depolarized plateau state where they fire action potentials. Moreover, studies have shown that the effects of DA
vary depending on the state of the membrane potential at which it is administered. In particular, DA and D1 agonists cause an
increase in excitability of PFC neurons in the depolarized state but not at the hyperpolarized state (118 ). Furthermore, studies
combining in vivo microdialysis administration of drugs with intracellular recording (119 ) found that DA could potentiate glutamate-
driven bistable states of PFC neurons (Fig. 9.5 ). Therefore, the state of the membrane may significantly influence the response to
DA observed.

FIGURE 9.5. In vivo intracellular recordings from a

pyramidal neuron in the frontal cortex of a chloral hydrate
anesthetized rat is illustrated. The neuron was located
near a microdialysis probe implanted to deliver the
compounds to be tested by reverse dialysis to the
environment of the cell. Administration of NMDA (20 μM)
increases the number of spikes evoked by brief
depolarizing pulses. Following washout, administration of
NMDA + DA (30 μM) greatly increases the spikes evoked per
unit current. This occurs despite the fact that DA did not
appear to significantly affect current threshold. In the case
of NMDA alone, the increase in the number of spikes per
unit current occurs with (and may be secondary to) a
decrease in current threshold; that is, the cell is simply
more excitable.

In the case of DA added to NMDA, the cell fires more spikes during the current-induced depolarization, but the current
threshold is not further decreased; therefore, DA allowed the cell to respond maximally to the NMDA input without altering
the threshold for spiking. (From Moore et al., 1998b, with permission.)

Ventral Pallidum
The ventral pallidum (VP) receives a DA innervation from the midbrain (120 ), and is believed to play a significant role in several of
the behavioral aspects of DA system function (121 ), particular related to drug sensitization (122 ). In vivo dopamine iontophoresis is
known to: (a) increase and decrease VP neuronal firing (120 ,123 ); (b) potentiate or attenuate the excitatory effects of glutamate
iontophoresis (124 ); and (c) modulate the firing rate enhancements produced in VP neurons by activating the amygdala (123 ) and
attenuating the excitatory influences of the amygdala on pallidal cell firing at local concentration that are below that required to
alter spontaneous firing (123 ). Dopamine receptors for the D1 and D2 class have been identified in the ventral pallidum (120 ), and
electrophysiologic and behavioral evaluations have revealed that these two classes operate in opposition in this region (which
contrasts the “enabling” effects reported for striatal regions and other pallidal regions). Local D1 activation induces a robust
attenuation of cell firing (125 ) and enhances locomotor activity (126 ), whereas D2 activation slightly attenuates or has no effect on
firing (125 ) and is largely without influence on motor activity (126 ).
 P.127

Because the VP is positioned anatomically at the crossroads of the limbic and extrapyramidal system, DA modulation in this area has
the ability to potently influence motivated behavior by its actions in this region (121 ).

Mediodorsal Thalamus
Anatomic studies have revealed the presence of a DA innervation of the mediodorsal (MD) thalamic nucleus arising from the
midbrain. Using in vitro intracellular recordings, DA was found to alter MD neuron activity via a D2-mediated effect. In particular,
quinpirole was found to increase membrane excitability and enhance the low threshold spike in these neurons (127 ). This was
mediated at least in part via an alteration in potassium conductances. By increasing the low threshold spike, DA was found to
facilitate oscillatory activity within the MD, which would potently impact thalamocortical information processing in this region.

Basolateral Amygdala
The basolateral nucleus of the amygdala (BLA) exhibits a substantial innervation from the midbrain DA neurons. The effects
produced by DA in the BLA are dependent on the type of neuron recorded (128 ). DA causes an overall decrease in the firing rate of
presumed projection neurons by two mechanisms: (a) a direct effect on the projection neuron, and (b) an activation of the firing of
putative interneurons, which may be analogous to the interactions occurring in the PFC. In addition, DA produced effects on afferent
drive of these neurons that was dependent on the origin of the projection system. Thus, DA attenuates afferents from limbic
structures such as the PFC and MD thalamus, whereas afferent input from auditory association cortex (Te3) is potentiated (Fig. 9.6 ).
Intracellular recordings revealed that this was a consequence of a D1-mediated decrease in PFC-evoked EPSP amplitude, combined
with a D2-mediated increase in BLA input resistance that potentiated Te3 afferent drive (129 ). PFC stimulation also caused an
excitation of BLA interneurons, which lead to a subsequent attenuation of input arising from Te3; however, in the presence of DA
stimulation, the ability of the PFC stimulation to attenuate responses from Te3 was diminished (129 ). These data suggest that the
PFC is normally capable of attenuating amygdala responses to sensory inputs, which could be a mechanism for decreasing emotional
responses to familiar or nonthreatening stimuli. However, with excessive DA stimulation, the ability of the PFC to suppress
amygdala-mediated emotional responses may be lost.

FIGURE 9.6. DA attenuates prefrontal cortex (PFC) modulation of basolateral amygdala (BLA) neuronal responses. The PFC
provides a direct drive of BLA projection neurons and interneurons, whereas inputs from the sensory association cortex project
only to the output neurons. As a result, the PFC inhibits the ability of the sensory association cortex to activate BLA neuron
firing. However, the PFC inputs are attenuated by elevated DA levels in the BLA, removing a source of inhibition on BLA
projection neurons. Furthermore, elevated DA levels in the BLA increase the input resistance of BLA projection neurons,
leading to augmentation of nonsuppressed inputs to BLA neurons. Thus, DA receptor activation enables sensory-driven
amygdala-mediated affective responses by removal of regulatory inputs and augmenting sensory inputs. (From Rosenkranz and
Grace, 2001, with permission.)

Substantia Nigra
In addition to the effects of DA on DA neuron autoreceptors within the substantia nigra, there are also DA receptors
 P.128

located on striatonigral afferents to this region. Locally evoked IPSCs in neurons of the substantia nigra zona reticulata (ZR) are
GABAergic in nature, and are believed to arise from striatal afferents. These IPSCs are depressed by DA acting on D1 but not D2
receptors. The fact that this depression was accompanied by increased paired-pulse facilitation and not by a change in membrane
potential or conductance indicates that the effect is likely presynaptic in origin (130 ). It is interesting to note that the striatonigral
neurons that exhibit terminal D1 receptors do not exhibit D1 receptors on their local collaterals within the striatum (77 ). This
suggests that these neurons can selectively traffic presynaptic receptors to long projection sites.

BEHAVIORAL CORRELATES OF DA SYSTEM FUNCTION

Part of "9 - Dopamine "

DA is known to play an important role in working memory and response sequencing in the PFC. In particular, DA acting on D1
receptors has been shown to exert dual actions on these types of behaviors. Thus, D1 agonist administration into the PFC of rats
with poor performance on attentional function tasks significantly improved their performance, whereas impairing performance in
rats that had higher baseline attentional skills (131 ). This is consistent with studies suggesting that optimal DA levels are required
to maintain function in the PFC, with both too high or low D1 stimulation leading to impaired working memory function (132 ,133 ).

Several studies have shown that the DA system is activated by rewarding stimuli, such as food (134 ,135 ); however, it is becoming
evident that DA is not the reward signal per se, but instead is necessary for the acquisition of reinforcing stimuli. In some cases, DA
has been described as a type of error signal (136 ), in which the predicted occurrence of reward does not correlate with the
behavioral response emitted to generate this reward. Thus, when a task is well learned, DA neuron firing no longer is a necessary
correlate of the reward signal. But if reward is absent, DA neuron firing appears to decrease (137 ). Studies of DA overflow in the
nucleus accumbens show that DA is released when the DA cell bodies are stimulated electrically. However, when the stimulation is
contingent on a bar press by the rat, the DA overflow does not occur even in the presence of the electrical stimulus (138 ). These
data suggest that the lack of DA system activation during a well-learned contingent reinforcement task is not simply a failure to
activate DA neuron firing, but instead may represent an offsetting inhibitory influence over the DA system, either at the level of the
DA cell body or the terminal. Indeed, the reports of an anticipatory increase in extracellular DA in the accumbens prior to self-
administration of a DA drug such as cocaine (139 ) could potentially increase extracellular DA sufficiently to inhibit phasic DA release
occurring via stimulation of the DA cell bodies (44 ).

Overall, studies support the suggestion that DA actions in the PFC may have a greater involvement in the regulation of novel
circumstances, with the striatum involved more in expression of learned behaviors (140 ). This model is consistent with the
physiologic studies cited that show that DA can selectively activate circuits within frontal cortex and striatal complex, potentially
facilitating information flow along new pathways when a change occurs, but playing less of a role once a new stable steady state is
achieved at which the internal representation is at equilibrium with the predicted external events.

SUMMARY OF DA ACTIONS
Part of "9 - Dopamine "

It is clear from the preceding that DA exerts multiple actions at each level of integration within the cortico-striato-pallido-thalamo-
cortical loop. The actions exerted at each stage of this loop appear to have marked differences, however. For example, DA acting on
primary inputs to this circuit (e.g., the amygdala and PFC) affect both primary neurons and interneurons, with the net effect being a
selective potentiation of particular afferent drive sources. Within the striatum, DA exerts actions on presynaptic terminals
containing glutamate, as well as affecting the actions of glutamate on postsynaptic neurons. Combined with the reciprocal feedback
interactions between glutamate and DA terminals, this system appears to be designed to facilitate rapid changes in input states
while attenuating any long-term alterations that may occur. Moreover, the effects of DA on cellular coupling provide a type of
reversible hardwiring, which may facilitate performance of well-learned motor actions (141 ). Within the VP and MD, DA has effects
that would alter the behavioral output by changing the state of neuronal activity within these structures. Therefore, DA could
enable multiple state transitions within these regions, selecting among competing inputs, facilitating information transfer, and
altering states that would ultimately feed back via the thalamus to reinforce cortical activity that is most pertinent to the task at
hand (58 ,141 ).

The actions of DA may best be described not in terms of inhibition or excitation, but rather as related to the gating of inputs and
modulation of states of neuronal elements. This modulation of information integration is then further influenced at the network
level via the actions of DA on interneurons or cell coupling. Such a description is consistent with the behavioral actions of this
transmitter as well, in that it does not directly produce a motor output or reward signal, but instead modulates inputs and adjusts
the states of the organism in order to redirect the stimulus-response output to achieve the most effective behavioral strategy. Given
these constraints, one could imagine how dysfunctions in such a system could produce the profound pathologic states that have been
attributed to DA.

ACKNOWLEDGMENTS
Part of "9 - Dopamine "
 P.129

Dr. Grace has received research support from Pharmacia-Upjohn and Warner-Lambert/Parke Davis, as well as travel support for presenters at a scientific panel he co-organized for
Pharmacia-Upjohn.

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10
Astrocytes

Pierre J. Magistretti

Bruce R. Ransom

Pierre J. Magistretti: Institut de Physiologie, University of Lausanne Medical School, Lausanne, Switzerland

Bruce R. Ransom: Department of Neurology, University of Washington Medical School, Seattle, Washington 98195

The astrocyte is a ubiquitous type of glial cell that is defined in part by what it lacks: axons, action potentials, and synaptic
potentials. Astrocytes greatly outnumber neurons, often 10:1 and occupy 25% to 50% of brain volume (1 ,2 and 3 ). Although these
cells are anatomically obvious, their functions have been difficult to determine. Discoveries in the last 25 years, however, have
revealed some of their functions and established the essential nature of interactions between neurons and astrocytes for normal
brain function. We briefly review several basic facts about astrocytes and then selectively survey some of their functions,
particularly emphasizing recent findings about metabolic interactions between astrocytes and neurons. We also discuss features of
astrocyte function as they relate to synaptic plasticity and emerging concepts in the pathophysiology of psychiatric disorders.

 STRUCTURAL AND PHYSIOLOGIC PROPERTIES

 FUNCTIONS
 GLIAL CELL FUNCTION: IMPLICATIONS FOR PSYCHIATRY

STRUCTURAL AND PHYSIOLOGIC PROPERTIES

Part of "10 - Astrocytes "

The form of astrocytes is important in thinking about their functions. Astrocytes are stellate cells (hence their name) with multiple
fine processes. Astrocytes in white matter are complex cells with 50 to 60 long branching processes that radiate from the cell body
and terminate in end-feet at the pial surface, on blood vessels, or freely among axons; white matter astrocytes are usually called
fibrous astrocytes (4 ). Astrocytes in gray matter, called protoplasmic astrocytes, have profuse, short stubby processes that contact
blood vessels and the pial surface, and surround neurons. Astrocytic end-feet cover the entire surface of intraparenchymal
capillaries (5 ). These end-feet express glucose transporters of the GluT 1 type (6 ) and are a likely site of glucose uptake. In gray
matter, astrocytic processes ensheath virtually every synapse; the ensheathing membranes constitute about 80% of total membrane
surface and are devoid of organelles (7 ). Thus, astrocytes are polarized cells with some processes contacting cells of mesodermal
origin (i.e., endothelial cells of the capillary or fibroblasts of the pia mater), whereas other processes are intimately intertwined
with neuronal processes and synapses (4 ,7 ).

Astrocytes are the only cells in the brain that contain the energy storage molecule glycogen (8 ). The importance of this is discussed
elsewhere in this chapter. They also contain distinctive 9-nm intermediate filaments composed of a unique protein called glial
fibrillary acidic protein (GFAP). Fibrous astrocytes contain more of these filaments than protoplasmic astrocytes. Recent work has
assessed the functional significance of this defining astrocytic protein using genetic knockout experiments (9 ,10 and 11 ).
Astrocytes in GFAP knockout animals have disturbed neuronal plasticity manifest as a loss of long-term depression (10 ), late-onset
dysmyelination (9 ), and increased susceptibility to ischemia (12 ). It is not known how GFAP deficiency causes these changes.

Astrocytes are strongly coupled to one another by gap junctions (13 ), aqueous pores that are permeable to ions and other molecules
with a molecular weight less than 1,000. A broad range of biologically important molecules, including nucleotides, sugars, amino
acids, small peptides, cAMP, Ca2+ and inositol triphosphate (IP3) have access to this pathway. Such intercellular communication is
believed to mediate the coordinated action of adjacent but individual cells in terms of electrical and biochemical activity (13 ), and
equalizes their intracellular ion concentrations (14 ). Gap junction permeability is strongly reduced by intracellular acidification or
large increases in intracellular [Ca2+].

The membrane potential (Vm) of astrocytes is more negative than that of neurons. For example, astrocytes have a Vm of about -85
mV, whereas neuronal membrane potential is about -65 mV. Although glial cells express a variety of K+ channels, inwardly rectifying
K+ channels seem to be important in setting the resting potential (15 ). These channels are voltage sensitive and are open at
membrane potentials more negative than about -80 mV, close to the observed resting potential of astrocytes. Astrocytes express
many other voltage-activated ion channels, previously thought to be restricted to neurons (15 ). The significance
 P.134

of voltage-activated Na+ and Ca2+ channels in glial cells is unknown. Because the ratio of Na+ to K+ channels is low in adult astrocytes,
these cells are not capable of regenerative electrical responses like the action potential.
One consequence of the high K+ selectivity of astrocytes, compared to neurons, is that the membrane voltage of astrocytes is more
sensitive to changes in extracellular [K+] ([K+]o). For example, when [K+]o is raised from 4 to 20 mM, astrocytes depolarize by ˜25 mV,
compared to only ˜5 mV for neurons (16 ). This relative insensitivity of neuronal resting potential to changes in [K+]o in the
“physiologic” range may have emerged as an adaptive feature that stabilizes the resting potential of neurons in the face of the
transient increases in [K+]o that accompany neuronal activity. In contrast, natural stimulation, such as viewing visual targets of
different shapes or orientations, can cause depolarizations of up to 10 mV in astrocytes of the visual cortex (17 ). The accumulation
of extracellular K+ that is secondary to neural activity may serve as a signal to glial cells that is proportional to the extent of the
activity. For example, small increases in [K+]o cause breakdown of glycogen (18 ), perhaps providing fuel for nearby active neurons
(see later).

Neurons and glial cells do not make functional synaptic or gap junction contacts with one another; therefore, interactions between
these cell types must occur via the narrow extracellular space (ECS) between them (16 ). There may be rare exceptions to this rule
(19 ,20 ). In the mammalian central nervous system (CNS), the ECS is a uniform and very small compartment formed by adjacent cell
membranes that are, on average, separated by approximately by 0.02 μm. Brain ECS is a dynamic compartment in terms of its ionic
contents and even its dimensions (15 ,21 ). Because of the extreme narrowness of the ECS, molecules released from one cell diffuse
almost instantly to adjacent cells. Glial cells interact with neurons by influencing the contents (e.g., ions, energy metabolites,
neurotransmitters, etc.) of the ECS. It should be emphasized that nearly every neuron in the brain shares common ECS with adjacent
astrocytes, and astrocytic processes entirely surround synapses. It has been surprising to discover that glial cells release and express
receptors for a wide range of informational molecules, including neurotransmitters (22 ); this greatly expands the possibilities for
glial–neuronal interactions. Indeed, astrocytes are in a position to sense and modulate synaptic transmission through the pervasive
lamellar processes that surround synaptic contacts (7 ).

FUNCTIONS
Part of "10 - Astrocytes "

Ion Homeostasis
One of the best-established functions of astrocytes is regulation of brain [K+]o. Astrocytes are also likely to participate in the
regulation of extracellular pH, but this aspect of astrocyte function is still evolving and is not considered further here (23 ,24 ).
Neural activity can rapidly increase [K+]o, which is tightly regulated to a resting level of about 3 mM (25 ). A single action potential
increases the instantaneous [K+]o by ˜0.75 mM (26 ). The increase in [K+]o is proportional to the intensity of neural activity but has a
so-called “ceiling” level of accumulation of 10 to 12 mM (27 ,28 ), which is only exceeded under pathologic conditions (29 ). If
diffusion alone were responsible for dissipating K+ released from neurons, it is easily calculated that extracellular K+ accumulation
would exceed 10 mM during normal neural activity, whereas measured increases in [K+]o are in the range of 1 to 3 mM indicating
powerful control mechanisms (30 ). Homeostatic control of [K+]o is needed because brain [K+]o can influence transmitter release (31 ),
cerebral blood flow (32 ), ECS volume (33 ,34 ), glucose metabolism (35 ), and neuronal activity (36 ). Unchecked increases in [K+]o
act as an unstable positive feedback loop increasing excitability.
Astrocytes expedite the removal of evoked increases in [K+]o and limit its accumulation to a maximum level of 10 to 12 mM, the
ceiling level seen with intense activity such as epileptic discharge (37 ,38 ). Neurons, and perhaps blood vessels, also participate in
[K+]o regulation, but glial mechanisms are probably most important. Two general mechanisms of astrocyte K+ removal have been
proposed (39 ): 1) net K+ uptake into astrocytes (by transport mechanisms and/or Donnan forces) and 2) K+ redistribution through
astrocytes, which is known as K+ spatial buffering. The relative importance of these two mechanisms of [K+]o regulation remains an
open question and may depend on the nature of the [K+]o increase as well as brain region (38 ).
If glial cells take up K+ during neural activity and release it thereafter, a transient increase in glial [K+]i should result. Astrocyte [K+]i
does transiently increase during neural activity and has a similar time course to the K+ lost from active neurons and the increase in
[K+]o, indicating that the K+ released from neurons is passing by way of the ECS into glial cells (40 ,41 and 42 ). Uptake of K + into glial
cells depends on the glial Na+ pump (38 ,42 ,43 and 44 ), an anion transporter that cotransports K+ and Na+ with Cl- (43 ) and Donnan
forces that propel KCl into glial cells in the face of elevated [K+]o (42 ) (Fig 10.1 ). It has not been determined with certainty which
of these mechanisms is quantitatively most important for K+ uptake. The astrocyte Na+ pump, however, is exquisitely sensitive to
elevations of [K+]o. Even a 1 mM increase in [K+]o activates the Na+ pump in these cells indicating, perhaps, that this is the major
mechanism of K+ sequestration (44 ). Neurons, of course, must eventually reaccumulate K+ lost during activity using their Na+ pump,
but only glial cells show net accumulation of K+ (Fig. 10.1 ). It is interesting to note that the neuronal Na+ pump is not sensitive to
small increases in [K+]o and is probably activated mainly by increases in intracellular [Na+] (45 ).

FIGURE 10.1. Schematic representation of mechanisms of

K+ uptake in astrocytes. K+ released by firing neurons is
actively accumulated by astrocytes in three ways. The
sodium pump and an anion transporter both take up K+;
the sodium pump relies directly on the availability of
ATP, whereas the anion transporter is indirectly powered
by the energy stored in the transmembrane Na+ gradient.
The presence of channels for Cl- and K+, allow Donnan
forces to produce KCl influx. These mechanisms along
with K+ spatial buffering (see text), prevent [K+]o from
exceeding ˜12 mM. Increases in [K+]i are seen during
neural activity as [K+]o increases.
 P.135

The idea that focal increases in [K+]o could be redistributed by glial cells was introduced by Kuffler and colleagues (46 ). They
realized that the selective K+ permeability of glia coupled with their low-resistance intercellular connections (mediated by gap
junctions), would permit them to transport K+ from focal areas of high [K+]o, where a portion of the glial network would be
depolarized, to areas of normal [K+]o, where the glial network would have a near normal membrane potential (46 ). Experiments
suggest that under conditions of focal increases in [K+]o, five times as much K+ moves by way of glial cells as through the ECS, except
where only very localized K+ gradients are involved (25 ). A further specialization that contributes to spatial buffering is a
nonuniform distribution of K+ channels on a single cell. The density of K+ channels on the cell membrane of retinal Müller cells, which
are specialized astrocytes, is highest on the cell’s end-foot. Because the end-foot of the Müller cell, which abuts the vitreous humor
of the eye, has the highest density of K+ channels, accumulated [K+]o is preferentially transported to the vitreous, which acts as a
disposal site. It is not known if nonuniform K+ channel distribution is a general feature of astrocytes.

Anoxia/ischemia causes rapid increases in [K+]o in both gray matter (to ˜60 to 80 mM) and white matter (to ˜12 to 15 mM in vitro) of
the brain (27 ,47 ). The increases in [K+]o result because energy-dependent ion gradients can no longer be maintained and K+ entering
the ECS can no longer be taken up by glial cells, which also depend on ATP (48 ). In fact, under conditions of diminished energy
supply, glial cells actually contribute K+ to the ECS, rather than take it up (49 ).

Transmitter Synthesis
Glutamate is one of the most common amino acids in the brain, present at millimolar concentrations in brain tissue homogenate. It
is also the predominant excitatory neurotransmitter (50 ). Only a small fraction of total brain glutamate is packaged for synaptic
release and astrocytes are intimately involved in the synthesis of this crucial vesicular pool of glutamate.

Although glutamate can be derived from neuron glucose metabolism, carbon-labeling experiments reveal that astrocyte-derived
glutamine is the principal precursor of synaptically released glutamate (51 ,52 ). The synthesis and release of glutamine by
astrocytes is part of a biochemical shuttle mechanism called the glutamate-glutamine cycle (53 ) (Fig 10.2 ). After release from the
presynaptic terminal, glutamate is taken up primarily by astrocytes (54 ,55 ). In the glial cell, glutamate is converted to glutamine
through the ATP-dependent enzyme glutamine synthetase, located exclusively in astrocytes (56 ). In fact, glutamine synthetase is
localized to astrocytic processes surrounding glutamatergic synapses (57 ).
 P.136

Glutamine is released by the glial cells and taken up by the neurons through specific uptake carriers. In the presynaptic terminal,
glutamine is converted to glutamate through glutaminase, a phosphate-dependent enzyme preferentially localized to synaptosomal
mitochondria (58 ,59 ). The newly synthesized glutamate is then packed into vesicles and becomes available for release. The
glutamate-glutamine cycle is a clear and important example of cooperativity between astrocytes and neurons (Fig. 10.2 ). It
mediates removal of potentially toxic excess glutamate from the extracellular space and provides the neuron with a synaptically
inert precursor for resynthesis of glutamate. (Glutamine does not bind to neurotransmitter receptors.)

FIGURE 10.2. Scheme showing how astrocytes are

involved in glutamate metabolism and uptake. Only
astrocytes contain the enzyme glutamine synthetase,
which converts glutamate to glutamine in an ATP-
requiring reaction. Glutamine is transported to
nearby presynaptic terminals where it is converted to
glutamate for synaptic release. Finally, the released
glutamate is recaptured by astrocytes via a high
affinity glutamate uptake system. Although
glutamate transporters are present in neurons,
astrocytes are the most active in removing glutamate
(see text). In the absence of the normal
transmembrane Na+ gradient, maintained by the ATP-
dependent Na+ pump, the glutamate transporter
ceases to remove glutamate and can run in reverse so
that it pumps glutamate into the extracellular space
(ECS).

The cycle is surprisingly rapid. After a 6-min incubation of slices of rabbit hippocampus in [14C] glutamine, half of the radioactivity
was in the form of glutamate. Removal of glutamine from the bathing solution of the hippocampal slices decreased glutamate efflux
by 60% to 80% after only 6 min (52 ).
Not all of the glutamate taken up by astrocytes is directly converted to glutamine. Glutamate can also enter the TCA cycle through
its conversion to α-ketoglutarate (KG). Three enzymatic reactions can yield KG: one catalyzed by aspartate amino transferase and
another by alanine aminotransferase, both reactions involving the transfer of an α-amino group. The third reaction is the direct
conversion of glutamate to KG via the action of glutamate dehydrogenase (60 ) (Fig. 10.2 ). Theoretically, therefore, neurons might
not get back in the form of glutamine (from astrocytes) all of the glutamate that they release for two reasons: (a) some of the
glutamate diffuses away or is taken up by postsynaptic neurons, or (b) not all of the glutamate that enters astrocytes becomes
glutamine. Two possibilities can be considered for stabilizing the pool of vesicular glutamate in neurons. First, contrary to the
preceding premise, astrocytes might be able to compensate neurons for their loss of glutamate by appropriate adjustments in
glutamine export. This would be possible because the pool of cytosolic glutamate in astrocytes is in equilibrium with TCA cycle
intermediates, which in turn can be replenished by the carboxylation of pyruvate derived from glucose. The signal for more
glutamine export could be extracellular (glutamine), which would fluctuate in response to the needs of glutamatergic neurons.
Second, recent data have demonstrated that neurons can generate glutamate directly from pyruvate obtained from glucose or
lactate (61 ,62 ). Indeed, lactate produced by astrocytes in response to synaptically released glutamate (see the following) appears
to be taken up by neurons and could be a substrate for glutamate formation.
GABA, the most common inhibitory neurotransmitter in the brain, is synthesized from glutamate. Consequently, glutamine and α-
ketoglutarate are used for GABA synthesis as well (63 ). Depolarization-released GABA is preferentially synthesized from glutamine
supplied by astrocytes (64 ). Inhibition of astrocyte glutamine synthetase with methionine sulfoximine produces a significant
decrease in GABA production both in vivo and in brain slices (65%); however, because a 90% decrease in glutamine did not fully
suppress GABA synthesis, an additional metabolic source is considered to be likely (65 ).
Astrocytes, it would seem, are essential for normal glutamate- and GABA-mediated synaptic transmission. Indeed, selective
inhibition of glial cells in the guinea pig hippocampus using the glial-specific metabolic blocker, fluoroacetate, decreases
transmission at glutamate synapses (66 ). Intracellular recordings verified the integrity of neurons in fluoroacetate-treated slices and
the persistence of normal responses to glutamate applied iontophoretically. A modulatory role of astrocytes in excitatory synaptic
transmission is supported by this study.

Transmitter Removal
In addition to being the most important excitatory neurotransmitter in the brain, glutamate is also a potent neurotoxin and has been
implicated in stroke, amyotrophic lateral sclerosis, and epilepsy. Highly efficient glutamate transporters remove synaptically
released glutamate and also keep the extracellular concentration of this amino acid at about 2 μM (67 ). Glutamate transporters are
expressed in oligodendrocytes, neurons, microglia, and astrocytes, but transporters in astrocytes are quantitatively the most
important in regulating glutamate at synapses and in the extracellular space (Fig. 10.2 ). (See ref. 55 for review.)
Five main types of glutamate transporters have been described: GLAST (EAAT 1), GLT-1 (EAAT 2), EAAC 1 (EAAT 3), EAAT 4, and
EAAT 5 (55 ). The latter two appear to be predominantly localized in cerebellum and retina, respectively. All have been cloned,
functionally characterized, and their localization and distribution at the regional, cellular, and subcellular levels in the CNS are
known (55 ,68 ). A detailed review of this fertile field of research is beyond the scope of this chapter. EAAC 1 transporters are
neuronal, mostly localized on the cell body and dendrites, whereas GLAST and GLT-1 are predominantly glial (68 ). There are
regional differences in the expression of GLAST and GLT-1; GLAST is more heavily expressed in the cerebellum and GLT-1 is more
prevalent in the forebrain.
Glutamate uptake into astrocytes is driven by the electrochemical gradients of Na+ and K+, with a stoichiometry of 3 Na+ and 1 H+ in
and 1 K+ out with the uptake of each glutamate anion (55 ,69 ). The resulting increase in [Na+]i must be corrected by a cycle of the
Na+ pump and ATP consumption (70 ). One advantage of a system where astrocytes take up most of the glutamate is that the
metabolic burden of this work is offloaded from neurons (55 ).
Several lines of evidence support that astrocytes play an essential role in glutamate uptake in the brain (55 ): (a) Astrocytes
preferentially accumulate glutamate transporter
 P.137

substrate (71 ). (b) In the absence of astrocytes, neurons are 100-fold more vulnerable to glutamate toxicity (72 ). (c) Genetic down-regulation of GLAST or GLT-1, but not the neuronal
subtype EAAC1, causes elevated extracellular levels of glutamate and neurotoxicity (73 ).

As emphasized, astrocytes form a ubiquitous part of all glutamatergic synapses. Astrocyte membrane facing a glutamate synapse expresses higher levels of GLAST than membrane
facing other structures such as pia mater or capillaries (74 ). Most of the glutamate released at synapses appears to be taken up by the adjacent astrocytes (75 ), although there may
be some regions in the brain where up to 20% of glutamate is transported into the postsynaptic neuron (55 ). The impact of astrocytic glutamate uptake at synapses is most
emphatically detected when uptake is blocked. This increases both the amplitude and the duration of the excitatory postsynaptic current (76 ).

Metabolic Coupling with Neurons

Coupling of Synaptic Activity to Glucose Utilization

The cytoarchitecture of astrocytes is of particular relevance in a discussion about the coupling of synaptic activity to glucose utilization. As mentioned, astrocyte end-feet surround
virtually all brain capillaries, whereas other astrocytic processes ensheath synaptic contacts (5 ,7 ) or end among axons (4 ). In addition, astrocytes possess receptors and reuptake sites
for a variety of neurotransmitters, including the excitatory neurotransmitter glutamate (77 ), whereas astrocytic end-feet are enriched in the specific glucose transporter GLUT-1 (6 ).
Thus, astrocytes possess the necessary features to sense synaptic activity, through receptors and reuptake sites for neurotransmitters, and to couple it with the entry of glucose into
the brain parenchyma (78 ). Experimental evidence supporting this function is reviewed here.

Glutamate, the main excitatory neurotransmitter released by activated circuits, is a potent stimulator of glycolysis; that is, of glucose uptake and lactate production, in primary
astrocyte cultures. (See refs. 60 and 79 for review.) The metabolic effect of glutamate is not mediated by receptors, but rather by glutamate transporters selectively expressed in
astrocytes, in particular GLAST (80 ). These observations suggest a mechanism whereby astrocytes contribute to the uptake of glucose from the circulation into brain parenchyma in
register with synaptic activity: The release of glutamate from synaptically active neurons stimulates glucose uptake in nearby astrocytes. The extent of glucose uptake would be
proportional to the extent of activity, thereby “coupling” neuronal activity to glucose utilization (80 ). The Na-K-ATPase is critical for this coupling. Thus, ouabain, a specific inhibitor
of the Na-K-ATPase, completely inhibits the glutamate-evoked glycolysis in astrocytes (80 ). Glutamate stimulates astrocytic Na-K-ATPase (81 ) by increasing intracellular Na+
concentration ([Na+]i) via Na+-dependent glutamate uptake by glutamate transporters (55 ,69 ) (Fig. 10.2 and Fig. 10.3 ). The overall stoichiometry of the molecular steps involved in
the coupling between glutamate uptake and glucose utilization are as follows: one glutamate is taken up with 3 Na+, whereas one glucose consumed through glycolysis produces two
ATPs. One ATP is used by the Na+/K+ ATPase for the extrusion of 3 Na+; the other ATP could be used for the synthesis of glutamine from glutamate by glutamine synthase (Fig. 10.3 )
(82 ). The glutamate-stimulated glycolytic processing of glucose results in approximately two lactate molecules produced per one glucose molecule; that is, an expected stoichiometric
relationship between glucose and lactate. This scheme is, of course, primarily relevant for synaptic regions of the brain (i.e., gray matter). The mechanism that “couples” axonal
activity to glucose utilization in white matter is not established. Because axonal activity produces proportional increases in [K+]o (28 ) and increases in [K+]o increase astrocyte glucose
utilization (35 ,83 ), it is tempting to speculate that activity-dependent changes in [K+]o in white matter play an analogous role to glutamate release in gray matter.

FIGURE 10.3. Schematic representation of the

mechanism for glutamate-induced glycolysis in
astrocytes during physiologic activation and its
relevance for functional brain imaging. At
glutamatergic synapses, presynaptically
released glutamate depolarizes postsynaptic
neurons by acting at specific receptor subtypes.
The action of glutamate is terminated by an
efficient glutamate uptake system located
primarily in astrocytes. Glutamate is
cotransported with Na+, resulting in an increase
in the intra-astrocytic concentration of Na+,
leading to an activation of the astrocyte Na+/K+
ATPase. Activation of the Na+/K+ ATPase
stimulates glycolysis (i.e., glucose utilization
and lactate production). Lactate, once released
by astrocytes, can be taken up by neurons and
serve them as an adequate energy supply. The
proposed model of glutamate-induced glycolysis
in astrocytes implies that the activity-linked
uptake of 18FDG monitored with PET, reflects
primarily an astrocyte-based signal. Because
neuronally released glutamate triggers the
cascade of events that leads to glucose uptake,
the 18FDG-PET signal faithfully reflects
activation of neuronal circuits. The model is
also consistent with the transient lactate peak
detected during activation by MRS in humans
and by microdialysis and electrochemical
detection in animals (see text for details).

Is the lactate released by astrocytes in this model used as fuel by neurons? A vast array of experimental data indicate that in vitro, lactate in the absence of glucose can adequately
maintain synaptic (84 ,85 and 86 ) or axonal activity (87 ,88 ). If astrocytes transfer lactate to neurons as a fuel source (Fig. 10.3 ), several conditions must be met. There must be
appropriate enzymes for the creation of lactate in astrocytes and its use in neurons, and appropriate transport mechanisms for the movement of lactate. Indeed one isoform of lactate
dehydrogenase (LDH5), which is enriched in lactate-producing glycolytic tissues such as skeletal muscle, is predominantly localized in astrocytes in the human brain, whereas the other
isoform, LDH1, expressed in highly oxidative tissues such the heart, which utilize lactate, is mostly found in neurons (89 ). Monocarboxylate transporters (MCTs) mediate the exchange
of lactate between astrocytes and neurons. These transporters show a cell-specific distribution, with MCT1 predominantly present in astrocytes, whereas MCT2 is enriched in neurons
(90 ,91 ).

Glutamate-mediated neuron–glia metabolic interactions provide an initial basis to better understand the cellular and molecular steps involved in neuro metabolic coupling (Fig. 10.3 ).
In particular, the model proposed according to which the activity-dependent synaptic release of glutamate triggers glucose uptake into the brain parenchyma coupled with a transient
production of lactate, provides a possible basis for functional brain imaging techniques such as positron emission tomography (PET) (78 ,82 ). Indeed, a host of PET human studies have
indicated an activity-dependent partial uncoupling between glucose utilization (18F-deoxyglucose PET) and oxygen consumption (15O PET), whereas magnetic resonance spectroscopy
(MRS) analyses show a transient
P.138
 P.139

lactate production. (See ref. 79 for review.) In addition, recent MRS studies provide strong support for a tight coupling between
glutamate-mediated synaptic activity and glucose utilization. Thus, the simultaneous measurements, over a range of synaptic
activity, of glucose oxidation and the cycling of glutamate to glutamine (a process that occurs exclusively in astrocytes) using 15C
MRS has revealed a striking stoichiometric relationship of 1:1 between glutamate cycling (a reflection of synaptic activity) and
glucose utilization (92 ). According to these data, for each glutamate released from active terminals and taken up by astrocytes one
glucose would be oxidized.

Implications for Imaging

The model proposed on the basis of studies at the cellular level suggests an initial glycolytic processing of glucose occurring in
astrocytes during neuronal activation, resulting in a transient lactate overproduction, followed by a recoupling phase during which
lactate would be oxidized by neurons (Fig. 10.3 ).

Results obtained in a variety of in vivo paradigms both in laboratory animals and humans, support the existence of such a transient
lactate production during activation. Thus, microdialysis studies in rats indicate a marked increase in the concentration of lactate in
the dialysate in striatum and hippocampus during physiologic sensory stimulation (93 ). Interestingly, this activity-linked lactate
peak is completely inhibited when the glutamate uptake inhibitor THA is present in the perfusate, thus providing further supporting
evidence for the existence of glutamate stimulated glycolysis during activation (93 ). In humans, MRS reveals a transient lactate
peak in primary visual cortex during physiologic activation of the visual system (94 ). Thus, microdialysis and MRS data in vivo
support the notion of a transient glycolytic processing of glucose during activation. In addition, some PET studies have shown that
oxygen consumption does not increase commensurately with blood flow and glucose utilization in activated brain areas (95 ),
suggesting the occurrence of an activity-dependent glycolytic processing of glucose. In contrast, using 13C-glucose MRS, recent data
are consistent with a significant increase in oxygen utilization during activation (96 ). These contrasting views relevant to the degree
of oxygen utilization during activation can be reconciled by the model proposed (80 ), which suggests that glucose imported into the
brain parenchyma during activation undergoes a transient glycolysis in astrocytes, resulting in the production of lactate that is then
oxidized by neurons. This latter process would imply a metabolic recoupling with increased oxygen consumption. The spatial and
temporal “window” during which a transient glycolysis occurs and a lactate peak can be detected, may depend on the rapidity and
degree of recoupling existing between astrocytic glycolysis and neuronal oxidative phosphorylation.

This operational model for coupling is consistent with the notion that the signals detected during physiologic activation in humans
with FDG PET may result from signaling and metabolic exchanges between neurons (glutamate release) and astrocytes (glycolysis)
(82 ). This conclusion does not question the validity of these techniques for monitoring neuronal function because the triggering
event is neuronal glutamate release; rather, this conclusion provides a cellular and molecular basis for these functional brain-
imaging techniques (Fig. 10.3 ).

Glycogen
Another facet of neuron–glia metabolic interactions concerns glycogen. Glycogen, the storage form of glucose, is the largest energy
reserve of the brain and it is almost exclusively localized in astrocytes. Although the levels of glycogen are low compared to muscle
or liver, they appear to vary in register with synaptic activity and are tightly regulated by a variety of neurotransmitters (97 ). For
example, somatosensory stimulation readily mobilizes glycogen in the corresponding somatosensory cortex as well as in subcortical
relays (98 ). In contrast, during anesthesia glycogen levels increase dramatically. Plastic adaptations in glycogen regulation appear
to occur in astrocytes as a consequence of acute or slow neuronal loss; indeed, glycogen deposits are often observed in reactive
astrocytes present in acutely lesioned areas, as well as at sites of slow neurodegeneration, such as those observed in Alzheimer’s
disease (97 ). These latter observations suggest that impaired synaptic activity associated with neuronal loss results in a glycogen-
sparing situation in astrocytes; active mechanisms driving glycogen metabolism toward increased resynthesis may also be operative
under such conditions. The mechanisms that underlie the regulation of glycogen metabolism at the cellular and molecular levels
have been partially characterized. (See ref. 97 for review.) Whereas the role of brain glycogen remains to be fully elucidated,
recent results indicate that astrocytic glycogen in white matter is readily available to axons, mainly as lactate, and sustains their
function during glucose withdrawal (99 ). It also seems likely that glycogen mobilization by certain neurotransmitters is an
adaptation designed to provide additional energy substrate to synaptically active neurons (see the following).

It is appealing to think that astrocytic glycogen serves to provide fuel to the brain when glucose is in short supply. Indeed, astrocytic
glycogen in vitro is rapidly degraded when glucose is withdrawn (100 ), and glycogen falls rapidly in vivo during ischemia, with a
time course that is closely related to (87 ) ATP depletion and accumulation of lactate (101 ). Some experimental evidence supports
this concept. Neurons grown in astrocyte-rich cultures are less severely injured by glucose withdrawal than are neurons in astrocyte-
poor cultures (102 ). This benefit appeared to derive from the presence of greater amounts of glycogen in the astrocyte-rich cultures
because depleting glycogen negated the benefit (102 ).
 P.140

Two possible mechanisms for this outcome were suggested but not tested: (a) Astrocytes themselves utilize the energy from
glycogen breakdown to prevent the accumulation of toxic levels of glutamate (removing it by a sodium-gradient-dependent
transporter); or (b) Glycogen provides fuel to neurons to sustain their energy metabolism.

The role of astrocytic glycogen in CNS white matter has been analyzed using the rat optic nerve, a representative white matter tract.
Optic nerve function, measured as the compound action potential (CAP), persists for about 40 min in the absence of glucose
(87 ,103 ), suggesting the presence of an intrinsic energy reserve such as astrocytic glycogen. The theory was tested that during
glucose withdrawal, astrocytic glycogen is converted to lactate and transported into axons to act as an energy source. Glycogen
content of the optic nerve falls during glucose withdrawal, compatible with rapid use in the absence of glucose. Up-regulation of
glycogen content increases latency to CAP failure and improves CAP recovery after glucose withdrawal, whereas down-regulation of
glycogen content has the opposite effects (99 ). Lactate can replace glucose as an energy source and appears to be transported by
MCT out of astrocytes and into axons in the absence of glucose. These results indicate that astrocytic glycogen in white matter is
readily available to axons, mainly as lactate, during glucose withdrawal (Fig. 10.4 ). Conceptually, they provide “proof of principle”
that astrocytic glycogen is an energy reserve that can be shared with, and thus can prolong the function of, neural elements in times
of need.

FIGURE 10.4. Schematic illustration of how astrocytic glycogen appears to fuel axons in the absence of glucose. Blood glucose
first encounters astrocytic end-feet as it is transported into the brain. In the absence of glucose, astrocytic glycogen is broken
down to lactate, which is transported to the extracellular space via a monocarboxylate transporter (MCT). It is then taken up
by an MCT in axons and is oxidatively metabolized to produce energy needed to sustain excitability. LDH5 preferentially
reduces pyruvate to lactate, whereas LDH1 preferentially oxidizes lactate to pyruvate. Neurotransmitters such as
norepinephrine, vasoactive intestinal peptide, and adenosine, promote glycogenolysis. This scheme recognizes that astrocytes
can subsist, at least transiently, on glycolytic energy metabolism, whereas axons require oxidative metabolism.

Glycogen levels in astrocytes are tightly regulated by various neurotransmitters. Several monoamine neurotransmitters, namely
noradrenaline, serotonin, and histamine, are glycogenolytic in the brain, in addition to certain peptides such as vasoactive intestinal
peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP), as well as adenosine and ATP (104 ,105 ). The effects of
all these neurotransmitters are mediated by specific receptors coupled to second messenger pathways such as adenylate cyclase, for
the β-adrenergic, VIP/PACAP and adenosine A2 receptors, or phospholipase C for α-1 adrenergic receptors (106 ). The initial rate of
glycogenolysis activated by VIP and noradrenaline is between 5 and 10 nmol/mg prot/min (106 ), a value that is remarkably close to
glucose utilization of the gray matter as determined by the 2-DG autoradiographic method (107 ). This correlation raises the
possibility that the glycosyl units mobilized in response to the glycogenolytic neurotransmitters are in register with the energy
demands of the neuropil.

In addition to the rapid (within seconds) glycogenolysis, VIP, noradrenaline, and adenosine induce a long-lasting plastic response
resulting in massive glycogen resynthesis (108 ). This effect is expressed after several hours and is transcriptionally regulated,
involving the expression of new genes: two immediate-early and two late genes, all being induced in a cAMP-dependent manner in
astrocytes. The immediate-early genes, C/EBP β and δ are members of a family of transcription factors called CCAAT/enhancer
binding protein. This family of transcription factors is predominantly involved in two types physiologic responses: inflammation,
through the control of expression of several acute phase response genes, and energy metabolism, in particular through the
regulation of cAMP-sensitive genes controlling glucose metabolism in peripheral tissues (109 ). The late genes regulated by VIP and
NA are glycogen synthase (110 ) and protein targeting to glycogen (PTG) (111 ). The physiologic function of PTG appears to be that
of a chaperone protein coordinating the activity and compartmentalization of glycogen-synthesizing enzymes (111 ).

Glycogen metabolism in astrocytes appears to be under the dynamic control of at least two neurotransmitters, VIP and NA, with the
balance between short-term (glycogenolysis) and transcriptionally regulated long-term effects (glycogen resynthesis) setting the
intracellular levels of glycogen.

Calcium Waves and Glial Signaling

The phenomenon of inducible waves of increased [Ca2+]i moving through adjacent astrocytes was first reported in tissue culture by
Cornell-Bell and associates (112 ). Such waves, stimulated by glutamate application or a mechanical
 P.141

stimulus, are also known to occur in other cell types (113 ). It is of special interest that astrocyte Ca2+ waves may be elicited by
activity in adjacent neurons (114 ). The mechanism of these waves, which move through cells at a rate of 10 to 20 μm/sec, is not
entirely understood (115 ). It appears to involve the intracellular formation and intercellular transmission of inositol-1,4,5-
trisphosphate (IP3), and the release of an extracellular messenger substance, perhaps ATP (115 ). This phenomenon has become
more interesting with the discovery that it is seen in intact brain tissue such as the retina (116 ).

The function of intracellular Ca2+ waves in astrocytes could be to coordinate the activity of these glial cells. A more intriguing
possibility would be that the Ca2+ wave could influence neurons in its vicinity. Indeed, Ca2+ elevation in astrocytes can cause
increases in neuronal [Ca2+]i (117 ) and induce action potentials in hippocampal neurons (118 ,119 ). The most compelling
demonstration to date of glia-to-neuron signaling mediated by Ca2+ waves, however, has come from studies in the rat retina (116 ).
Mechanical stimulation of astrocyte Ca2+ waves led to changes in light-induced ganglion cell firing, usually inhibition, when the Ca2+
wave reached the neuron. The likely mechanism was the release of glutamate from the astrocytes with stimulation of inhibitory
interneurons projecting to the ganglion cells. The importance of this fascinating observation for the normal operation of the nervous
system remains unclear, but it suggests a unique form of glial modulation of neuronal activity.

Astrocytes and Synaptic Plasticity

Considerable attention has been given in recent years to the mechanisms of synaptic plasticity. It is now clear that plastic events
outlast the early stages of nervous system development and are maintained, to different degrees, throughout adult life, providing
the basis for the processes of learning and memory (120 ). Most experimental paradigms both in vivo and in vitro, such as the study
of long-term potentiation in brain slices, have thoroughly explored the mechanisms of plasticity occurring as a consequence of
intense activation of neuronal circuits purportedly associated with the learning process (12 ). For example, structural
rearrangements at synapses characterized by an enhanced number of axodendritic synaptic contacts have been demonstrated (121 ),
and the relative contribution of presynaptic adaptations (increased neurotransmitter release) versus postsynaptic ones (increased
responsivity of the target elements) have been thoroughly investigated (122 ). The involvement of a variety of molecules, including
glutamate and its receptors, nitric oxide, arachidonic acid, certain neurotrophins, and cell adhesion molecules, has been proposed
(123 ).

However, few studies have explored the possible adaptations that may occur in glial elements, in particular at the astrocytic profiles
that ensheath synapses, as a consequence of learning paradigms known to affect synaptic plasticity. The structural rearrangements
occurring at synaptic contacts are likely to affect the morphology of the associated astrocytic profiles. Such an astrocytic structural
plasticity has been well documented in the hypothalamus. Here, on physiologic stimulation (lactation, dehydration) the astrocytic
profiles surrounding the soma and dendrites of oxytocin-containing neurons retract, allowing a marked increase in membrane
surface available for synaptic contacts (124 ). This structural modification, in which a clear role for certain cell-adhesion molecules
(e.g., PSA-NCAM, F3, and tenascin) has been shown (125 ), is reversible, being associated with the period of stimulation. A similar
structural astrocytic plasticity has been shown in the arcuate nucleus during the estrous cycle (124 ). Evidence for activity-
dependent astrocytic plasticity is beginning to be demonstrated also in extrahypothalamic areas of the brain. Striking structural
modifications of astrocyte morphology surrounding synaptic contacts occur in parallel to neuronal plastic adaptations in brain areas
activated by simple behavioral paradigms of learning. Thus, in animals reared in complex environments, the size and the number of
GFAP-immunoreactive astrocytes was found to be increased in the visual cortex as compared to animals raised in normal laboratory
cage environment (126 ,127 and 128 ). In addition, the extent of contact between astrocytic processes and synapses is increased
under the same conditions (129 ). Similar astrocytic modifications are also observed in the cerebellum following synaptic plasticity
induced by motor-skill learning (130 ). Following a spatial learning task (the Morris water maze test), an increase in the density of
GFAP-immunoreactive astrocytes is observed in the hippocampus (131 ). This increase in GFAP is correlated with enhanced
expression of basic-fibroblast growth factor. Considering the purported role of LTP in spatial learning (132 ), it is worth noting that
induction of LTP in vivo by repeated high-frequency stimulation of the perforant pathway in the hippocampus causes a similar
increase in numerical density, higher surface density, and closer apposition of astrocyte processes to the synaptic clefts, or
dendritic spines in the potentiated synapses (133 ). The concept that glial cells, and in particular astrocytes, could contribute to
plastic changes occurring after learning and memory or developmental paradigms has already been the subject of reviews (134 ).

In some of these learning paradigms involving structural plastic responses in astrocytes, long-lasting adaptations in local energy
metabolism have been reported. Thus, increases in capillary formation, capillary branching, and surface area were reported in visual
cortex following complex experience (127 ). Angiogenesis was also demonstrated in the cerebellum after motor skill learning (135 ),
thus suggesting that a long-lasting enhancement in the supply of energy substrates (mostly glucose and O2) is required in the
activated area. Several studies have reported long-lasting changes in glucose utilization following various learning and memory tasks
(136 ,137 ,138 and 139 ).
 P.142

Spatial discrimination training was reported to cause persistent increases in glucose utilization, as measured with the 2-
deoxyglucose autoradiographic technique in regions such as the hippocampus and various cortical areas (140 ).

GLIAL CELL FUNCTION: IMPLICATIONS FOR PSYCHIATRY

Part of "10 - Astrocytes "
From the overview presented in the preceding paragraphs, it clearly appears that all facets of intercellular communication in the
brain are relevant to glial cell, and specifically astrocyte function. Thus, the presence on astrocytes of receptors for all chemical
classes of neurotransmitters (amino acids, monoamines, and peptides) indicates that the receptor-mediated effects of psychoactive
drugs can involve astrocytes. To take a simple example, increasing synaptic levels of noradrenaline with certain classes of
antidepressants, by activating β-adrenergic receptors on astrocytes, is likely to result in induction of genes that regulate glycogen
metabolism. Plasticity is no longer exclusive to neurons, providing the conceptual background for considering the activity- and drug-
induced adaptations observed in the function of specific circuits as also involving astrocyte-based mechanisms. As recently reviewed,
alterations in excitatory neurotransmission, which appear to be involved in neuropsychiatric disorders such as schizophrenia or
Alzheimer’s disease, may well include an astroglial component (141 ). Recent neuroimaging studies have shown that the volume of
the subgenual prefrontal cortex is reduced in familial forms of major depressive and in bipolar disorders. This decrease in cortical
volume could be ascribed to a marked decrease in the number of glial cells (142 ). Functional imaging in patients suffering from
both clinical conditions show a significant decrease in glucose utilization determined by 18F-deoxyglucose PET (143 ). In addition to
providing indirect evidence for the involvement of glia in certain psychiatric disorders, this set of observations is consistent with the
proposed role of astrocytes in the 18F-deoxyglucose PET signal (80 ). Chronic treatment with neuroleptics has been associated with
increased glial density in the primate prefrontal cortex (144 ). Although these examples still need validation in terms of their
relevance in the pathophysiology of certain neuropsychiatric disorders, they provide, along with advances in the understanding of
glial cell biology, sufficient grounds for opening the scope of psychiatric neuroscience to the study of what Joseph Coyle and Robert
Schwarcz have recently called “mind glue” (the term glia, meaning glue in German) (141 ). This is a major step indeed from
“neuron-centric” dominated psychiatric theory and one that is justified by the available facts.

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11
Synaptic Plasticity

Robert C. Malenka

Robert A. Malenka: Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Palo Alto,
California.

The most fascinating and important property of the mammalian brain is its remarkable plasticity, which can be thought of as the
ability of experience to modify neural circuitry and thereby to modify future thought, behavior, and feeling. Thinking simplistically,
neural activity can modify the behavior of neural circuits by one of three mechanisms: (a) by modifying the strength or efficacy of
synaptic transmission at preexisting synapses, (b) by eliciting the growth of new synaptic connections or the pruning away of existing
ones, or (c) by modulating the excitability properties of individual neurons. Synaptic plasticity refers to the first of these
mechanisms, and for almost 100 years, activity-dependent changes in the efficacy of synaptic communication have been proposed to
play an important role in the remarkable capacity of the brain to translate transient experiences into seemingly infinite numbers of
memories that can last for decades. Because of its fundamental importance, there has been an enormous amount of work describing
the many forms of synaptic plasticity and their underlying mechanisms.

Synaptic transmission can either be enhanced or depressed by activity, and these alterations span temporal domains ranging from
milliseconds to enduring modifications that may persist for days or weeks and perhaps even longer. Transient forms of synaptic
plasticity have been associated with short-term adaptations to sensory inputs, transient changes in behavioral states, and short-
lasting forms of memory. More lasting changes are thought to play important roles in the construction of neural circuits during
development and with long-term forms of memory in the mature nervous system. Given these diverse functions, it is not surprising
that many forms and mechanisms of synaptic plasticity have been described. In this chapter, I provide a brief overview of some of
the forms of synaptic plasticity found at excitatory synapses in the mammalian brain, focusing on long-term potentiation (LTP) and
long-term depression (LTD).

 SHORT-TERM SYNAPTIC PLASTICITY

 LONG-TERM SYNAPTIC PLASTICITY
 CONCLUSIONS
 ACKNOWLEDGMENTS

SHORT-TERM SYNAPTIC PLASTICITY

Part of "11 - Synaptic Plasticity "

Virtually every synapse that has been examined in organisms ranging from simple invertebrates to mammals exhibits numerous
different forms of short-term synaptic plasticity that last on the order of milliseconds to a few minutes (for detailed reviews, see 1
and 2 ). In general, these result from a short-lasting modulation of transmitter release that can occur by one of two general types of
mechanisms. One involves a change in the amplitude of the transient rise in intracellular calcium concentration that occurs when an
action potential invades a presynaptic terminal. This occurs because of some modification in the calcium influx before transmitter
release or because the basal level of calcium in the presynaptic terminal has been elevated because of prior activity at the terminal.
A second mechanism occurs downstream of calcium elevation in the presynaptic terminal and involves some modulation of the
biochemical processes involved in synaptic vesicle exocytosis.

Paired-Pulse Facilitation and Depression

When two presynaptic stimuli are delivered within a short interval, the synaptic response to the second stimulus can be either
enhanced or depressed relative to the first stimulus. Paired-pulse depression is commonly observed at all synapses at short (less
than 20 milliseconds) interstimulus intervals. It may result from inactivation of voltage-dependent sodium or calcium channels or
from a transient depletion of the synaptic vesicles that are “docked” adjacent to the presynaptic plasma membrane, waiting to be
released. Many synapses at longer interstimulus intervals (20 to 500 milliseconds) exhibit paired-pulse facilitation that is thought to
result from the influx of calcium that occurs in response to the first action potential. One simple idea is that the “residual” calcium
left over from the first action potential combines with the calcium influx during the second action potential, and because the
relationship between calcium concentration in the terminal and release is highly nonlinear, this small increase in resting calcium
may cause substantial facilitation. However, with a single action potential, the increase in resting calcium concentration is very
small, and
 P.148

thus additional mechanisms are likely involved. Currently, there is much interest in the possibility that transient modulation, by
activation of protein kinases, of some of the presynaptic phosphoproteins that are known to be involved in the control of transmitter
release may play an important role in very short-term synaptic plasticity. For example, knockout mice lacking one or more of the
synapsins (3 ,4 ), or lacking the small guanosine triphosphate–binding protein rab3A (5 ,6 ), exhibit abnormal short-term synaptic
plasticity.

Whether a specific synapse displays paired-pulse facilitation or depression depends on the initial state of the synapse and its recent
history of activation. Because these forms of plasticity largely result from changes in the probability of transmitter release, synapses
that begin with a very high probability of release tend to show depression, whereas those with a low probability of release exhibit
facilitation. Consistent with this idea, activation of presynaptic receptors that cause a decrease in transmitter release almost always
causes an increase in the magnitude of paired-pulse facilitation (or even a conversion of paired-pulse depression to paired-pulse
facilitation).

Facilitation and Depression Following Trains of Stimuli

Longer-lasting forms of plasticity are observed following repetitive or tetanic stimulation of synapses with prolonged (approximately
200-millisecond to 5-second) trains of stimuli applied at high frequencies (10 to 200 Hz). Augmentation and posttetanic potentiation
refer to enhancements of transmitter release that can last anywhere from seconds (augmentation) to several minutes (posttetanic
potentiation). They are thought to result in large part to the buildup of calcium concentration in the presynaptic terminal during the
trains of stimuli. This residual calcium may both combine with the calcium influx elicited by subsequent single action potentials and
lead to biochemical modifications of proteins in the presynaptic terminal. At some synapses, repetitive activation leads to
depression that can last for several seconds or even minutes. As in paired-pulse depression, this generally occurs at synapses that
exhibit a high probability of release and is thought to result, at least in part, from a transient depletion of the synaptic vesicles that
are poised to be released by an action potential.

In large part because of these short-term forms of synaptic plasticity, the strength of communication between pairs of neurons can
be modified even during short bursts of presynaptic activity (e.g., five to ten action potentials at 20 to 50 Hz) (7 ). The functional
relevance of such short-term synaptic dynamics has received much less attention than long-lasting forms of synaptic plasticity and is
just beginning to be explored (8 ). One potential role of these short-term forms of synaptic plasticity is to transform incoming
information in the temporal domain into a spatially distributed code (9 ,10 ). Furthermore, given that presynaptic proteins that may
be involved in short-term plasticity may be abnormal in neuropsychiatric disorders (11 ), it is not unreasonable to speculate that
abnormal short-term synaptic dynamics in specific neural circuits may contribute to the pathophysiology of any number of mental
illnesses.

LONG-TERM SYNAPTIC PLASTICITY

Part of "11 - Synaptic Plasticity "

During the last decade, there was enormous interest in elucidating the mechanisms responsible for activity-dependent long-lasting
modifications in synaptic strength. The great interest in this topic is largely based on the simple idea that external and internal
events are represented in the brain as complex spatiotemporal patterns of neuronal activity, the properties of which result from the
pattern of synaptic weights at the connections made between the neurons that are contributing to this activity. The corollary to this
hypothesis is that new information is stored (i.e., memories are generated) when activity in a circuit causes a long-lasting change in
the pattern of synaptic weights. This simple idea was put forth by Ramon y Cajal almost 100 years ago, but experimental support for
such a process was lacking until the early 1970s, when it was demonstrated that repetitive activation of excitatory synapses in the
hippocampus caused an increase in synaptic strength that could last for hours or even days (12 ,13 ). This long-lasting synaptic
enhancement, LTP, has been the object of intense investigation because it is widely believed that LTP provides an important key to
understanding the molecular mechanisms by which memories are formed (14 ,15 ) and, more generally, by which experience
modifies behavior. Furthermore, the activity- and experience-dependent refinement of neural circuitry that occurs during
development shares features with learning, and thus a role for LTP in this process has been proposed (16 ,17 and 18 ).

Long-Term Potentiation
No form of synaptic plasticity has generated more interest and has been more extensively studied than LTP in the CA1 region of the
hippocampus. The excitement surrounding this phenomenon derives mainly from four sources. First, there is compelling evidence
from studies in rodents and higher primates, including humans, that the hippocampus is a critical component of a neural system
involved in various forms of long-term memory (19 ). Second, several properties of LTP make it an attractive cellular mechanisms for
information storage (20 ,21 ). Like memories, LTP can be generated rapidly and is prolonged and strengthened with repetition. It is
also input specific in that it is elicited at the synapses activated by afferent activity and not at adjacent synapses on the same
postsynaptic cell. This feature dramatically increases the storage capacity of individual neurons
 P.149

that, because synapses can be modified independently, can participate in the encoding of many different bits of information. Third,
LTP is readily generated in in vitro preparations of the hippocampus, thus making it accessible to rigorous experimental analysis.
Indeed, much of what we know about the detailed mechanisms of LTP derives from studies of LTP at excitatory synapses on CA1
pyramidal cells in hippocampal slices. Fourth, LTP has been observed at virtually every excitatory synapse in the mammalian brain
that has been studied. Table 11.1 gives a list of the brain regions in which LTP has been demonstrated, and it can be seen that
regions thought to be particularly important for various forms of learning and memory are prominent. Although LTP is not a unitary
phenomenon, most synapses appear to express a form of LTP that is identical or highly analogous to the LTP found at excitatory
synapses on CA1 pyramidal cells. Thus, this form of LTP is the focus of the remainder of this section.

TABLE 11.1. AREAS OF BRAIN IN WHICH LTP HAS BEEN DEMONSTRATED

Triggering of LTP: A Critical Role for NMDA Receptors and Calcium

It is well established that the triggering of LTP requires synaptic activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors,
a subtype of ionotropic glutamate receptor (see Chapter 6 ) and postsynaptic depolarization, which is accomplished experimentally
by repetitive tetanic stimulation of synapses or by directly depolarizing the cell while continuing low-frequency synaptic activation
(a so-called “pairing protocol”). How do these requirements explain the properties of LTP? During basal low-frequency synaptic
transmission, synaptically released glutamate binds to two different subtypes of ionotropic glutamate receptor, termed AMPA (α-
amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and NMDA receptors, which are often, but not always (see later), co-localized
on individual dendritic spines. The AMPA receptor has a channel that is permeable to monovalent cations (Na+ and K+), and activation
of AMPA receptors provides most of the inward current that generates the excitatory synaptic response when the cell is close to its
resting membrane potential (Fig. 11.1 ). In contrast, as described in Chapter 6 , the NMDA receptor exhibits a strong voltage
dependence because of the block of its channel at negative membrane potentials by extracellular magnesium. As a result, NMDA
receptors contribute little to the postsynaptic response during basal synaptic activity. However, when the cell is depolarized,
magnesium dissociates from its binding site within the NMDA receptor channel and allows calcium as well as sodium to enter the
dendritic spine (Fig. 11.1 ). The resultant rise in intracellular calcium is a necessary and perhaps sufficient trigger for LTP. This local
source of calcium within the dendritic spine accounts for the input specificity of LTP.

FIGURE 11.1. Model for the induction of long-term potentiation (LTP). During normal synaptic transmission (left), synaptically
released glutamate acts on both NMDA and AMPA receptors. Na+ flows through the AMPA receptor channel but not through the
NMDA receptor channel because of the Mg2+ block of this channel. Depolarization of the postsynaptic cell (right) relieves the
Mg2+ block of the NMDA receptor channel and allows Na+ and Ca2+ to flow into the cell. The resultant rise in Ca2+ in the dendritic
spine is a necessary trigger for the subsequent events leading to LTP.

The evidence in support of this model for the initial triggering of LTP is compelling. Specific NMDA receptor antagonists have
minimal effects on basal synaptic transmission but block the generation of LTP (22 ,23 ). Preventing the rise in calcium by loading
cells with calcium chelators blocks LTP (24 ,25 ), whereas directly raising intracellular calcium in the postsynaptic cell mimics LTP
(25 ,26 ). Furthermore, imaging studies have demonstrated that NMDA receptor activation causes a large increase in calcium level
within dendritic spines (see 23 for references). The exact properties of the calcium signal that is required to trigger LTP are
unknown, but a transient signal lasting only 1 to 3 seconds appears to be sufficient (27 ). Whether additional sources of calcium,
such as release from intracellular stores, are required for the generation of LTP is unclear. It is also uncertain whether additional
factors provide by synaptic activity are required. Various neurotransmitters found in the hippocampus such as acetylcholine and
norepinephrine can modulate the ability to trigger LTP, and such modulation
 P.150

may be of great importance for the functional in vivo roles of LTP. However, there is no compelling evidence to suggest that any
neurotransmitter other than glutamate is required to trigger LTP.

Signal Transduction Mechanisms in LTP

A bewildering array of signal transduction molecules has been suggested to play a role in translating the calcium signal that is
required to trigger LTP into a long-lasting increase in synaptic strength (28 ). However, for only a few of these has compelling
evidence of a mandatory role in LTP been presented. A major limitation of much of the work on this topic is that investigators have
not adequately distinguished molecules that are key components of the signal transduction machinery absolutely required for LTP
from biochemical processes that modulate the ability to generate LTP or play a permissive role. For example, any manipulation that
modifies the activity of NMDA receptors may affect LTP. Therefore, several requirements must be met for a signaling molecule to be
considered a key component of the biochemical machinery that triggers LTP. First, it must be activated or produced by stimuli that
trigger LTP but not by stimuli that fail to do so. Second, inhibition of the pathway in which the molecule participates should block
the generation of LTP. Third, activation of the pathway should lead to LTP.

Strong evidence indicates that calcium/calmodulin–dependent protein kinase II (CaMKII) fulfills these requirements and is a key
component of the molecular machinery for LTP. Inhibiting its activity pharmacologically by directly loading postsynaptic cells with
CaMKII inhibitors or genetic knockout of a critical CaMKII subunit blocks the ability to generate LTP (29 ,30 and 31 ). Conversely,
acutely increasing the postsynaptic concentration of active CaMKII increases synaptic strength and occludes LTP (32 ,33 ).
Furthermore, CaMKII undergoes autophosphorylation after the triggering of LTP (34 ,35 ). That this autophosphorylation is required
for LTP was demonstrated by the finding that genetic replacement of endogenous CaMKII with a form lacking the
autophosphorylation site prevented LTP (36 ).

Several other protein kinases have also been suggested to play roles in the triggering of LTP, but the experimental evidence
supporting their role is considerably weaker than for CaMKII. Activation of the cyclic adenosine monophosphate–dependent protein
kinase (PKA), perhaps by activation of a calmodulin-dependent adenylyl cyclase, has been suggested to boost the activity of CaMKII
indirectly by decreasing competing protein phosphatase activity (37 ,38 ). This presumably happens by phosphorylation of inhibitor-1,
an endogenous protein phosphatase inhibitor (see section on LTD later). Protein kinase C may play a role analogous to CaMKII,
whereas the tyrosine kinases Fyn and Src may indirectly modulate LTP by affecting NMDA receptor function (see 23 for references).
The mitogen-activated protein kinase (MAPK) has also been suggested to be important for LTP, albeit in unknown ways.

Expression Mechanisms and LTP

In the 1990s, tremendous confusion and controversy surrounded the seemingly simple issue of whether LTP is caused primarily by
presynaptic or postsynaptic modifications. The great challenge to answering this question largely stemmed from the great technical
difficulties inherent in examining the changes the occur at individual synapses that are embedded in a complex network in which
each cell receives 10,000 or more synapses. Most neurobiologists studying this question agree that the simplest postsynaptic change
that could cause LTP would be a change in AMPA receptor function or number, whereas the simplest presynaptic change would be an
increase in the probability of neurotransmitter release.

Most studies examining this issue have used electrophysiologic assays, and most of these are inconsistent with the hypothesis that
the release of glutamate increases significantly during LTP (23 ,39 ). For example, changes in transmitter release probability
invariably influence various forms of short-term synaptic plasticity such as paired-pulse facilitation, yet these phenomena are not
affected by LTP. To measure glutamate release more directly, two approaches were used. One took advantage of the finding that
glial cells tightly ensheath synapses and respond to synaptically released glutamate by activation of electrogenic transporters that
generate a current directly proportional to the amount of glutamate released (40 ,41 ). The other took advantage of use-dependent
antagonists of the NMDA receptor or of a mutant AMPA receptor that lacks the GluR2 subunit. These antagonists decrease synaptic
currents at a rate that is directly proportional to the probability of transmitter release (42 ,43 ). LTP had no discernible effect on
these measures, even though they were affected in the predicted fashion by manipulations known to increase transmitter release.

In addition to these negative findings, certain electrophysiologic and biochemical measures were found to increase during LTP. An
increase in the amplitude of miniature electrophysiologic synaptic currents (mEPSCs), which represent the postsynaptic response to
the spontaneous release of a single quantum of neurotransmitter, normally indicates an increase in the number or function of
postsynaptic neurotransmitter receptors. Such an increase occurs during LTP (44 ), as well as after manipulations that load dendritic
spines with calcium (45 ,46 ). A more direct way of monitoring changes in AMPA receptors is to measure the postsynaptic response to
direct application of agonist,
 P.151

and such responses have also been reported to increase, albeit gradually (47 ).

That LTP is caused by a modification of AMPA receptors is supported by the finding that LTP causes an increase in the
phosphorylation of the AMPA receptor subunit GluR1 at the site that is known to be phosphorylated by CaMKII (as well as PKC)
(35 ,48 ,49 ). Using expression systems, this phosphorylation has been shown to increase the single-channel conductance of AMPA
receptors (50 ). Because an increase in single-channel conductance of AMPA receptors has been reported to occur during LTP (51 ),
one mechanism that seems likely to contribute to LTP is CaMKII-dependent phosphorylation of GluR1. Consistent with this idea,
genetic knockout of GluR1 has been found to prevent the generation of LTP (52 ).

Silent Synapses and Quantal Synaptic Transmission

Although the evidence presented thus far makes a strong case for postsynaptic changes contributing to LTP, there remained one
reproducible experimental result that was difficult to reconcile with this idea. This result derived from experiments that took
advantage of the finding that the action potential-dependent release of quanta of neurotransmitter at individual synapses is
probabilistic, and therefore release occurs only 10% to 40% of the time. Therefore, if a single synapse or a very small number of
synapses is activated once every few seconds, on some of the trials no postsynaptic response is recorded, that is, a so-called failure
occurs. An extensively replicated finding is that LTP causes a decrease in the proportion of failures that occur (see 53 for review).
Because these failures were assumed to result from failures of neurotransmitter release, it was concluded that LTP involves an
increase in the probability of transmitter release.

How can this result be reconciled with all the evidence suggesting that LTP is caused by modulation of AMPA receptors and is not
accompanied by an increase in glutamate release? One straightforward idea to explain this apparent discrepancy is the silent
synapse hypothesis (54 ), which predicts that some synapses express only NMDA receptors, whereas others express both AMPA and
NMDA receptors (Fig. 11.2 ). Synapses with only NMDA receptors would be functionally silent at hyperpolarized membrane potentials,
and thus, when transmitter is released, they would not yield a response. However, LTP at such silent synapses could occur by the
rapid expression of AMPA receptors, and such a mechanism would account for the apparent change in failure rate.

FIGURE 11.2. Diagram of the silent synapse hypothesis. A synapse is functionally silent when it expresses NMDA receptors but
not AMPA receptors in its plasma membrane (bottom). The induction of LTP causes the insertion of AMPA receptors (top)
from a putative cytosolic pool. To the right of each diagram are the synaptic currents (i.e., EPSCS) that would be recorded
from the corresponding synapse.

There is now strong evidence to support this model of LTP. First, it is possible to record EPSCs that are mediated solely by NMDA
receptors, and applying an LTP induction protocol at such synapses causes the rapid appearance of AMPA receptor-mediated EPSCs
(55 ,56 ). Second, immunocytochemical analysis demonstrates that AMPA receptors are not found at a significant percentage of
hippocampal synapses, whereas all synapses appear to contain NMDA receptors (see 23 for references). Third, LTP has been shown
to cause the delivery of green fluorescent protein (GFP)-tagged AMPA receptors to dendritic spines and the insertion of recombinant
AMPA receptors into the synaptic plasma membrane (57 ,58 ). Fourth, AMPA and NMDA receptors interact with different proteins at
the synapse (59 ), a finding suggesting that they are regulated independently. Fifth, interference with membrane fusion and
presumably exocytosis in the postsynaptic cells impairs LTP (60 ) and AMPA receptors can interact with proteins involved in
membrane fusion (61 ). These findings are consistent with the idea that membrane fusion may be an important mechanism for the
delivery of AMPA receptors to the synaptic plasma membrane.

Virtually all the data presented thus far are consistent with the simple model that LTP, at least initially, is caused by the
phosphorylation of AMPA receptors and the delivery or clustering of AMPA receptors within the synaptic plasma membrane (23 ).
These events will presumably occur both at synapses that already contain functional AMPA receptors and ones that are functionally
silent. As discussed later, LTD appears to involve the converse process, that is, the removal or endocytosis of AMPA receptors. At the
end of this chapter,
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I discuss how these changes in the number of AMPA receptors at individual synapses may lead to more permanent structural changes,
which, in turn, may mediate long-lasting forms of experience-dependent plasticity.

Long-Term Depression
Like LTP, LTD has been demonstrated in a large number of different brain regions and comes in a variety of different forms (62 ,63
and 64 ). This section focuses on the NMDA receptor-dependent form of LTD found at excitatory synapses on CA1 pyramidal cells and
that appears to result, in large part, from a reversal of the processes that mediate LTP.

Triggering of LTD: A Critical Role for NMDA Receptors and Calcium

LTD is normally generated by prolonged (3- to 15-minute) low-frequency (1- to 5-Hz) afferent stimulation or by a pairing protocol
during which the cell is held at approximately -50 mV. It shares many features with LTP including input specificity, and it can
completely reverse LTP, a process often termed depotentiation. Surprisingly, the triggering of LTD requires NMDA receptor
activation and an increase in postsynaptic calcium concentration (65 ,66 ). This can occur because at resting membrane potentials,
the voltage-dependent block of the NMDA receptor channel by magnesium is not 100% effective, and thus, each stimulus will cause a
very small amount of calcium entry. Current evidence suggests that the specific properties of the intracellular calcium signal dictate
whether LTP or LTD is generated by a specific pattern of synaptic activity, with LTD requiring a modest rise in calcium (67 ) and LTP
requiring a large rise beyond some critical threshold value (68 ). The temporal characteristics of this calcium signal may also be
important.

Signal Transduction Mechanisms in LTD: A Role for Protein Phosphatases

If calcium is the critical triggering signal for LTD, it must be capable of activating biochemical processes that reverse LTP. Because
LTP results, at least in part, from activation of protein kinases, a reasonable hypothesis is that LTD is caused by preferential
activation of protein phosphatases, several of which are known to be found at excitatory synapses (69 ). This idea was first proposed
in a theoretic article (70 ) that presented a specific model that accounted for the bidirectional control of synaptic strength by
calcium (Fig. 11.3 ). It proposed that a balance between CaMKII activity and protein phosphatase 1 (PP1) influenced synaptic
strength by controlling the phosphorylation state of unidentified synaptic phosphoproteins. Small rises in calcium favored activation
of PP1, whereas large rises were required to increase CaMKII activity. Because PP1 is not directly influenced by calcium, a well-
established calcium-dependent phosphatase cascade was invoked to translate the calcium signal into increased PP1 activity (69 ).
This cascade (Fig. 11.3 ) begins with activation of the calcium/calmodulin–dependent phosphatase calcineurin (also known as protein
phosphatase 2B or PP2B). PP2B then dephosphorylates inhibitor 1 (I1), a phosphoprotein that, in its phosphorylated state, is a potent
inhibitor of PP1. Thus, activation
 P.153

of PP2B causes an increase in PP1 activity through a mechanism of disinhibition. An attractive feature of this model is that the
affinity of PP2B for calcium/calmodulin is significantly greater than that of CaMKII. Therefore, PP2B will be preferentially activated
by small increases in synaptic calcium levels. Furthermore, a large rise in calcium will preferentially increase protein kinase activity
not only by directly activating CaMKII but also by leading to the activation of PKA, which phosphorylates I1 and thereby further
inhibits PP1.

FIGURE 11.3. Model for the signaling cascades

responsible for LTD and LTP. LTD is generated
when a low rise in Ca2+ binds to calmodulin (cam)
and activates calcineurin (PP2B). Calcineurin then
dephosphorylates inhibitor 1 (I1), which therefore
no longer inhibits protein phosphatase 1 (PP1).
Active PP1 may act on any number of substrates
including camkii or AMPA receptors (left). LTP is
generated when a high rise in Ca2+ activates camkii.
Other protein kinases that may also be involved in
triggering LTP are protein kinase C (PKC), cAMP-
dependent protein kinase (PKA), the tyrosine
kinase src (Src), and MAP kinase (MAPK)(right).

Several experimental results are consistent with this model, which currently remains the leading hypothesis for the triggering of LTD.
Pharmacologic inhibitors of PP1 or PP2B, when loaded directly into CA1 pyramidal cells, prevent the generation of LTD (71 ,72 ).
Furthermore, loading cells with the phosphorylated form of I1 blocked LTD. However, although the results of such inhibitor studies
are consistent with an important role for protein phosphatases in triggering LTD, other interpretations are possible, and more
experimental work testing this hypothesis needs to be performed. Most notably, if PP1 plays a role in LTD analogous to that played
by CaMKII in LTP, it should be possible to increase PP1 activity directly in postsynaptic cells and to mimic LTD.

Expression Mechanisms of LTD: A Role for Endocytosis of AMPA Receptors

The silent synapse hypothesis discussed earlier suggested that LTP involves the insertion of AMPA receptors into the synaptic plasma
membrane. Consistent with the idea that LTD is a reversal of LTP, there is now considerable evidence that LTD involves removal
(i.e., endocytosis) of synaptic AMPA receptors. The first direct evidence that the synaptic localization of AMPA receptors could be
rapidly modified was the demonstration that, in cultured hippocampal neurons, short application of glutamate receptor agonists
caused rapid a loss of synaptic AMPA receptors with no significant effect on the synaptic localization of NMDA receptors (73 ). This
agonist-induced loss of synaptic AMPA receptors was subsequently shown to result from dynamin-dependent endocytosis (74 ).
Perhaps more important, synaptically triggered LTD in the cultured neurons was accompanied by a decrease in the number of
synaptic surface AMPA receptors with no discernible effect on the distribution of NMDA receptors (75 ). Consistent with these
findings, loading CA1 pyramidal cells with inhibitors of endocytosis prevented the generation of LTD (76 ). These inhibitors also
caused a gradual increase in the size of the synaptic responses, whereas inhibitors of exocytosis caused a gradual decrease (76 ).
These results suggest that there is a pool of AMPA receptors that cycle into and out of the synaptic plasma membrane fairly rapidly
and that LTP and LTD may involve a modification of the kinetics of these processes.

Structural Changes and Long-Term Synaptic Plasticity

How are the changes in synaptic strength that occur following the triggering of LTP or LTD maintained for periods lasting weeks or
perhaps even years? Although the answer to this question is unknown, recent evidence suggests that the mechanisms described
previously may be the initial steps in a more profound anatomic restructuring of synapses, including perhaps the growth of new
synapses and the pruning away of preexisting ones. Dendritic spines, the postsynaptic sites that presynaptic boutons contact, have a
complex ultrastructure and come in a large variety of shapes (77 ). With technical advances in microscopy and the use of
recombinant fluorescent proteins such as GFP, it has become possible to image individual spines in living neurons. Such experiments
have shown that spines are not static but can undergo rapid shape changes (78 ,79 ) that are influenced by activity (80 ).
Furthermore, strong synaptic activation of the type that triggers LTP causes an NMDA receptor-dependent growth of spines as well
as filopodia, which may be the precursors of spines (81 ,82 ). Prolonged application of NMDA to cultured neurons also can cause the
loss of spines (83 ), a finding indicating that, like synaptic strength, the growth and loss of dendritic spines may be under the control
of NMDA receptors.

Changes in synapse structure in response to activity also have been extensively explored using more standard electron microscopic
techniques. One specific morphologic modification repeatedly associated with increased neuronal activity involves a reorganization
of the postsynaptic density (PSD), the electron-dense thickening that contains synaptic glutamate receptors. Specifically, it has been
suggested that LTP is associated with an increase in the fraction of synapses that contain discontinuities in their PSDs, termed
perforated synapses (see 84 for references). This idea is strongly supported by studies in which the synapses activated by strong
tetanic stimulation were identified in electron microscopic sections and were found to have larger total PSD surface areas and a
larger proportion of perforated synapses (85 ,86 ). Several lines of evidence suggest that this growth of PSDs and their eventual
perforation may be initiated by increasing the number of AMPA receptors in the PSD (see 84 for references).

The insertion of new AMPA receptors in the PSD and the generation of perforated synapses may also be early events in the
generation of new synapses by a process of splitting or duplication of existing spines (87 ,88 ). Consistent with this hypothesis, LTP
may be associated with an increase in spine density (84 ), as well as the frequency of multiple-spine synapses in which two adjacent
spines arising from the same dendrite contact a single presynaptic bouton (86 ).
 P.154

These types of observations have led to a model (Fig. 11.4 ) (84 ) that proposes a sequence of events by which the insertion of AMPA
receptors leads to a growth and perforation of the PSD and eventually to multiple-spine synapses. Subsequently, retrograde
communication perhaps involving cell adhesion molecules would cause appropriate presynaptic structural chances such that a
completely new, independent synapse is formed. An attractive corollary to this hypothesis is that LTD involves a shrinkage of the
PSD and eventually leads to a complete loss of the dendritic spine and its corresponding presynaptic bouton. However, minimal work
on the structural changes associated with LTD has been performed.

FIGURE 11.4. Model for sequence of events leading to structural changes following triggering of LTP. Within 10 minutes of LTP
induction, AMPA receptors are phosphorylated and inserted into the postsynaptic membrane. This process leads to an increase
in the size of the postsynaptic density (PSD) and the production of perforated synapses within 30 minutes. By 1 hour, some
perforated synapses split and form multispine synapses. Eventually, retrograde communication, perhaps involving cell-adhesion
molecules, leads to presynaptic structural changes and the production of new synapses.

An attractive feature of incorporating structural changes into the mechanisms of long-term synaptic plasticity is that it provides a
straightforward means by which the activity generated by experience can cause very long-lasting modifications of neural circuitry.
Structural changes also may explain the well-known requirement of long-lasting forms of synaptic plasticity for new protein synthesis
and gene transcription (see 89 ,90 and 91 for reviews).

CONCLUSIONS
Part of "11 - Synaptic Plasticity "

This is a brief review of some of the most common forms of synaptic plasticity found at excitatory synapses throughout the
mammalian brain. Although relatively little is known about the functional roles of these phenomena, such changes in synaptic
function and structure remain the leading candidates for some of the fundamental mechanisms by which experiences of any type
cause the reorganization of neural circuitry and thereby modify thoughts, feelings, and behavior. One hopes that it is also apparent
why understanding the mechanisms of synaptic plasticity has important implications for many branches of clinical neuroscience. For
example, the development of many pathologic behaviors, such as drug addiction, likely depends on the maladaptive use of neural
mechanisms that normally are used for adaptive learning and memory (92 ). Similarly, the recovery of function following brain injury
or the successful pharmacologic and behavioral treatment of mental illness also certainly must result from the reorganization of
neural circuitry that is in part achieved by synaptic plasticity mechanisms. Thus further elucidation of the mechanisms of
phenomena such as LTP and LTD will continue to have implications for all those interested in the neural basis of cognition and
behavior.

ACKNOWLEDGMENTS
Part of "11 - Synaptic Plasticity "

I am currently supported by grants from the National Institute of Mental Health, the National Institute of Drug Abuse, and the
National Institute on Aging, and I also acknowledge past support from the National Alliance for Research on Schizophrenia and
Depression, the McKnight Endowment Fund for Neuroscience, and the Human Frontier Science Program. Thanks to Roger Nicoll,
Steven Siegelbaum, Christian Lüscher and Dominique Muller,
 P.155

with whom I have written previous reviews that provided much of the material for the present chapter.

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12
Gaba
Richard W. Olsen

Richard W. Olsen: Department of Molecular and Medical Pharmacology, University of California Los Angeles School of Medicine, Los Angeles, California.

 GABA IS THE MAJOR INHIBITORY NEUROTRANSMITTER IN THE NERVOUS SYSTEM

 PHYSIOLOGY AND PHARMACOLOGY OF GABAA, GABAB, AND GABAC RECEPTORS
 STRUCTURE AND FUNCTION OF GABAA RECEPTORS
 GABAA RECEPTORS ARE THE SITES OF ACTION OF BENZODIAZEPINES AND BARBITURATES
 GABAA RECEPTORS ARE THE TARGETS OF ALCOHOL, GENERAL ANESTHETICS, AND NEUROSTEROIDS
 GABAA RECEPTORS ARE THE TARGETS OF MANY CNS EXCITANTS
 GENETIC ENGINEERING AND PSYCHOPHARMACOLOGY
 ACKNOWLEDGMENTS

GABA IS THE MAJOR INHIBITORY NEUROTRANSMITTER IN THE

NERVOUS SYSTEM
Part of "12 - Gaba "

Several amino acids are found in high concentrations in brain, and some have been established as neurotransmitters. L-Glutamic acid (glutamate) is the major neurotransmitter for fast
excitatory synaptic transmission, whereas γ-aminobutyric acid (GABA) is the major neurotransmitter for fast inhibitory synaptic transmission. Glycine is a secondary rapid inhibitory
neurotransmitter, especially in the spinal cord (1 ,2 ). Because of the widespread presence and utilization of glutamate and GABA as transmitters, one could say that they are involved
in all functions of the central nervous system (CNS), as well as in all diseases. At any point in the CNS, one is either at a cell that uses or responds to glutamate and GABA or no more
than one cell removed. Many clinical conditions including psychiatric disorders appear to involve an imbalance in excitation and inhibition, and therapeutics thus involve attempts to
restore the balance. The GABA system is the target of a wide range of drugs active on the CNS, including anxiolytics, sedative-hypnotics, general anesthetics, and anticonvulsants (3 ).
See the chapters on GABA in previous editions of this book (1 ,4 ).

Since its discovery in the CNS in the early 1950s (5 ,6 ), GABA was shown to fulfill the criteria for establishment as a neurotransmitter (Fig. 12.1 ). It is synthesized by a specific enzyme,
L-glutamic acid decarboxylase (GAD), in one step from L-glutamate. Thus, in addition to its role in protein synthesis, in cofactors such as folic acid and in hormones such as
thyrotropin-releasing hormone, and its action as a neurotransmitter itself, glutamate must be available in certain nerve endings for biosynthesis of GABA. Much of the glutamate and
GABA used as neurotransmitter is derived from glial storage pools of glutamine (2 ,6 ). Two genes for GAD have been cloned, and the two forms of the enzyme are proposed to differ in
their affinity for the cofactor pyridoxal phosphate and the subcellular localization (7 ). GABA was shown to be released from electrically stimulated inhibitory nerve cells (8 ), and a
mechanism of rapid removal from the synaptic release site was demonstrated by identification of high-affinity transporter proteins (9 ,10 ). The application of GABA and structural
analogues to cells innervated by GABAergic neurons produces effects on that target cell identical to those produced by stimulating the inhibitory innervation (11 ).

FIGURE 12.1. Schematic GABA synapse. Diagram showing the main features of the GABA synapse.
Transporters are indicated by oval symbols, receptors and ion channels by rectangular symbols. A:
Transporters: GAT-1, GAT-3, plasma membrane GABA transporters; VGAT, vesicular GABA transporter. B:
Receptors: GABA-A, ionotropic GABA receptor; GABA-B, G-protein–coupled GABA receptor; KAINATE,
presynaptic kainate receptor; MGLUR, metabotropic glutamate receptor. C: Ion channels: GIRK2, G-
protein–coupled inwardly rectifying K+ channel; VDCC: voltage-dependent calcium channel. D: Enzymes:
GABA-T, GABA transaminase; GAD, glutamic acid decarboxylase; GS, glutamate synthetase. (Courtesy of
O.P. Ottersen; design G. Lothe.)

PHYSIOLOGY AND PHARMACOLOGY OF GABA , GABA , AND GABA A B C

RECEPTORS
Part of "12 - Gaba "

GABA-mediated synaptic inhibition involves rapid, less than 100-millisecond, inhibitory postsynaptic potentials and slower, more than 100-millisecond, inhibitory postsynaptic
potentials. The former were shown by voltage clamp to involve increased chloride ion permeability and to be blocked by the plant convulsant drug picrotoxin, as seen with GABA action
in invertebrates, such as crayfish muscle and nerve preparations (12 ). The rapid chloride current defined a physiologic receptor mechanism termed the GABAAreceptor, also
pharmacologically defined by the antagonist bicuculline, as well as picrotoxin, and the agonist muscimol (Fig. 12.2 ). Thus, the GABAA receptor is a chloride channel regulated by GABA
binding, and it is now grouped in the superfamily of ligand-gated ion channel receptors, which includes the well-characterized nicotinic acetylcholine receptor, present at the skeletal
neuromuscular junction (13 ,14 ).

FIGURE 12.2. Chemical structures of GABAA receptor drugs.

Chloride channel gating is generally inhibitory on a neuron by virtue of stabilizing the membrane potential near the resting level. However, under conditions of high intracellular
chloride, for example, in immature neurons with low capacity to maintain a chloride gradient, increasing chloride permeability can depolarize the membrane potential. This
depolarization could be sufficient to fire the cell, and it would be likely to activate certain voltage-gated ion channels, including calcium, that can, in turn, regulate other cellular
events. Variable permeability to bicarbonate ions for some subtypes of GABAA receptor (GABAR) could
 P.160

also play a role in depolarization (15 ). Such depolarizing GABAR action has been proposed as an important excitatory system in
developing brain (16 ), and it may explain the well-known trophic action of GABA to promote both survival and differentiation during
development (17 ).

The slow inhibitory polysynaptic potentials were shown to be insensitive to GABAA drugs such as bicuculline, but to be activated by
β-chlorophenyl GABA (the antispastic drug baclofen) and to be mediated by a G-protein–coupled receptor that increases potassium
conductance (18 ), now called the GABAB receptor. A further inhibitory GABA response was observed in some cells to be “non-A, non-
B,” neither bicuculline nor baclofen sensitive and sometimes called GABAC (19 ), and generally sensitive to the GABA analogue cis-
aminocrotonic acid. GABAC–type inhibition was shown to involve a rapid chloride conductance, as with GABAA receptors; however, it
was not only insensitive to bicuculline, but also not modified by other GABAA drugs, such as benzodiazepines and anesthetics (19 ).
The eventual cloning of a retinal-specific subunit cDNA ρ that produced bicuculline-insensitive GABA chloride channels appeared to
account for GABAC receptors (20 ). However, because of the structural and functional homology with GABAA receptors, the
International Union of Pharmacology subcommittee on nomenclature recommended that these ρ receptors not be called GABAC
receptors, but rather a subtype of pharmacologically unique GABAA receptors (21 ).

GABAB receptors were shown to mediate presynaptic inhibition on some nerve endings and postsynaptic inhibition on some cell
bodies or dendrites. The coupling mechanism depends on the cell location, because several G-protein–coupled effectors can be used,
involving negative modulation of adenylate kinase and negative modulation of inositol tris phosphate production. These lead to
activation of potassium channels or inhibition of voltage-gated calcium channels (22 ). Presynaptic inhibition of GABA release
 P.161

by GABA involves GABAB autoreceptors, and their activation would be overall excitatory, as opposed to inhibition of glutamate
release, which would be overall inhibitory. Considerable effort was therefore expended to determine whether different GABAB
receptors could mediate these very different functions, possibly allowing the development of receptor subtype-specific drugs.
Although some classic pharmacology studies supported this hypothesis (18, 22), it was the long-awaited cloning of the GABAB
receptor (23 ) that established the true situation. The first receptor exists as two splice variants, and additional clones for GABAB
receptor subtype genes have been isolated. Surprisingly, the GABAB receptors appear to exist as heterodimers, previously unknown
for G-protein–coupled receptors. The dimers produce the diverse pharmacologic specificity for the GABA site and the diverse
coupling mechanisms observed in nature (24 ). It seems that the pharmacology of GABAB receptors is in a very promising infancy.

STRUCTURE AND FUNCTION OF GABA RECEPTORS A

Part of "12 - Gaba "

The GABARs are the major players in CNS function and relevance to psychopharmacology. These receptors, defined by
pharmacologists using electrophysiologic and other techniques (14 ,22 ), were identified in brain homogenates by radioligand binding
(25 ), and are shown to have the correct specificity for GABA analogues expected from the neuropharmacology (26 ,27 ). The GABAR
protein (Fig. 12.3 ) also contains binding sites for benzodiazepines, picrotoxin, barbiturates, and other anesthetics, all of which
allosterically interact with each other (28 ). One or more polypeptides of 45 to 60 kd on sodium dodecylsulfate–polyacrylamide gel
 P.162

electrophoresis were identified in brain homogenates as constituents of the GABAR by photoaffinity labeling with the radioactive
benzodiazepine flunitrazepam (29 ,30 ), and monoclonal antibodies were developed to the partially purified bovine receptor, which
recognized the photolabeled peptides using Western blotting (31 ).

FIGURE 12.3. Schematic GABAa receptor structure. The chloride channel is shown as a
pore in the center of five equivalent subunits, each with four membrane-spanning
domains (see the isolated subunit at the bottom). Because of the existence of subunit
families, many such heteropentamer combinations are possible, each with multiple
drug sites. Ligand sites: GABA: agonists (muscimol), antagonists (bicuculline);
Benzodiazepine: agonists (flunitrazepam), antagonists (flumazenil), inverse agonists
(DMCM); Picrotoxin/Convulsant (TBPS); Barbituate (phenobarbitol); Steroid
(alphaxalone, allopregnanolone); Volatile Anesthetic (halothane). (Modified from
Olsen RW, Tobin AJ. Molecular biology of GABAA receptors. FASEB J 1990;4:1469–1480,
with permission.)

The GABAR proteins were purified using benzodiazepine affinity chromatography (32 ), which allowed partial protein sequencing and
expression cloning of two receptor genes (13 ). GABA-activated currents were demonstrated in Xenopus oocytes using cDNAs for two
polypeptides that contained the partial sequences within their coded sequence, and these were designated α and β. At first, these
were thought (incorrectly) to correspond to the two bands seen in the purified protein (32 ). These two subunits were related to
each other and also to the nicotinic acetylcholine receptor family of subunits, a finding indicating a superfamily of receptor
polypeptide genes and a likely heteropentameric structure (Fig. 12.3 ) (13 ,14 ). These two cDNAs were used as probes to clone
additional family members with more or less sequence homology to the first two. Those with high homology were named with the
same Greek letter, whereas those with less homology were given other Greek letters. The current repertoire involves α1 to 6, β1 to
3, γ1 to 3, δ, ε, θ, π, and ρ1 to 3 (21 ). There are also a few splice variants; for example, γ2 exists in two forms differing in an
eight-amino acid insert in the intracellular loop that includes a substrate serine for protein kinase C (33 ). All the subunits are
related to each other and have molecular weights of about 50 kd. The purified receptor protein thus actually contains about a dozen
subunit polypeptides, of varying amount (6 ). Hydropathy plots show that they have a long extracellular N-terminal domain, which
has glycosylation sites and is believed to carry the GABA binding site. They have four membrane-spanning domains (M1 to M4) of
about 25 residues each, a long intracellular loop between M3 and M4, and a short extracellular C-terminal tail. These subunits are
arranged as heteropentamers (Fig. 12.3 ), several of which are common in nature, but whose expression varies with both age and
brain region. The different receptor subtypes have biological differences, such as location, affinity for GABA, and channel properties,
as well as pharmacologic heterogeneity. Most receptors contain two copies of one type of α subunit, two copies of one type of β
subunit, and a γ subunit. Rarely, another subunit (δ, ε, θ) can substitute for γ (30 ,33 ). The presence of a γ subunit is needed for
benzodiazepine sensitivity, and other subunits affect the detailed specificity. For example, the α subunits define the
benzodiazepine pharmacology, and some subunits α4 and α6 do not bind classic benzodiazepine agonists; the detailed pharmacology
depends on the small differences in polypeptide sequence for the various subunits (6 ,34 ,35 and 36 ). Because of the unique
location of receptor subtypes, and thus unique functions of the circuits involved, great hope for new drugs of improved
pharmacologic profile has been expressed. The GABAR strategy has certainly not been exhausted.

GABA RECEPTORS ARE THE SITES OF ACTION OF BENZODIAZEPINES

A

AND BARBITURATES
Part of "12 - Gaba "
The actions of several classes of CNS depressant drugs had for some time been suggested to involve enhancement of inhibitory
synaptic transmission. In particular, the anxiolytic effects of benzodiazepines were shown probably to result from potentiation of
GABA action (37 ,38 ). When the benzodiazepine receptors were discovered using radioligand binding to brain homogenates
(1 ,4 ,39 ,40 ), it was quickly determined that the benzodiazepine binding sites were physically present on the GABAA receptor–
chloride channel complex (28 ,41 ). The various types of drug binding site on the GABAA receptor allosterically interact with each
other in the test tube. Barbiturates and related sedatives also enhance GABAA receptor–mediated inhibition, and their pharmacologic
spectrum overlaps with that of the benzodiazepines and related substances, such as zolpidem, zopiclone, and abecarnil (Fig. 12.4 ).
The selective actions of benzodiazepines not shown by barbiturates or vice versa are believed to arise from heterogeneity in GABA
receptor sensitivity to the drugs, and corresponding heterogeneity in brain regions, circuits, and functions. Further, some GABARs
are insensitive to benzodiazepines but not to barbiturates, as well as additional nonoverlapping, nonGABA actions of high doses,
especially barbiturates. In addition, the two classes of drugs have a different mechanism of action at the molecular channel level;
barbiturates prolong the lifetime of GABA currents, in addition to gating channels directly at high concentrations, whereas
benzodiazepines increase the frequency of opening of GABAR channels and do not directly open channels in the absence of GABA
(3 ,42 ).

FIGURE 12.4. Chemical structures of drugs active at the

benzodiazepine site on the GABAA receptor–chloride channel
complex.
 P.163

The classical benzodiazepines such as diazepam (Valium) have had a tremendous history in psychopharmacology, reaching
tremendous sales, primarily for clinical anxiety (38 ,43 ,44 and 45 ). Other uses of benzodiazepines include sedation, muscle
relaxation, and a significant utilization for treatment of panic (1 ,45 ). Various structural analogues were developed by numerous
firms, with slight variations in pharmacokinetics and other details, and quite a few nonbenzodiazepine structures were discovered
that act at the benzodiazepine site on the GABAR to enhance GABA-mediated inhibition (Fig. 12.4 ) (46 ). This group includes
compounds called β-carbolines, some of which were isolated from biological tissues (47 ). However, neither the β-carbolines nor any
peptides have been demonstrated to act as biological ligands at benzodiazepine receptor sites (45 ). Surprisingly, some β-carbolines,
and indeed, benzodiazepines and other types of chemical structures active on the benzodiazepine site, were found to have the
opposite pharmacologic efficacy as classic benzodiazepine ligands such as diazepam; that is, they are anxiogenic and proconvulsant
in animals and inhibit GABAR function in cells, while binding to the same sites as agonist benzodiazepine site ligands. These
compounds were given the name inverse agonists (48 ,49 ).

Given this spectrum of efficacy, it would be expected that compounds with true antagonist efficacy would exist, and these were
found, for example, Ro15-1788, or flumazenil (50 ). This compound does not affect GABAR function on its own, but it blocks the
actions of both agonists to enhance GABAR function and inverse agonists to inhibit GABAR function. In animals, it also reverses the
pharmacologic actions of both agonists and inverse agonists (50 ). Thus, an antagonist can be used to treat overdose of agonist, or
inverse agonist, and it triggers withdrawal in individuals treated on a long-term basis with agonists (45 ,51 ). Flumazenil
administration to rats after long-term administration of diazepam was found to reverse tolerance rapidly and permanently, and
treated animals showed no long-lasting effects but resembled treatment-naive animals (52 ,53 ). This finding suggested that
benzodiazepine antagonists may be useful in reversing benzodiazepine dependence and also potentially for other GABAR drugs, such
as ethanol. This has not proved effective so far, however (43 ,45 ).

Certain benzodiazepines have considerable success in the treatment of some types of epilepsy (38 ). Every emergency medical cart
contains injectable benzodiazepine (diazepam, clonazepam, lorazepam) for convulsions and status epilepticus. However, long-term
therapy of epilepsy with benzodiazepines is often prevented by the development of tolerance to the anticonvulsant actions, without
change in blood levels (54 ). The development of tolerance to long-term administration of benzodiazepines, and also of withdrawal
signs (43 ,45 ,55 ), is consistent with the development of psychological and physical dependence with these drugs. The potential for
abuse with CNS depressant drugs in general and benzodiazepines in particular is well known, as is the interaction with ethanol. This
has led to a considerable drop in prescriptions of these agents for routine anxiety. Because the danger of fatal overdose with
benzodiazepines is lower than that of ethanol and barbiturates, and because withdrawal symptoms are less dangerous for
benzodiazepines than for alcohol, benzodiazepines reached considerable popularity in treatment of alcoholism. However, the two
drugs show cross-tolerance and cross-dependence, so substitution of benzodiazepines for ethanol is merely substituting one
addiction for another (55 ).

Conversely, an interesting observation was made with the benzodiazepine partial inverse agonist Ro15-4513. This compound was
found to antagonize the behavioral effects of ethanol (49 ), as well as the in vitro action of ethanol to enhance GABAR function (56 ).
(Ethanol and GABA are discussed further later.) Moreover, the action of Ro15-4513 to antagonize ethanol occurred under conditions
of assay, such as behavior, tissue, or species, in which Ro15-4513 itself did not exhibit inverse agonist activity or inhibit GABAR
function, nor did it reverse the actions of pentobarbital (56 ,57 ,58 and 59 ). Thus, this compound or one like it had potential as an
“alcohol antidote” in humans, by reducing intoxication and perhaps withdrawal and craving. Unfortunately, the ethical decisions
involved in prescribing such a drug were made moot by discovery that Ro15-4513 was tremorigenic and proconvulsant in nonhuman
primates, as well as other animals (60 ).

Understanding the mechanism of tolerance development has been a research topic of high interest, especially for epilepsy
treatment, but also because of the relevance to brain plasticity. Whereas long-term administration of benzodiazepines may produce
tolerance in part by down-regulation of receptor levels, considerable evidence suggests that receptors are not removed, but rather
are altered in some way to produce tolerance (61 ,62 ,63 and 64 ). Besides tolerance development to long-term use of agonist
benzodiazepines, sensitization to the actions of inverse agonists is observed; that is, excitatory benzodiazepine receptor ligands
become more efficacious (65 ). This may resemble the kindling process seen with long-term administration of inverse agonists; that
is, repeated administration of nonconvulsant doses of inverse agonists eventually leads to convulsions to that dose. This resembles
the electrical kindling model of epilepsy, in which repeated electrical stimuli with nonconvulsive amplitude eventually evoke a
seizure (66 ). Thus, long-term administration of benzodiazepine agonists or inverse agonists may shift the set point of the GABAR
toward the excitatory or lower functional end of the spectrum (65 ,67 ). Dependence on benzodiazepines and alcohol resulting from
long-term administration (abuse) may be exacerbated by a kindling-like development of increased severity of withdrawal symptoms,
with an increased risk of relapse (68 ). Another aspect of the tolerance model is the possibility of replacing one type of GABAR
subunit with another that still responds to GABA
 P.164

but not to the chronically administered modulatory drug (69 ,70 ).

GABA RECEPTORS ARE THE TARGETS OF ALCOHOL, GENERAL

A

ANESTHETICS, AND NEUROSTEROIDS

Part of "12 - Gaba "

Alcohols are CNS depressants with a pharmacologic spectrum of action overlapping those of the benzodiazepines and barbiturates,
known to act by enhancement of GABAR. Long-chain alcohols have anesthetic activity, as does ethanol at high doses (greater than
100 mM), whereas the intoxicating effects at lower concentrations (10 to 100 mM) have been suggested to involve blockade of N-
methyl-D-aspartate (NMDA)–type glutamate receptors (71 ) or enhancement of GABAR (72 ,73 and 74 ). Because the latter effect
varies considerably among, for example, laboratories, preparations, assays, and brain regions, unique ethanol-sensitive subtypes of
GABAR were suggested, but they have not been established. Alternatively, and most popular currently, is the hypothesis that
ethanol acts on GABAR indirectly to produce important aspects of its pharmacologic actions in cells and in animals (75 ). For
example, ethanol may interact with membrane signaling proteins that regulate GABAR and NMDA receptors.

GABAA receptor function appears to be modulated by an endogenous substance: not a benzodiazepine-like or a picrotoxin-like
peptide, but a barbiturate-like steroid. The neurosteroids are endogenous steroid hormone metabolites that have direct and rapid
actions on cells not involving steroid hormone receptors or regulation of gene expression. Progesterone was shown to produce rapid
sedative activity, a finding that led to the development of the clinical intravenous steroid anesthetic, alphaxalone. Progesterone has
anxiolytic and anticonvulsant activity; discontinuation after long-term administration leads to withdrawal signs that are clearly CNS
mediated: these actions are mediated by the progesterone metabolite, produced primarily in the adrenals but to some extent in
brain, 3α-hydroxy-5-α-pregnane-20-one (76 ,77 and 78 ). The neuroactive steroids act principally by binding directly to membrane
GABAA receptors and enhancing their function in a manner resembling the barbiturates (79 ,80 ).

Many related steroid compounds have been developed as lead compounds for potential use as antiepileptics, anxiolytics, and
sedative-hypnotics (81 ). Whether these compounds are biologically relevant is uncertain, but this is suggested by considerable
evidence. Endogenous steroids reach levels sufficient to modulate GABAA receptors during conditions of stress and anxiety, and
during pregnancy (82 ,83 ). These compounds are probably involved in CNS plasticity responses to chronic stress and possibly
epileptogenesis, and even drug dependence (84 ,85 ). The progesterone metabolite is the endogenous steroid that appears to be the
most likely to be biologically relevant, but metabolites of testosterone and cortisone are also active (77 ,81 ). Pregnenolone sulfate,
a biosynthetic intermediate in the synthesis of all the steroid hormones, present in high levels in the CNS, has weak activity as an
antagonist of GABA function, but this appears to involve another mechanism and is unlikely to be biological (85 ). Neurosteroid
action apparently has relevance to alcohol action. GABA-active steroids can substitute for ethanol in discriminative stimulus testing
in rats and monkeys, and neurosteroids are synthesized in brain in response to ethanol administration and may mediate some of the
pharmacologic actions (86 ). The neurosteroid-GABA connection potentially may be fruitful for new applications in
psychopharmacology. As the endogenous functions of neurosteroids in stress control, seizure protection, attention and learning, and
possibly even sleep, become better delineated, additional therapeutic approaches may arise.

Enhancement of GABAA receptor-mediated inhibition is currently the major candidate molecular mechanism for a generalized theory
of general anesthesia. Everyone agrees that the anesthetic action of the steroid alphaxalone occurs by enhancement of GABAR
(84 ,85 ), and many investigators believe that the actions of high-dose ethanol and other alcohols as anesthetics probably do also
(75 ,87 ). Further, the sedative-hypnotic effects, and possibly anesthetic effects, of barbiturates and related drugs are considered to
act through GABAR (88 ). Anesthetics are now believed to have a greater effect on membrane ion channels than on many other
biological systems and to affect synaptic transmission more potently than nerve conduction. Ligand-gated ion channels, especially
receptors for glutamate, glycine, and GABA, are most sensitive (89 ). All general anesthetics enhance GABA function at anesthetic
concentrations (36 ,75 ). The ketamine-phencyclidine category of dissociative anesthetics enhances some GABA synapses, but these
agents probably inhibit NMDA receptors more potently; further, they produce a different sort of anesthesia (90 ).

The Meyer-Overton hypothesis shows a high correlation for many drugs with respect to potency as a general anesthetic and partition
in an oil-water biphasic system. The Meyer-Overton correlation has been found wanting, because of the existence of compounds
with identical lipid solubility (oil-water partition coefficient), boiling point, and dipole moment, such as halogenated cyclobutane
isomers, that differ in anesthetic potency: only the anesthetic isomers enhance GABAA receptors (91 ). Volatile anesthetics and
alcohols (87 ), as well as intravenous agents such as barbiturates, propofol, neuroactive steroids, and etomidate, are all able at
anesthetic concentrations to modulate GABAA receptor binding assays in vitro as well as to enhance GABAA receptor function in cells
(36 ,88 ).

GABA RECEPTORS ARE THE TARGETS OF MANY CNS EXCITANTS

A

Part of "12 - Gaba "

 P.165

Many naturally occurring and synthetic convulsive agents are blockers of GABA-mediated inhibition (46 ). The prototypic GABAA
channel blocker picrotoxinin (Fig. 12.2 ) is isolated from plants of the moonseed family, Menispermaceae, and its close relatives
tutin and coriamyrtin, from the New Zealand tutu plant Coriaria arborea (92 ), known as a loco weed, which causes occasional
poisonings in cows and even in people. A major category of synthetic potent neurotoxic chemicals (93 ), comprising the cage
convulsants, was discovered to consist of noncompetitive GABAA receptor antagonists acting at the picrotoxinin site (93 ,94 and 95 ).
One of these drugs, t-butyl bicylcophosphorothionate (Fig. 12.2 ), is a major research tool used to assay GABA receptors by
radioligand binding (96 ). Synthetic butyrolactones with depressant and excitatory actions have also been described for the
picrotoxinin site (97 ). In addition, this drug target appears to be the site of action of the experimental convulsant pentylenetetrazol
(PTZ) and numerous polychlorinated hydrocarbon insecticides, including dieldrin, α-endosulfan, and lindane (93 ). The
monoterpenoid thujone is the active constituent of oil of wormwood, the major ingredient of the famous green liqueur, absinthe,
outlawed in about 1910. Absinthe was reputed to have hallucinogenic action and to be an inspiration for fin de siècle French artists
and poets (92 ). Oil of wormwood has a history as a medicinal herb for treating intestinal worms and killing insects, and thujone is
known to cause convulsions in high doses; thujone was demonstrated to be a GABAA receptor channel blocker like picrotoxinin (98 ).
It remains anecdotal whether thujone/wormwood/absinthe produces psychic actions additional to those of the ethanol in the
liqueur.

GABA-blocking agents thus have potential pharmacologic utility as excitants. Although at one time listed in the Merck Index and in
pharmacology textbooks as a “barbiturate overdose antidote,” picrotoxin is too dangerous as a convulsant to attempt to find an
appropriate dose in the clinic. PTZ and related agents are known to show anxiogenesis in low doses, but also proabsence seizures.
An alerting, attention-activation mechanism may figure to promote learning and memory in certain tasks, that is, nootropism.
Partial inverse agonists at the benzodiazepine site, such as Ro15-4513, have been considered as candidates for memory
enhancement (38 ,99 ), as well as for actions as antagonists and possible anticraving, antiwithdrawal agents for the treatment of
addiction to benzodiazepines, alcohol, and many other drugs of abuse, as discussed earlier (1 ,60 ,69 ).

GENETIC ENGINEERING AND PSYCHOPHARMACOLOGY

Part of "12 - Gaba "

Gene targeting and transgenic mice have demonstrated several important roles for GABA in the CNS. Knockouts of both GAD67 and
GABAA receptor subunit β3 lead to cleft palate and early neonatal lethality (100 ,101 and 102 ). GAD65 knockout mice show
increased anxiety, increased sensitivity to benzodiazepines, and impaired developmental plasticity in the cortex (103 ,104 ).
Epilepsy results from knockout of GAD65, GABAR β3, and GABAR δ subunit. Other phenotypic deficits include motor incoordination,
movement disorders, cognitive defects, and other CNS circuitry problems resulting from lack of inhibitory synaptic transmission. In
particular, the GABAR β3 subunit is implicated in the human genetic disease Angelman syndrome, associated with mutation in
maternal chromosome 15q and typified by severe mental retardation, epilepsy, motor incoordination, and sleep disorder (105 ).
Mice targeted for this subunit have a phenotype remarkably similar to Angelman syndrome, especially the epilepsy, but also
including the cognitive, motor, and sleep impairment (106 ).

The γ2 subunit knockout shows early neonatal lethality (107 ), without cleft palate, involving impaired clustering of GABAA receptors
at synapses (108 ). Even heterozygotes, with presumably a partial deficit of γ2-containing GABAR, have impaired synapses and
overanxious and paranoid behavior (109 ). Because GABARs are important drug targets, some GABAR subunit knockout mice have
impaired sensitivity to drugs, such as decreased response to benzodiazepines in γ2 homozygous knockouts (107 ). Increased response
to benzodiazepines is seen in γ2 heterozygous knockouts or in γ2L null mutants (109, 110). Reduced sensitivity to anesthetics was
seen in β3 but not α6 knockouts (102 ), and reduced sensitivity to neuroactive steroids is observed in the δ subunit knockout (111 ).
This finding may be interesting in light of the apparent biological role of the neurosteroids in normal CNS. Gene targeting in mice
also has been employed to “knock in” a mutation of the α1 subunit H101N, which prevents benzodiazepine binding to GABAR
containing this subunit (112 ). The resulting animals have greatly impaired sensitivity to the sedative but not the anxiolytic actions
of the benzodiazepines, whereas anticonvulsant activity is partially reduced. This finding indicates that the subtypes of GABAR
containing the α1 subunit and the brain circuits in which they function are the substrates for benzodiazepine-stimulated sedation,
whereas other GABARs, containing α2, α3, and α5, with α2 the most abundant and the major candidate, subserve specifically the
role of GABARs in anxiety pathways sensitive to benzodiazepine therapy. (The observations of Rudolph et al., 1999 (112 ) were
verified by McKernan et al., 2000 (113 ) for the role of the α1 subunit in the sedative actions of benzodiazepines, and extended by
Low et al., 2000 (114 ) for the role of the α2 subunit in the anxiolytic actions of benzodiazepines.) Thus, new biotechnology applied
to drug development is continuing to make new advances in psychopharmacology based on this now relatively “old” or at least well-
known neurotransmitter system, GABA.

ACKNOWLEDGMENTS
Part of "12 - Gaba "
 P.166

This work was supported by National Institutes of Health grant NS35985.

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 P.169

13
Substance P and Related Tachykinins

Nadia M.J. Rupniak

Mark S. Kramer

Nadia M.J. Rupniak: Department of Pharmacology, Merck Sharp & Dohme, Harlow, Essex, United Kingdom.

M.S. Kramer: Department of Clinical Neuroscience, Merck & Co., West Point, PA; University of Pennsylvania, Philadelphia,
Pennsylvania.

Substance P belongs to a family of neuropeptides known as tachykinins that share the common C-terminal sequence: Phe-X-Gly-Leu-
Met-NH2. The three most common tachykinins are substance P, neurokinin A (NKA), and neurokinin B (NKB); their biologic actions
are mediated through specific cell-surface receptors designated NK1, NK2, and NK3, with substance P the preferred agonist for NK1
receptors, NKA for NK2 receptors, and NKB for NK3 receptors.

Preclinical studies with substance P antagonists have been complicated not only by phylogenetic differences in central nervous
system (CNS) localization of tachykinin receptors, but also by species variants in NK1 receptor pharmacology. This situation greatly
complicates preclinical evaluation of selective substance P receptor antagonists because most of these have only low affinity for the
rat receptor, which is the most commonly used preclinical species. Substance P and the NK1 receptor have a widespread distribution
in the brain and are found in brain regions that regulate emotion (e.g., amygdala, periaqueductal gray, hypothalamus). They are
also found in close association with 5-hydroxytryptamine (5-HT) and norepinephrine-containing neurons that are targeted by the
currently used antidepressant drugs.

The effects of substance P antagonists in preclinical assays for analgesic, antiemetic, antipsychotic, anxiolytic, and antidepressant
drugs is reviewed. The process of elucidating the clinical uses of substance P antagonists raises certain fundamental issues that will
apply to other novel neurotransmitter ligands in future. The difficulty of predicting clinical efficacy from preclinical data, and of
testing novel therapeutic drugs in patients with psychiatric disorders, is discussed.

Substance P, NKA, and NKB are related neuropeptides that are widely distributed in the peripheral nervous system and the CNS.
With the development of selective nonpeptide receptor antagonists, it has become possible to investigate the physiologic roles of
these peptides and to explore their use as novel treatments for neurologic and psychiatric disorders. Because the substance P–
preferring NK1 receptor is the predominant tachykinin receptor expressed in the human brain, most compounds that have been
developed for clinical use are substance P–preferring (NK1) receptor antagonists.

 TACHYKININ FAMILY OF PEPTIDES

 SPECIES DIFFERENCES IN THE DISTRIBUTION OF NEUROKININS AND THEIR RECEPTORS IN THE NERVOUS SYSTEM
 PHYLOGENETIC DIFFERENCES IN TACHYKININ RECEPTOR PHARMACOLOGY
 POTENTIAL FOR USE OF TACHYKININ RECEPTOR ANTAGONISTS TO TREAT PSYCHIATRIC AND NEUROLOGIC DISORDERS
 CONCLUSIONS AND IMPLICATIONS FOR FUTURE STUDIES OF NEUROKININ ANTAGONISTS IN PSYCHIATRIC AND NEUROLOGIC
DISORDERS

TACHYKININ FAMILY OF PEPTIDES

Part of "13 - Substance P and Related Tachykinins "

Substance P belongs to a family of neuropeptides known as tachykinins that share the common C-terminal sequence: Phe-X-Gly-Leu-
Met-NH2. Two other mammalian tachykinins are NKA and NKB (Table 13.1 ). Their biologic actions are mediated through specific G-
protein–coupled neurokinin receptors designated NK1, NK2, and NK3, with substance P the preferred agonist for NK1 receptors, NKA
for NK2 receptors, and NKB for NK3 receptors. However, the receptor selectivity of these peptides is relatively poor, and it is possible
that their actions could be mediated by activation of their less preferred receptors. Indeed, this possibility is suggested by the
mismatch between tachykinin-containing neurons and fibers and their corresponding receptor that is seen in certain brain regions.
This is particularly apparent in the case of NKA, because NK2 receptor expression appears to be extremely low in the adult
mammalian brain (1 ).

TABLE 13.1. MAMMALIAN TACHYKININS

Substance P was originally discovered in 1931 by von Euler and Gaddum as a tissue extract that caused intestinal contraction in vitro;
its biologic actions and tissue distribution were further investigated over subsequent decades. NKA (previously known as substance K
or neuromedin L) and NKB (originally known as neuromedin K), were isolated from porcine spinal cord in 1983 and were also found
to stimulate intestinal contraction (2 ). Substance P and NKA are produced from a polyprotein precursor after differential splicing of
a single precursor gene, preprotachykinin
 P.170

A (3 ). NKB is produced from a distinct precursor protein encoded by preprotachykinin B (4 ).

The existence of several neurokinin receptors was originally suggested by the differential contractile responses elicited in various
tissues by mammalian and nonmammalian tachykinins (5 ). Subsequently, specific binding sites labeled by Bolton Hunter substance P,
NKA, and eledoisin were identified in the CNS (6 ), a finding suggesting that at least three receptors mediated the actions of
tachykinins. This was confirmed by cloning of three distinct functional cDNA constructs corresponding to NK1, NK2, and NK3 receptor,
which preferentially bound substance P, NKA, and NKB, respectively (7 ,8 and 9 ). However, the endogenous neurokinins exhibit a
high degree of cross-reactivity with these tachykinin receptors.

SPECIES DIFFERENCES IN THE DISTRIBUTION OF NEUROKININS AND

THEIR RECEPTORS IN THE NERVOUS SYSTEM
Part of "13 - Substance P and Related Tachykinins "

The substance P–preferring NK1 receptor has attracted most interest as a CNS drug target because it is the predominant tachykinin
receptor expressed in the human brain, whereas NK2 and NK3 receptor expression is extremely low or absent (10 ,11 and 12 ).
Therefore, it appears that the central actions of all tachykinins may be mediated predominantly through the NK1 receptor in humans.
However, understanding the role of substance P in the brain has been greatly complicated by marked differences in the distribution
of tachykinin receptor subtypes in rodent species that are normally used for such studies. For example, in the rat and guinea pig
brain, both NK1 and NK3 receptors are expressed (10 ), findings suggesting that the CNS functions mediated by NK1 receptors in the
human brain may be subserved by NK1 and/or NK3 receptors in rodents. NK2 receptors appear to be absent in the adult mammalian
brain of all species examined (10 ). For these reasons, interpretation of the effects of selective tachykinin receptor antagonists in
preclinical assays requires great caution. If such compounds either succeed or fail to exhibit activity in rodent assays for psychiatric
and neurologic disorders, this may merely reflect different roles of tachykinin receptors in rodent versus human brain. Hence there
is a risk of both false-positive and false-negative extrapolations from preclinical species to humans.

Substance P is widely distributed throughout the CNS and in primary sensory neurons. The demonstration of substance P
immunoreactivity in the cell bodies of dorsal root ganglia, in sensory nerve fibers, and in the dorsal horn of the spinal cord led to
early speculation that substance P is involved in pain perception (13 ). Substance P and the NK1 receptor have a widespread
distribution in the brain and are found in brain regions that regulate emotion (e.g., amygdala, periaqueductal gray, hypothalamus)
(14 ,15 ). They are also found in close association with major catecholamine-containing nuclei, including the substantia nigra and
the nucleus tractus solitarius (16 ), as well as with 5-HT- and norepinephrine-containing neurons that are targeted by currently used
antidepressant drugs. NKA and NKB are also expressed in varying ratios in the CNS and spinal cord (17 ,18 ) and in the rodent (but
not human) brain, and NK3 receptors and mRNA have also been demonstrated in various regions, including the substantia nigra,
raphe nuclei, and locus ceruleus (19 ,20 and 21 ).

An interesting aspect of the neuroanatomic localization of substance P is that that it is coexpressed with 5-HT in approximately 50%
of ascending dorsal raphe neurons in the primate brain (22 ,23 ). In contrast, coexpression of substance P and 5-HT in ascending
raphe neurons is not seen in the rat brain (24 ). These findings provide further illustrations of the marked species differences in the
neuroanatomy, and possibly physiology, of neurokinin systems. The functional significance of substance P and 5-HT coexpression in
the human brain is not known, but it suggests that both neurotransmitters may be coreleased in certain brain regions receiving
terminal innervation.

Other evidence suggests that substance P and NKB may also modulate ascending norepinephrine systems. NK1 receptors (25 ) have
been shown to be expressed on tyrosine hydroxylase–positive cell bodies in the rat locus ceruleus, and both substance P and senktide
(a selective NK3 receptor agonist) excite the firing of locus ceruleus neurons in rats and guinea pigs (26 ,27 ).

PHYLOGENETIC DIFFERENCES IN TACHYKININ RECEPTOR

PHARMACOLOGY
Part of "13 - Substance P and Related Tachykinins "

Preclinical studies with NK1 receptor antagonists have also been complicated by species variants in NK1 receptor pharmacology
(28 ,29 ). Compounds such as CP-96,345 were found have high (nM) affinity for the NK1 receptor expressed in human, gerbil, rabbit,
guinea pig, cat, and monkey brain, but they had considerably lower affinity for the mouse and rat NK1 receptor. Subsequent
mutation analysis revealed that subtle differences in the amino acid sequence between the human and the rat NK1 receptor
dramatically alter antagonist binding affinity (30 ). This feature has greatly hindered preclinical evaluation of high-affinity human
NK1 receptor antagonists because most of these have considerably lower affinity for the rat receptor, the most
 P.171

commonly used preclinical species (Table 13.2 ). A few compounds have high affinity for the rat receptor (e.g., SR140333), but their
utility for in vivo studies may be severely limited by poor brain penetration (31 ). Although these difficulties may be overcome by
administering high doses of NK1 receptor antagonists to rats, unspecific pharmacologic effects are then frequently encountered,
mostly attributable to ion channel blockade. It has therefore been necessary to examine the preclinical pharmacology of these
compounds in species with humanlike NK1 receptor pharmacology (gerbils, guinea pigs, ferrets, hamsters) whenever possible.
Pharmacologic differences among human, guinea pig, and rat NK3 receptors also exist (32 ).

TABLE 13.2. SPECIES VARIANTS

IN NK, RECEPTOR
PHARMACOLOGY

POTENTIAL FOR USE OF TACHYKININ RECEPTOR ANTAGONISTS TO

TREAT PSYCHIATRIC AND NEUROLOGIC DISORDERS
Part of "13 - Substance P and Related Tachykinins "

The distribution of neurokinins in the central and peripheral nervous system has generated much speculation about the potential
therapeutic uses of selective tachykinin receptor antagonists. The major hypotheses that are supported by preclinical data and have
been investigated in clinical trials are considered here. Numerous clinical trials have now been conducted with NK1 receptor
antagonists to define their therapeutic potential in psychiatric and neurologic disorders. In all these studies, the compounds have
been extremely well tolerated, with no significant side effects. There are as yet no reports of clinical trials with NK2 or NK3 receptor
antagonists in patients with CNS disorders.

Pain
Radioligand-binding studies confirm the expression of tachykinin NK1 and NK3 (but not NK2) receptors in the dorsal horn of the spinal
cord (33 ,34 and 35 ). A role of spinal substance P and NKA in nociception is suggested by the reduction in response thresholds to
noxious stimuli by central administration of NK1 and NK2 (but not NK3) agonists (36 ,37 and 38 ). Based on these neuroanatomic and
functional studies, it was anticipated that NK1, and possibly NK2, receptor antagonists could be developed as analgesic drugs.

Electrophysiologic studies on anesthetized or decerebrate animals provide evidence of potent and selective inhibition of facilitated
nociceptive spinal reflexes by NK1 receptor antagonists. Responses of dorsal horn neurons to noxious or repetitive electrical
stimulation of a peripheral nerve was blocked by CP-96,345 (39 ); NK1 receptor antagonists also blocked the flexor reflex facilitation
produced by C-fiber–conditioning stimulation, but they did not affect protective nociceptive reflexes (40 ,41 ). NK1 receptor
antagonists have also been shown to inhibit the late-phase response to formalin in gerbils (42 ), to inhibit carrageenan and Freund
adjuvant–induced hyperalgesia in guinea pigs (J. Webb, S. Boyce, and N. Rupniak, unpublished observations; 43), and to attenuate
peripheral neuropathy in rats and guinea pigs (43 ,44 ). Overall, the profile of activity of NK1 receptor antagonists in a range of
assays is comparable to that seen with clinically used analgesic agents such as indomethacin (Table 13.3 ).

TABLE 13.3. PRECLINICAL

EVIDENCE OF AN ANALGESIC
PROFILE OF NK1 RECEPTOR
ANTAGONISTS

The first clinical trials with NK1 receptor antagonists were conducted in patients with various pain conditions. These trials uniformly
failed to confirm the analgesic efficacy of these compounds in humans and are reviewed in detail elsewhere (45 ,46 ). The patient
populations and compounds examined included the following: peripheral neuropathy, in which CP-99,994 had no analgesic effect
(47 ); molar extraction, in which MK-869 was ineffective (48 ); and postherpetic neuralgia, in which MK-869 was ineffective (49 ).
Further unpublished studies with other compounds support these conclusions. Thus, clinical studies to date indicate that NK1
receptor antagonists do not have major potential as analgesics.

Less is known about the profile of NK2 receptor antagonists in nociception assays. The NK2 antagonist MEN 10207 completely blocked
both facilitation and protective nociceptive reflex responses (40 ), and SR48968 reduced responses to both noxious and innocuous
pressure applied to
 P.172

the knee joint (50 ). In conscious rats, Sluka et al. found that pretreatment with SR48968 prevented the induction of hyperalgesia
induced by intraarticular injection of kaolin and carrageenan (51 ), but it was not effective after hyperalgesia had been established.

Migraine
The vasculature of meningeal tissues such as the dura mater is densely innervated by nociceptive sensory afferents that run in the
trigeminal nerve and contain substance P and other neuropeptides. The release of neuropeptides from these sensory fibers during a
migraine attack is thought to cause neurogenic inflammation within the meninges and activation of nociceptive afferents projecting
to the trigeminal nucleus caudalis (52 ). In rats, antidromic stimulation of the trigeminal nerve increases vascular permeability and
causes plasma protein extravasation in the meninges that is inhibited by NK1 receptor antagonists (53 ). These findings suggest that
if meningeal plasma extravasation and inflammation of the meninges is involved in the pathogenesis of migraine, then NK1 receptor
antagonists should provide an effective antimigraine therapy. In addition, because of their potential analgesic activity, CNS-
penetrant NK1 antagonists may also be able to alleviate headache by preventing activation of sensory neurons in the trigeminal
nucleus caudalis. However, this hypothesis was not confirmed in clinical trials in patients with migraine, in whom neither LY 303870
(54 ) nor GR205171 (55 ) gave headache relief.

Emesis
Substance P is present in the nucleus tractus solitarius and the area postrema (56 ), regions implicated in the control of emesis.
Local application of substance P in the area postrema causes retching in ferrets (57 ), a finding suggesting that NK1 receptor
antagonists may be antiemetic. Consistent with this proposal, these compounds have emerged as an important new class of
antiemetics in preclinical studies using ferrets. CP-99,994 completely abolished cisplatin-induced retching and vomiting and
exhibited broad-spectrum activity against peripheral and centrally acting emetogens (58 ,59 and 60 ). Importantly, CP-99,994
markedly attenuated both acute and delayed emesis induced by cisplatin, a profile that distinguishes NK1 receptor antagonists from
established antiemetics (61 ,62 ). The ability of CP-99,994 to block both peripherally and centrally acting emetogens and the
demonstration that direct injection of CP-99,994 into the region of the nucleus tractus solitarius inhibited cisplatin-induced emesis
in ferrets (63 ) suggest that the antiemetic activity of NK1 antagonists is centrally mediated. This proposal was confirmed by the use
of a poorly brain-penetrant quaternary NK1 receptor antagonist, L-743,310, which prevented cisplatin-induced retching in ferrets
when it was infused directly into the CNS, but not systemically (64 ).

Evaluation of NK1 receptor antagonists as antiemetics in patients has produced encouraging results. Three independent trials have
confirmed that CP-122,721 (65 ), CJ-11974 (66 ), and MK-869 (67 ) are extremely effective in the prevention of acute and delayed
emesis after cisplatin chemotherapy. CP-122,721 was also effective in preventing postoperative nausea and vomiting after
gynecologic surgery (68 ), a finding suggesting the utility of NK1 receptor antagonists as broad-spectrum antiemetics in humans.
There are no published studies examining the effects of selective NK2 and NK3 receptor agonists and antagonists on emesis.

Schizophrenia
A rationale that NK1 receptor antagonists may be useful as antipsychotic drugs has been built on evidence that substance P
modulates the activity of the mesolimbic dopamine system through which established antipsychotic drugs are thought to act.
Substance P–containing fibers have been shown to make synaptic contact with tyrosine hydroxylase–positive neurons in the ventral
tegmental area (VTA) from which the mesolimbic dopamine projection arises (69 ). Infusion of substance P agonists into the VTA
stimulates locomotor activity in rats, an effect attributed to the activation of dopamine neurons because this is accompanied by an
increase in dopamine turnover in the terminal projection area (nucleus accumbens) (70 ). Consistent with this interpretation, the
locomotor hyperactivity and changes in accumbens cell firing induced by intra-VTA infusion of substance P were blocked by the
dopamine receptor antagonist haloperidol, an antipsychotic drug (71 ).

The ability of a monoclonal antibody to substance P, injected into the nucleus accumbens, to attenuate the locomotor response to
amphetamine (72 ) was consistent with the proposal that endogenous substance P modulates the release of dopamine in the
mesolimbic system. A subsequent study appeared to support this interpretation because the NK1 receptor antagonist CP-96,345
reduced the firing of cells in the VTA in rats (73 ). However, other studies with NK1 receptor antagonists are not consistent with
these findings. Surprisingly, intra-VTA coinfusion of CP-96,345 was unable to block substance P agonist–induced locomotor activation
in rats (71 ), and amphetamine-induced hyperactivity in guinea pigs was not selectively inhibited by CP-99,994.

A possible explanation for the lack of effect of NK1 receptor antagonists in these studies is that the effects of substance P in the
rodent VTA may be mediated by stimulation of NK3, rather than NK1, receptors, as is suggested by anatomic (19 ), electrophysiologic
(74 ), and behavioral (75 ) evidence. Intra-VTA application of the NK3 receptor agonist senktide was shown to enhance markedly the
extracellular concentration of dopamine in the nucleus accumbens and
 P.173

prefrontal cortex of anesthetized guinea pigs, and this was blocked by the selective NK3 receptor antagonist SR142801 (76 ).
SR142801 (but not the NK1 receptor antagonist GR205171 or the NK2 antagonist SR144190) was able to antagonize the increase in
neuronal activity caused by acute administration of haloperidol in guinea pigs (77 ), a finding suggesting that NK3 receptors play a
key role in regulating midbrain dopamine neurons in this species.

Preliminary findings from an exploratory trial with MK-869 in patients with schizophrenia indicated that this compound did not
ameliorate the core symptoms of acute psychosis (46 ).

Anxiety and Depression

Substance P and its preferred NK1 receptor are highly expressed in brain regions that are critical for the regulation of emotion and
neurochemical responses to stress (14 ,15 ,24 ). Direct central injection of substance P agonists produces a range of fear-related
behaviors and defensive cardiovascular changes in animals (78 ,79 ,80 and 81 ). Neurochemical studies have revealed rapid
reductions in substance P content in the mesolimbic system, hippocampus, septum, periaqueductal gray, and hypothalamus of rats
after inescapable footshock (82 ,83 ) and immobilization stress (84 ). These findings indicate that activation of central substance P
pathways occurs in response to noxious or aversive stimulation and suggest that NK1 receptor antagonists may have anxiolytic or
antidepressant-like properties.

Substance P antagonists are capable of attenuating psychological stress responses in paradigms using neurochemical and behavioral
endpoints. This was first suggested by the demonstration that intra-VTA injection of a monoclonal antibody to substance P
prevented stress-induced activation of mesocortical dopamine neurons (85 ). More recently, the NK1 receptor antagonist GR205171
was shown to inhibit the stress-induced elevation in the dopamine metabolite DOPAC in the frontal cortex (86 ). Certain chemically
diverse NK1 receptor antagonists have also shown activity in a range of assays for anxiolytic and antidepressant drugs after
intracerebral or systemic administration. One of the earliest reported studies demonstrated a direct substance P–ergic projection
from the medial amygdala to the medial hypothalamus that regulates the expression of defensive rage in cats. Either systemic or
intrahypothalamic injection of CP-96,345 inhibited amygdaloid facilitation of defensive rage (87 ). A second study examined the role
of NK1 receptors in the caudal pontine reticular nucleus and showed that injection of CP-96,345 or CP-99,994 into this region blocked
potentiation of the acoustic startle response by footshock in rats (88 ). In the resident-intruder paradigm, L-760,735 reduced
aggression in singly housed hamsters in a dose-dependent manner resembling the effect of fluoxetine (J. Webb, E. Carlson, N.
Rupniak, unpublished observations) (Fig. 13.1 ). CGP 49823 has been reported to be active in the rat social interaction test for
anxiolytic activity (89 ,90 ) and the forced swim test for antidepressant drugs (89 ). In guinea pig pups, the vocalization response
elicited by maternal separation is inhibited by brain-penetrant NK1 receptor antagonists (L-773,060, L-760,735, GR205171), a
property also seen with clinically used antidepressant and anxiolytic drugs (91 ,92 ). The amygdala is a potential site of action for
this effect of NK1 receptor antagonists because separation stress caused internalization of NK1 receptors (reflecting the release of
substance P) in this brain region (91 ,93 ), and intraamygdala injection of L-760735 attenuated the neonatal vocalizations (93 ).
Further evidence for an antidepressant-like preclinical profile of substance P antagonists is suggested by preliminary findings with L-
733,060, which was active in the learned helplessness paradigm in rats (94 ), despite having only low affinity for the rat NK1 receptor.
These findings are summarized in Table 13.4 .

TABLE 13.4. PRECLINICAL

EVIDENCE OF AN
ANTIDEPRESSANT AND
ANXIOLYTIC-LIKE PROFILE OF
NK1 RECEPTOR ANTAGONISTS

FIGURE 13.1. Activity of L-760,735 in the hamster

residentintruder test. The subjects were individually
housed adult male hamsters. On test days, both resident
and intruder hamsters were dosed with the same drug and
were returned to their own cages for 30 minutes before
testing. Pretreatment with either fluoxetine (0.3 to 30
mg/kg intraperitoneally) or the selective NK1 receptor
antagonist L-760735 (0.3 to 10 mg/kg intraperitoneally)
caused a dose-dependent increase in the latency to initiate
an aggressive encounter.

The NK2 receptor antagonists SR48968, GR100679, and GR159897 have been reported to exhibit anxiolytic-like effects in several
preclinical assays (mouse light-dark box, rat social interaction test, rat elevated plus maze, and marmoset threat test) (95 ,96 and
97 ). However, these compounds were reported to be extremely potent, and the micrograms per kilogram anxiolytic dose range was
considerably lower
 P.174

than that required to block NK2 agonist–mediated effects in peripheral tissues (mg/kg dose range) (98 ,99 ). A second difficulty
concerns the failure to establish convincing expression of NK2 receptors in the adult rat brain (100 ).

In rodents, there is evidence that NK3 receptors are able to modulate monoaminergic neurotransmission. Because the clinical
efficacy of currently used antidepressant drugs is ascribed to their ability to increase the synaptic availability of 5-HT and
norepinephrine, modulation of these systems by NK3 receptor ligands may suggest an antidepressant-like profile. The ability of
central infusion of senktide to elicit a 5-HT behavioral syndrome (101 ) and to increase the release of norepinephrine in brain (27 )
indicates that monoamine systems can be activated by NK3 receptor agonists. The ability of senktide to increase locus ceruleus firing,
to increase norepinephrine release, and to decrease locomotor activity in animals was blocked by the selective NK3 receptor
antagonist SR142801 (102 ). These actions are not clearly indicative of an antidepressant-like profile of NK3 receptor antagonists,
and the low abundance of these receptors in human brain suggests that, like NK2 receptor antagonists, NK3 antagonists are less
attractive candidates for clinical development in psychiatry than NK1 receptor antagonists.

There is currently only one published study in which a tachykinin antagonist has been examined in patients with depression. The
clinical efficacy of the NK1 receptor antagonist MK-869 was comparable to that of paroxetine in outpatients with major depressive
disorder and moderately high anxiety. As in other clinical trials, MK-869 was extremely well tolerated (94 ). Further studies are
currently in progress with this and other NK1 receptor antagonists in patients with depression and anxiety disorders.

CONCLUSIONS AND IMPLICATIONS FOR FUTURE STUDIES OF

NEUROKININ ANTAGONISTS IN PSYCHIATRIC AND NEUROLOGIC
DISORDERS
Part of "13 - Substance P and Related Tachykinins "

The process of elucidating the potential clinical uses of tachykinin receptor antagonists raises several fundamental issues that will
apply to other novel neurotransmitter ligands in the future. Preclinical studies have suggested therapeutic potential of neurokinin
antagonists in certain neurologic and psychiatric disorders, including migraine, pain, schizophrenia, anxiety, and depression. Of
these, antagonists of tachykinin NK1 receptors are the most attractive agents because this is the predominant receptor expressed in
the human brain. However, expectations have been only partially fulfilled in clinical trials, and although preliminary findings suggest
efficacy of NK1 receptor antagonists in the control of emesis and depression, these compounds do not appear to possess analgesic or
antipsychotic activity. It was not possible to predict this outcome from preclinical evidence, in which interpretation was
complicated by species variants in tachykinin receptor pharmacology and possibly physiology, and this was coupled with uncertainty
about whether relevant aspects of human disease can be accurately modeled in animals.

This chapter has focused on the intricacies of prioritizing efforts to identify the therapeutic uses of neurokinin antagonists for CNS
disorders. However, there are many other potential uses for NK1, NK2, and NK3 antagonists that have not yet been fully explored.
These include inflammatory diseases such as cystitis and inflammatory bowel disease, asthma, cancer, glaucoma, ocular hypotension,
cardiac disorders, and psoriasis.

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attenuates neonatal vocalisation in guinea-pigs and mice. Neuropharmacology 2000;39:1413–1421.

93. Boyce S, Smith D, Carlson E, et al. Intra-amygdala Injection of the substance P (NK1 receptor) antagonist L-760735 inhibits
neonatal vocalisations in guinea-pigs. Neuropharmacology 2001;41:130–137.

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14
Histamine

Jean-Charles Schwartz

Jean-Michel Arrang

Jean-Charles Schwartz and Jean-Michel Arrang: Unité de Neurobiologie et Pharmacologie Moléculaire de l’INSERM, Centre Paul
Broca, Paris, France.

In a certain way, histaminergic systems have had a great, although indirect, historical importance in the development of
neuropsychopharmacology. Indeed, the discovery of both the neuroleptic agents and the tricyclic antidepressant drugs in the 1950s
was derived from the clinical study of behavioral actions of “antihistamines,” a class of antiallergic drugs now designated H1-
receptor antagonists.

Nevertheless, the histaminergic neuronal system in the brain, although already understood by the mid-1970s, has remained largely
unexploited in drug design. Thus, only the traditional brain-penetrating H1-receptor antagonists, used as over-the-counter sleeping
pills, are known to interfere with histaminergic transmissions in the central nervous system (CNS). This situation contrasts with the
emergence, in the 1990s, of detailed knowledge of the system that revealed that it shares many biological and functional properties
with other aminergic systems overexploited in CNS drug design.

Histamine and its receptors in the brain have been the subject of two comprehensive reviews (1 ,2 ). Therefore, to limit the length
of the present chapter, we have deliberately elected to summarize the detailed information that can be found in these reviews and
have added only more recent information and major references.

 ORGANIZATION OF THE HISTAMINERGIC NEURONAL SYSTEM

 MOLECULAR PHARMACOLOGY AND LOCALIZATION OF HISTAMINE RECEPTOR SUBTYPES
 HISTAMINERGIC NEURON ACTIVITY AND THEIR CONTROL
 PHYSIOLOGIC ROLES OF HISTAMINERGIC NEURONS
 ROLE OF HISTAMINERGIC NEURONS IN NEUROPSYCHIATRIC DISEASES
 CONCLUSION

ORGANIZATION OF THE HISTAMINERGIC NEURONAL SYSTEM

Part of "14 - Histamine "

One decade after the first evidence by Garbarg et al. of an ascending histaminergic pathway obtained by lesions of the medial
forebrain bundle (3 ), the exact localization of corresponding perikarya in the posterior hypothalamus was revealed
immunohistochemically, and the distribution, morphology, and connections of histamine and histidine decarboxylase-
immunoreactive neurons were determined. Data were comprehensively reviewed (4 , 5 , 6 and 7 ), and they are summarized only
briefly here.

All known histaminergic perikarya constitute a continuous group of mainly magnocellular neurons (about 2,000 in the rat), located in
the posterior hypothalamus and collectively named the tuberomammillary nucleus (Fig.14.1 ). It can be subdivided into medial,
ventral, and diffuse subgroups extending longitudinally from the caudal end of the hypothalamus to the midportion of the third
ventricle. A similar organization was described in humans, except histaminergic neurons are more numerous (approximately 64), and
occupy a larger proportion of the hypothalamus (8 ). Besides their large size (25 to 35 μm), tuberomammillary neurons are
characterized by few thick primary dendrites, with overlapping trees, displaying few axodendritic synaptic contacts. Another
characteristic feature is the close contact of dendrites with glial elements in a way suggesting that they penetrate the ependyma
and come in close contact with the cerebrospinal fluid, perhaps to secrete or receive still unidentified messengers. Neurons
expressing mRNAs for histidine decarboxylase (EC 4.1.1.22), the enzyme responsible for the one-step histamine formation in the
brain (2 ), were found by in situ hybridization in the tuberomammillary nucleus, but not in any other brain area (9 ).
Tuberomammillary neurons possess the vesicular monoamine transporter 2 (10 ), which accounts for the histamine-releasing effect
of reserpine (2 ).

The histaminergic neurons are characterized by the presence of an unusually large variety of markers for other neurotransmitter
systems: glutamic acid decarboxylase, the γ-aminobutyric acid (GABA–synthesizing enzyme; adenosine deaminase, a cytoplasmic
enzyme possibly involved in adenosine inactivation; galanin, a peptide co-localized with all other monoamines; (Met5)enkephalyl-
Arg6Phe7, a product of the proenkephalin A gene; and other neuropeptides such as substance P, thyroliberin, or brain natriuretic
peptide. Tuberomammillary neurons also contain monoamine oxidase B, an enzyme responsible for deamination of
telemethylhistamine, a major histamine metabolite in brain. Finally, a subpopulation of histaminergic neurons is able to take up and
decarboxylate exogenous 5-hydroxytryptophan,
 P.180

a compound that they do not synthesize, however (5 ). Discovering the functions of such a high number of putative cotransmitters in
the same neurons remains an exciting challenge.
Like other monoaminergic neurons, histaminergic neurons constitute long and highly divergent systems projecting in a diffuse
manner to many cerebral areas (Fig.14.1 ). Immunoreactive, mostly unmyelinated, varicose or nonvaricose fibers are detected in
almost all cerebral regions, particularly limbic structures, and it was confirmed that individual neurons project to widely divergent
areas. Ultrastructural studies suggest that these fibers make few typical synaptic contacts (6 ).

FIGURE 14.1. Localization of histaminergic perikarya (closed circles)

in tuberomammillary nucleus and disposition of main histaminergic
pathways (arrows) in rat brain. A: Frontal section of the caudal
hypothalamus. B: Sagittal section of the brain. AH, anterior
hypothalamic area; Arc, arcuate nucleus; cc, corpus callosum; Cer,
cerebellum; CG, central gray; CX, cerebral cortex; DR, dorsal raphe
nucleus; f, fornix; Hip, hippocampus; LS, lateral septum; MD,
mediodorsal thalamus; MMn, medial mammillary nucleus median part;
OB, olfactory bulb; Pn, pontine nuclei; Sol, nucleus of solitary tract;
Sox, supraoptic decussation; sum, supramammillary nucleus; TMdiff,
tuberomammillary nucleus diffuse part; TMVr, ventral tuberomamillary
subgroup rostral part; VDB, nucleus of vertical limb of diagonal band;
VMH, ventromedial hypothalamic nucleus.

Fibers arising from the tuberomammillary nucleus constitute two ascending pathways: one laterally, through the medial forebrain
bundle, and the other periventricularly. These two pathways combine in the diagonal band of Broca to project, mainly in an
ipsilateral fashion, to many telencephalic areas, for example, in all areas and layers of the cerebral cortex, the most abundant
projections being to the external layers. Other major areas of termination of these long ascending connections are the olfactory
bulb, the hippocampus, the caudate putamen, the nucleus accumbens, the globus pallidus, and the amygdaloid complex. Many
hypothalamic nuclei exhibit a very dense innervation, for example, the suprachiasmatic, supraoptic, arcuate, and ventromedial
nuclei.
Finally, a long descending histaminergic subsystem also arises from the tuberomammillary nucleus to project to various
mesencephalic and brainstem structures such as the cranial nerve nuclei (e.g., the trigeminal nerve nucleus), the central gray, the
colliculi, the substantia nigra, the locus ceruleus, the mesopontine tegmentum, the dorsal raphe nucleus, the cerebellum (sparse
innervation), and the spinal cord.
Several anterograde and retrograde tracing studies established the existence of afferent connections to the histaminergic perikarya,
namely, from the infralimbic cortex, the septum-diagonal band complex, the preoptic region, the hypothalamus, and the
hippocampal area (subiculum) (7 , 11 ). Sleep-active GABAergic neurons in the ventrolateral preoptic nucleus provide a major input
to the tuberomammillary nucleus (12 , 13 ). Histaminergic neurons also receive very dense orexin innervation originating from the
lateral hypothalamus (14 ). Electrophysiologic studies provided evidence of inhibitory and excitatory synaptic control of
tuberomammillary neuron activity by afferents from the diagonal band of Broca, the lateral preoptic area and the anterior lateral
hypothalamic area (15 ). Projections from the brainstem to the tuberomammillary nucleus have also been demonstrated. Retrograde
tracing studies combined with immunohistochemistry showed that monoaminergic inputs to the tuberomammillary nucleus originate
mainly from the ventrolateral and dorsomedial medulla oblongata and from the raphe nuclei, with a low innervation originating from
the locus ceruleus, the ventral tegmental area, and the substantia nigra (16 ).

MOLECULAR PHARMACOLOGY AND LOCALIZATION OF HISTAMINE

RECEPTOR SUBTYPES
Part of "14 - Histamine "
Three histamine receptor subtypes (H1, H2 and H3) have been defined by means of functional assays, followed by design of selective
agonists and antagonists and, more recently, cloning of their genes (1 ). All three belong to the superfamily of receptors with seven
transmembrane domains (TMs) and coupled to guanylnucleotide-sensitive G proteins (Table 14.1 ). In addition, histamine affects the
glutamatergic N-methyl-D-aspartate (NMDA) receptor (17 ,18 ).

TABLE 14.1. PROPERTIES OF THREE

HISTAMINE RECEPTOR SUBTYPES

Histamine H Receptor
1

The H1 receptor was initially defined in functional assays (e.g., smooth muscle contraction) and in the design of potent
 P.181

antagonists, the so-called antihistamines (e.g., mepyramine), most of which display prominent sedative properties. Biochemical and
localization studies of the H1 receptor were made feasible with the design of reversible and irreversible radiolabeled probes such as
[3H] mepyramine, [125I]iodobolpyramine, and [125I]iodoazidophenpyramine (19 ,20 ).

Various intracellular responses were found to be associated with H1-receptor stimulation: inositol phosphate release, increase in Ca2+
fluxes, cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate accumulation in whole cells and arachidonic acid
release (1 ).

The deduced amino acid sequence of a bovine H1 receptor was disclosed after expression cloning of a corresponding cDNA. The latter
was based on the detection of a Ca2+-dependent Cl- influx into microinjected Xenopus oocytes. After the transient expression of the
cloned cDNA into COS-7 cells, the identity of the protein as an H1 receptor was confirmed by binding studies (21 ).

Starting from the bovine sequence, the H1 receptor DNA was also cloned in the guinea pig (22 ), a species in which the pharmacology
of the receptor is better established, as well as from several other species including humans (1 ). Although marked species
differences in H1-receptor pharmacology had been reported (2 ), the sequence homology between the putative TMs of the proteins is
high (90%).

The “anatomy” of the H1 receptor, with a long third intracellular domain and a short C-terminal tail, is similar to that of other
receptors positively coupled to phospholipases A2 and C. Amino acid sequence homology between the TMs of the H1 and those of the
muscarinic receptors (approximately 45%) is higher than between those of H1 and H2 receptors (approximately 40%). H1-receptor
antagonists often display significant antimuscarinic activity but only limited H2-receptor–antagonist properties.

A single gene seems to encode the guinea pig H1 receptor, and mRNAs of similar size were detected in brain areas and peripheral
tissues (22 ).The structure of the human gene was disclosed (23 ). Like other receptors of this superfamily, it contains an intron in
the 5′ flanking untranslated region, close to the translation initiation codon, but the translated region is intronless.

When stably expressed in transfected fibroblasts, the guinea pig H1 receptor was found to trigger a large variety of intracellular
signals involving or not coupling to pertussis toxin-sensitive G proteins (Gi or Go), namely, Ca2+ transients, inositol phosphates, or
arachidonate release (24 ). H1 receptor stimulation potentiates cAMP accumulation induced by forskolin in the same transfected
fibroblasts, a response that resembles the H1 potentiation of histamine H2- or adenosine A2-receptor–induced accumulation of cAMP
in brain slices. All these responses mediated by a single H1 receptor were known to occur in distinct cell lines or brain slices, but
they could have resulted from stimulation of isoreceptors.

Constitutive activity of the recombinant human H1 receptor

 P.182

consisting in an agonist-independent increase in inositol phosphates accumulation in COS-7 cells was evidenced. Several H1-receptor
antagonists behaved as inverse agonists (i.e., reduced this constitutive activity), but the physiologic relevance of the process, such
as in brain, was not established (25 ).

The H1 receptor mediates various excitatory responses in brain (26 ). A reduction of a background leakage K+ current was implicated
in these responses, in cortical, striatal, and lateral geniculate relay neurons (27 ,28 ).

H1-receptor distribution in the guinea pig brain was established autoradiographically using [3H]mepyramine or the more sensitive
probe [125I]iodobolpyramine (20 ), and the information was complemented by in situ hybridization of the mRNA (22 ). For instance,
the high density of H1 receptors in the molecular layers of cerebellum and hippocampus seems to correspond to dendrites of Purkinje
and pyramidal cells, respectively, in which the mRNA is highly expressed. H1 receptors are also abundant in guinea pig thalamus,
hypothalamic nuclei (e.g., ventromedial nuclei), nucleus accumbens, amygdaloid nuclei, and frontal cortex but not in neostriatum
(20 ), whereas they are more abundant in the human neostriatum (29 ). The H1 receptor was visualized in the primate and human
brain in vivo by positron emission tomography using [11C]mepyramine (30 ).

Blockade of H1 receptors located in cerebral areas involved in wakefulness and cognition, and including those mediating excitation of
thalamic relay neurons (31 ), neocortical pyramidal neurons (28 ) and ascending cholinergic neurons (32 ,33 ), presumably accounts
for the sedative properties of “antihistamines” of the first generation.

Histamine H Receptor
2

The molecular properties of the H2 receptor remained largely unknown for a long time. Reversible labeling of the H2 receptor was
achieved using [3H]tiotidine or, more reliably, [125I]iodoaminopotentidine (2 ).

By screening cDNA or genomic libraries with homologous probes, the intronless gene encoding the H2 receptor was first identified in
dogs (34 ) and, subsequently, in other species including humans (1 ). The H2 receptor is organized like other receptors positively
coupled to adenylyl cyclase: it displays a short third intracellular loop and a long C-terminal cytoplasmic tail.

Using transfected cell lines, positive linkage of the H2 receptor with adenylyl cyclase was confirmed, and unexpected inhibition of
arachidonate release and stimulation of Ca2+ transients was evidenced (1 ). Hence H2 receptor stimulation can trigger intracellular
signals either opposite or similar to those evoked by H1 receptor stimulation. Parallel observations were made for a variety of
biological responses mediated by the two receptors in peripheral tissues.

Helmut Haas and colleagues showed that, in hippocampal pyramidal neurons, H2-receptor stimulation potentiates excitatory signals
by decreasing a Ca2+-activated K+ conductance, presumably by cAMP production (26 ). H2-receptor activation depolarizes thalamic
relay neurons slightly and increases apparent membrane conductance markedly, responses caused by enhancement of the
hyperpolarization-activated cation current Ih (27 ). In addition to these short-lasting effects, histamine also induces very long-lasting
increases in excitability in the CA1 region of the hippocampus through activation of H2 receptors and the cAMP/cAMP-dependent
protein kinase signal transduction cascade. This process is modulated by other receptors such as the H1 receptor (35 ).

The sole selective H2-receptor antagonist known to enter the brain is zolantidine, a compound used sometimes in animal behavioral
studies but not introduced in therapeutics (36 ). However, some tricyclic antidepressants are known to block H2-receptor–linked
adenylyl cyclase potently and interact with [125I]iodoaminopotentidine binding in a complex manner (37 ).

Autoradiographic localization of the H2 receptor in guinea pig (20 ) and human brain (29 ) shows it distributed heterogeneously. The
H2 receptor is found in most areas of the cerebral cortex, with the highest density in the superficial layers, the piriform, and the
occipital cortices, which contain low H1-receptor density. The caudate putamen, the ventral striatal complex, and the amygdaloid
nuclei (bed nucleus of the stria terminalis) are among the richest brain areas. In the hippocampal formation, the relative
localizations of the H2 receptor and its gene transcripts are similar to those observed for the H1 receptor: the gene transcripts are
expressed in all pyramidal cells of the Ammon horn and in granule cells of the dentate gyrus (38 ), whereas the H2 receptor is
expressed in the molecular layers of these areas, which contain the dendritic trees of the mRNA-containing neurons. The partial
overlap with the H1 receptor may account for their synergistic interaction in cAMP accumulation.

Histamine H Receptor
3

The H3 receptor was initially detected as an autoreceptor controlling histamine synthesis and release in brain. Thereafter, it was
shown to inhibit presynaptically the release of other monoamines in brain and peripheral tissues as well as of neuropeptides from
unmyelinated C fibers (39 ,40 ).

Reversible labeling of this receptor was first achieved using the highly selective agonist [3H](R)α-methylhistamine (2 ), then [3H]Nα-
methylhistamine, a less selective agonist, was also proposed (19 ), as well as, more recently, [125I]iodophenpropit and
[125I]iodoproxyfan, two antagonists (41 ).

The regulation of agonist binding by guanylnucleotides (39 ), and the sensitivity of several H3-receptor–mediated
 P.183

responses to pertussis toxin (42 ,43 ), suggested that the H3 receptor was Gi/Go protein coupled, a suggestion confirmed by the
cloning of the corresponding human (44 ) and rodent (45 ) cDNAs. The H3 receptor gene contains two introns in its coding sequence
and several splice variants such as H3L and H3S differing by a stretch of 30 amino acids in the third intracellular loop, were identified
(45 ). The existence of these variants may partly account for the apparent H3-receptor heterogeneity in binding or functional studies
(46 ).

Significant differences in the pharmacology of the human and rodent H3 receptor (47 ) could be assigned to differences in only two
amino acid residues in the third TM (48 ). In various cell lines, stimulation of the H3 receptor, like that of other Gi-protein–coupled
receptors, inhibits adenylate cyclase (44 ) or phospholipase C (42 ) and activates phospholipase A2 (48a ).

On neurons, the H3 receptor mediates presynaptic inhibitions of release of several neurotransmitters, including histamine itself
(2 ,39 ), norepinephrine, serotonin, dopamine, glutamate, GABA, and tachykinins (40 ), presumably by inhibiting voltage-dependent
calcium channels (39 ,43 ).

Several H3-receptor antagonists, such as thioperamide and ciproxifan, potently enhance histamine release in vitro and in vivo
(2 ,39 ,49 ).This response was originally attributed to blockade of the inhibitory effects of endogenous histamine and was therefore
used in many studies, such as behavioral studies, to delineate the functions of histaminergic neurons. However, these drugs were
shown to act, in fact, as inverse agonists, and the native H3 receptor in brain display high constitutive activity including in vivo
(48a ).

Autoradiography of the H3 receptor in rat (50 ,51 ) and monkey brain shows it highly concentrated in the neostriatum, the nucleus
accumbens, the cingulate and infralimbic cortices, the bed nucleus of the stria terminalis, and the substantia nigra pars lateralis. In
contrast, its density is relatively low in the hypothalamus (including the tuberomammillary nucleus), which contains the highest
density of histaminergic axons (and perikarya), a finding indicating that most H3 receptors are not autoreceptors. In agreement with
this concept, intrastriatal kainate strongly decreases H3 binding sites in the forebrain (as well as in the substantia nigra, consistent
with their presence in striatonigral neurons) (50 ,51 ). In the human brain, the high densities of H3 receptors found in the striatum
and globus pallidus (29 ) were lower in patients with Huntington disease, a finding suggesting that the H3 receptor is also located on
striatonigral projection neurons of the direct and indirect pathways (52 ). Consistent with the proposal that most H3 receptors are
not autoreceptors, a strong expression of H3-receptor mRNAs was observed not only within the tuberomammillary nucleus, but also
in various regions of the rat (44 ) and guinea pig (45 ) brain, including the cerebral cortex, the basal ganglia, and the thalamus.

Interaction with NMDA Receptors

Histamine potentiates NMDA-evoked currents in acutely dissociated and cultured hippocampal and cortical neurons, an effect that
could not be ascribed to activation of the known histamine receptors (17 ,18 ), but rather of a novel recognition site on NMDA
receptors containing the subunits NR1A/NR2B (53 ).

Histamine may play a role in modulating the functions of NMDA receptors in vivo. It facilitates the NMDA-induced depolarization of
projection neurons in cortical slices (54 ) and phase shifts the circadian clock by a direct potentiation of NMDA currents in the
suprachiasmatic nucleus (55 ). Histamine, presumably acting through NMDA receptors, facilitates the induction of long-term
potentiation and causes long-lasting increases of excitability in the CA1 region of rat hippocampal slices (35 ).

The histamine-induced modulation of NMDA responses is higher under slightly acidic conditions (56 ), which occur during hypoxia or
epileptiform activity. This may lead to enhancement of neurotransmission or histamine-mediated neuronal death such as that
observed in a rat model of Wernicke encephalopathy (57 ).

HISTAMINERGIC NEURON ACTIVITY AND THEIR CONTROL

Part of "14 - Histamine "

Electrophysiologic Properties
Cortically projecting histaminergic neurons share with other aminergic neurons certain electrophysiologic properties evidenced by
extracellular recording. They fire spontaneously slowly and regularly, and their action potentials are of long duration (26 ). Among
the pacing events that may contribute to their spontaneous firing, tuberomammillary neurons exhibit a tetrodotoxin-sensitive
persistent Na+ current (58 ), a Ca2+ current probably of the low-threshold type (59 ), and multiple high-voltage–activated Ca2+
currents (43 ). In addition, they exhibit inward rectification attributed to an Ih current that may increase whole-cell conductance
and may decrease the efficacy of synaptic inputs during periods of prolonged hyperpolarization, that is, when histaminergic neurons
fall silent (60 ).

Modulation of Histamine Synthesis and Release In Vitro

The autoreceptor-regulated modulation of histamine synthesis in, and release from, brain neurons is well documented (2 ). It was
initially evidenced in brain slices or synaptosomes after labeling the endogenous pool of histamine using the [3H]histamine precursor.
Exogenous histamine decreases the release and formation of [3H]histamine induced by depolarization, and analysis of these
responses led to the pharmacologic definition of H3 receptors. The autoregulation
 P.184

was found in various brain regions known to contain histamine nerve endings, a finding suggesting that all terminals are endowed
with H3 autoreceptors.

Regulation of histamine synthesis was also observed in the posterior hypothalamus (39 ), and somatodendritic H3 autoreceptors
inhibit the firing of tuberomammillary neurons (26 ) by modulating high-voltage–activated calcium channels (43 ).

Galanin, a putative cotransmitter of a subpopulation of histaminergic neurons, regulates histamine release only in regions known to
contain efferents of this subpopulation, that is, in hypothalamus and hippocampus but not in cerebral cortex or striatum (61 ). In
brain slices, galanin also hyperpolarizes and decreases the firing rate of tuberomammillary neurons (26 ). It is not known, however,
whether these galanin receptors behave as “autoreceptors” modulating galanin release from histaminergic nerve terminals,
inasmuch as the tuberomammillary nucleus receives a strong galaninergic innervation from the ventrolateral preoptic area (12 ,13 ).
Other putative cotransmitters of histaminergic neurons failed to affect [3H]histamine release from slices of rat cerebral cortex (62 ).
However, GABAergic inhibitory postsynaptic potentials are mediated by GABAA receptors located on histaminergic neurons (63 ). To
what extent these receptors play an autoinhibitory role is unclear. A subpopulation of histaminergic neurons contains GABA (5 ), but
the tuberomammillary nucleus also receives dense GABAergic innervation (12 ,13 ,15 ).

[3H]Histamine synthesis and release are inhibited in various brain regions by stimulation of not only autoreceptors but also α2-
adrenergic receptors, M1-muscarinic receptors, and κ-opioid receptors (2 ). Because these regulations are also observed with
synaptosomes (62 ), all these receptors presumably represent true presynaptic heteroreceptors. In contrast, histamine release is
enhanced by stimulation of nicotinic receptors in rat hypothalamus (64 ) and by μ-opioid receptors in mouse cerebral cortex (2 ).

Some molecular mechanisms regulating neuronal histamine dynamics remain unclear. N-methylation catalyzed by histamine N-
methyltransferase is the major process responsible for termination of histamine actions in the brain (2 ), and genetic polymorphisms
for the enzyme have been associated with altered levels of its activity (65 ). No histamine transporter could be evidenced, and
direct feedback inhibition of histidine decarboxylase by histamine has been excluded (2 ).

Changes in Histaminergic Neuron Activity In Vivo

Both neurochemical and electrophysiologic studies indicate that the activity of histaminergic neurons is high during arousal. In rat
hypothalamus, histamine levels are low, whereas synthesis is high during the dark period, a finding suggesting that neuronal activity
is enhanced during the active phase (2 ). In mouse cerebral cortex, striatum, and hypothalamus, telemethylhistamine levels are
doubled at the end of the dark phase of the cycle as compared with the beginning of the light phase (66 ). Histamine release from
the anterior hypothalamus of freely moving rats, evaluated by in vivo microdialysis, gradually increases in the second half of the
light period and is maintained at a maximal level during the active phase (67 ). Such state-related changes are also found in single-
unit extracellular recordings performed in the ventrolateral posterior hypothalamus of freely moving cats. Neurons with properties
consistent with those of histaminergic neurons exhibited a circadian rhythm of their firing rate, falling silent during deep slow-wave
or paradoxical sleep (2 ). An important determinant of this circadian rhythm of tuberomammillary histaminergic neuron activity is a
GABAergic inhibitory pathway originating in the ventrolateral preoptic area and activated during sleep (12 ,15 ).

A feeding-induced increase in the activity of histaminergic neurons has also been shown by microdialysis performed in the
hypothalamus of conscious rats (68 ). Histaminergic neurons are a target for leptin in its control of feeding. An enhancement of
histamine turnover was observed after intracerebroventricular infusion of leptin (70 ). Changes in the metabolism and release of
histamine observed in vivo after occlusion of the middle cerebral artery in rats suggest that the histaminergic activity is also
enhanced by cerebral ischemia (71 ).

Whereas H1 and H2 receptors are apparently not involved, inhibition mediated by the H3-autoreceptor constitutes a major regulatory
mechanism for histaminergic neuron activity under physiologic conditions. Administration of selective H3 receptor agonists reduces
histamine turnover (2 ) and release, as shown by microdialysis (72 ). In contrast, H3-receptor antagonists enhance histamine turnover
(2 ,49 ) and release in vivo (73 ,74 ), a finding suggesting that autoreceptors are tonically activated.

Agents inhibiting histamine release in vitro through stimulation of presynaptic α2-adrenergic or muscarinic heteroreceptors reduce
histamine release and turnover in vivo, but systemic administration of antagonists of these receptors does not enhance histamine
turnover, a finding suggesting that these heteroreceptors are not tonically activated under basal conditions.

Activation of central nicotinic receptors inhibits histamine turnover (75 ). Several types of serotonergic receptors are likely to
modulate histamine neuron activity. 5-Hydroxytryptamine (5-HT)1A–receptor agonists inhibit (76 ), whereas 5-HT2–receptor
antagonists enhance (77 ), histamine turnover in various brain regions. Stimulation of D2 (but not D3) dopamine receptors by
endogenous dopamine released by amphetamine increases histamine neuron activity (77 ,78 ).

Histamine turnover in the brain is rapidly reduced after administration of various sedative drugs such as ethanol, Δ9-
tetrahydrocannabinol, barbiturates, and benzodiazepines (2 ),
 P.185

presumably as a result of their interaction with GABA receptors present on nerve endings and on perikarya of histaminergic neurons
(63 ,79 ).

In contrast, stimulation of μ-opioid receptors enhances histamine turnover in brain (2 ). NMDA receptors increase in vivo release of
histamine from the anterior hypothalamus (80 ). Activation of NMDA and non-NMDA receptors in the diagonal band of Broca, the
lateral preoptic area, and the anterior hypothalamic area led to inhibition or enhancement of firing rates of tuberomammillary
neurons (15 ).

PHYSIOLOGIC ROLES OF HISTAMINERGIC NEURONS

Part of "14 - Histamine "

In spite of many different suggestions mainly derived from the observations of responses to locally applied histamine, only a few
physiologic roles of histaminergic neurons appear relatively well documented.

Arousal
Our initial proposal in 1977 (81 ) that histaminergic neurons play a critical role in arousal has been confirmed by data from a variety
of experiments mainly performed by Lin and Jouvet in cats (33 ) and Monti in rats (2 ). In agreement with this concept, ablation of
these neurons and inhibition of histamine synthesis, release, or action by the H1 receptor decrease wakefulness and increase deep
slow-wave sleep; conversely, inhibition of histamine methylation or facilitation of histamine release by H3 receptor blockade
increase arousal (49 ).

The arousing effect of histamine may result from H1 and H2-receptor–mediated depolarization of thalamic relay neurons that induces
a shift of their activity from burst firing (predominating in deep sleep during which they are poorly responsive to sensory inputs) to
single spike activity (predominating in arousal during which sensory information is more faithfully relayed) (31 ). Arousal may also
result from H1-receptor–mediated excitation of neocortical pyramidal neurons by the same mechanism as in thalamus, that is,
reduction of a background leakage potassium current (28 ). Finally, arousal may occur indirectly by H1-receptor–mediated excitation
of ascending cholinergic neurons within the nucleus basilis or mesopontine tegmentum (32 ,33 ), which also induces cortical
activation.

All these cellular actions of histamine, together with observations that tuberomammillary neuron firing is maximal during
wakefulness, suggest that histaminergic systems make an important contribution to the control of arousal. The circadian changes in
histaminergic neuron activity seem to be directed by two major neuronal inputs arising from the anterior hypothalamus. The first
ones are slow-wave sleep-activated inhibitory GABA- and galanin-containing neurons arising from the ventrolateral preoptic area
(12 ,13 ); in contrast, neurons releasing the neuropeptide orexin that emanate from the lateral hypothalamus appear to exert
opposite actions because disruption of the orexin gene is associated with narcolepsy in dogs and knockout mice (14 ,82 ). Other
monoaminergic neurons participating in control of sleep and wakefulness states as well as GABA/galanin ventrolateral preoptic
neurons also receive inputs from orexin neurons, which are, themselves, likely influenced by photic signals from the suprachiasmatic
nucleus. In turn, neurons from the suprachiasmatic nucleus and the preoptic area seem to be influenced in a complex manner by
histaminergic inputs (83 ,84 ). Hence a complex neuronal network in the hypothalamus with reciprocal influences involving
histaminergic neurons seems to control wakefulness.

The major part played by the H1 receptor in these processes, confirmed in mutant mice lacking this receptor (85 ), accounts for the
sedating effects of the first generation of “antihistamines,” that is, antagonists that easily enter the brain and are still ingredients
of over-the-counter sleeping pills (86 ). It may also account for the sedative side effects of many antipsychotic or antidepressant
drugs that are potent H1 antagonists.

Cognitive Functions
The idea that histaminergic neurons may improve cognitive performance is consistent with projections of these neurons to brain
areas such as the prefrontal and cingulate cortices or hippocampus, their excitatory influences therein, and their positive role in
wakefulness.

Ciproxifan, a potent and selective H3-receptor antagonist (or inverse agonist), which strongly enhances histamine turnover in brain,
improved attentional performances in the rat five-choice test under conditions similar to those of drugs enhancing cholinergic
transmissions (49 ). Various H3 antagonists facilitate various forms of learning. They improve short-term social memory in rats (87 ),
reverse the scopolamine- or senescence-induced learning deficit in a passive avoidance test in mice (88 ), and facilitate retention in
a footshock avoidance test in mice (89 ).

Generally, H3 agonists exert opposite effects, and the effects of H3-antagonists are reversed by H1 antagonists, a finding that
suggests that these effects are attributable to enhanced histamine release. In contrast to the large body of experiments indicating a
“procognitive” role of tuberomammillary neurons, Huston and coworkers repeatedly showed that excitotoxic lesions aimed at these
neurons ablation result in improvement of learning in a variety of tests (e.g., ref. 90 ). The discrepancy with data from
pharmacologic approaches may result from the difficulty to achieve selective histamine neuron ablation.

Control of Pituitary Hormone Secretion

Histamine affects secretion of several pituitary hormones (2 ,91 ). Magnocellular neurons of the supraoptic and paraventrical
 P.186

nuclei are typically excited, an essentially H1-receptor–mediated response resulting in enhanced blood levels of vasopressin and
oxytocin. Histaminergic neurons are activated during dehydration, parturition, and lactation, and histamine release onto
magnocellular neurons participates in the control of these physiologic processes by the neurohypophysial hormones (92 ,93 ).

Histaminergic neurons may also participate in the hormonal responses to stress. In agreement with this concept, they are activated
during various forms of stress and heavily project to hypothalamic or limbic brain areas (e.g., the amygdala or bed nucleus of the
stria terminalis) involved in these responses. Various pharmacologic studies have shown the participation of endogenous histamine
by H1- and H2-receptor stimulation in the adrenocorticotropic hormone–corticosterone, prolactin, or renin responses to stressful
stimuli such as restraint, endotoxin, or dehydration (94 ). Many histaminergic neurons contain estrogen receptors, project to
luteinizing hormone–releasing hormone neurons in preoptic and infundibular regions, and may constitute, by H1-receptor stimulation,
an important relay in the estradiol-induced preovulatory luteinizing hormone surge (95 ).

Satiation
Weight gain is often experienced by patients receiving H1 antihistamines as well as by patients taking antipsychotics or
antidepressants displaying potent H1-receptor antagonist properties. These effects reflect the inhibitory role of endogenous
histamine on food intake mediated by the H1 receptor, namely, on the ventromedial nucleus (97 ). Histamine neurons projecting to
the hypothalamus may be responsible for the food intake suppression induced by leptin (70 ).

Seizures
The anticonvulsant properties of endogenous histamine were initially suggested from the occurrence of seizures in patients with
epilepsy, particularly children, after administration of high doses of H1-receptor antagonists crossing the blood–brain barrier, even
those agents devoid of anticholinergic activity (86 ). These drugs, by completely occupying the H1 receptor, as assessed by positron
emission tomography studies (30 ), could block the histamine-induced reduction of a background-leakage K+ current.

Drug-induced changes in histamine synthesis, release or metabolism confirmed the role of the endogenous amine acting through the
H1 receptor in preventing seizure activity elicited by pentetrazol, transcranial electrical stimulation, or amygdaloid kindling.
Acquired amygdaloid kindling susceptibility appears associated with reduced histamine synthesis in limbic brain areas (97 ). In
addition, kainic acid–induced limbic seizures are accompanied by up-regulation of the H1-receptor mRNA in striatum and dentate
gyrus, a finding consistent with a regulatory role of this receptor in seizure activity (98 ).

Nociception
The antinociceptive effects of histidine loads, H3-receptor antagonists, and histamine N-methyltransferase inhibitors, as well as
opposite effects of histamine synthesis inhibitors or H3 agonists, support the idea that brain histamine inhibits nociceptive responses
such as the mouse hot plate jump (99 ). In contrast, peripherally acting H3-receptor agonists prevent nociceptive responses such as
mouse abdominal constriction by inhibiting sensory C-fiber activity (100 ).

ROLE OF HISTAMINERGIC NEURONS IN NEUROPSYCHIATRIC DISEASES

Part of "14 - Histamine "

Among the various approaches that tend to establish the implication of other neuronal systems in neuropsychiatric diseases, so far
only a few have been applied to histamine.

Histamine, Schizophrenia, and Antipsychotic Drug Actions

Overdose of various classic H1 antagonists was repeatedly reported to result in toxic psychoses with hallucinations resembling
schizophrenia, and the hallucinogenic potential of these drugs has even led to abuse (86 ). Conversely, metamphetamine, a drug
with hallucinogenic potential and to which patients with schizophrenia seem hyperresponsive, releases histamine in rodent brain
areas, an indirect effect mediated by stimulation of D2 and not D3 dopamine receptors (77 ,78 ). Even more, endogenous dopamine
appears to exert a tonic stimulation of histamine neurons because typical neuroleptics, such as haloperidol, decrease their activity.
In contrast, atypical neuroleptics, such as clozapine, enhance histamine turnover, an effect related to 5-HT2 receptor blockade and
possibly underlying their procognitive properties (77 ). The locomotor activation elicited in rodents by amphetamine and other
dopaminergic agonists is attenuated by H3-receptor blockade (101 ). Repeated amphetamine administration to rodents that results in
behavioral sensitization to dopamine agonists, a cardinal feature of schizophrenia, is accompanied by enhanced histamine release, a
finding that presumably reflects an enhanced tonic dopaminergic influence on histaminergic neurons (77 ,78 ). In one comprehensive
study, an enhanced level of t-methylhistamine, the major histamine metabolite, was detected in the cerebrospinal fluid of patients
with schizophrenia, who were either treated or untreated with neuroleptic agents (102 ).

In several open studies, famotidine, an H2 antagonist, was found to improve schizophrenia in patients, a finding
 P.187

that remains to be confirmed in control studies. A previous claim of association between polymorphisms of the H2-receptor gene and
schizophrenia could not be confirmed (103 ).
These various observations, although not readily forming a coherent picture, suggest that histaminergic neuron activity is enhanced
in patients with schizophrenia, and blockade of H2 or H3 receptors could be useful in the treatment of this disease.

Histamine and Alzheimer’s Disease

Neuropathologic studies have documented a deficit in histaminergic neurotransmission in Alzheimer’s disease. In some, but not all,
cortical areas (e.g., the frontal or temporal cortex) of brains affected by Alzheimer’s disease, there is a decrease of histamine and
histidine decarboxylase levels that may reach up to approximately 50% (104 ), and the expression of the hdc gene in neurons of the
tuberomammillary nucleus is also reduced (S. Trottier, personal communication). Decreased histaminergic input may affect
cholinergic neuron activity in the nucleus basilis (32 ) and acetylcholine release in cortical areas.
If one takes into account an additional direct positive influence of histamine on attention and memory, this indicates that enhancing
histaminergic neurotransmission may constitute a novel symptomatic therapeutic approach to Alzheimer’s disease. The drug tacrine
was even more potent in inhibiting histamine-N-methyltransferase, the main histamine-metabolizing enzyme, than
acetylcholinesterase (105 ).

Histamine and Other Neuropsychiatric Disorders

Anxiety may be increased by endogenous histamine acting at the H1 receptor. In agreement with this concept, H1-receptor knockout
mice display significantly less anxiety in the elevated maze test (106 ). However, the utility of H1-receptor antagonists in anxiety
disorders is not established.
Patients with attention-deficit disorders may benefit from enhanced histamine release, as suggested by the therapeutic effect of
amphetamine in children and the attention-enhancing effects of an H3-receptor antagonist in the rat (49 ).
Antidepressant-like effects in the mouse forced swim test result from enhanced histamine release and H1-receptor activation (107 ).

CONCLUSION
Part of "14 - Histamine "
This chapter describes how our knowledge of the molecular neurobiology of cerebral histaminergic systems and their implications in
physiologic functions, such as arousal or hormonal regulations, have progressed over the years. In contrast, little is known, so far,
about their possible implications in neuropsychiatric diseases and the therapeutic utility of psychotropic drugs to affect their
activity. H3-receptor antagonists (or inverse agonists) that markedly enhance brain histamine release are currently undergoing
clinical trials. It seems likely that the next edition of this book will see their place in therapeutics established.

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102. Prell GD, Green JC, Kaufmann CA, et al. Histamine metabolites in cerebrospinal fluid of patients with chronic
schizophrenia: their relationship to levels of other aminergic transmitters and ratings of symptoms. Schizophr Res 1995;14:93–
104.

103. Ito C, Morisset S, Krebs MO, et al. Histamine H2 receptor gene variants: lack of association with schizophrenia. Mol
Psychiatry 2000;5:159–164.

104. Panula P, Rinne J, Kuokkanen K, et al. Neuronal histamine deficit in Alzheimer’s disease. Neuroscience 1998;82:993–997.

105. Morisset S, Traiffort E, Schwartz JC. Inhibition of histamine versus acetylcholine metabolism as a mechanism of tacrine
activity. Eur J Pharmacol 1996;315:R1–R2.

106. Yanai K, Son LZ, Endou M, et al. Behavioural characterization and amounts of brain monoamines and their metabolites in
mice lacking histamine H1 receptors. Neuroscience 1998;87:479–487.

107. Lamberti C, Ipponi A, Bartolini A, et al. Antidepressant-like effects of endogenous histamine and of two histamine H1
receptor agonists in the mouse forced swim test. Br J Pharmacol 1998;123:1331–1336.
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15
Purinergic Neurotransmission

Michael Williams

Michael Williams: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University School of Medicine,
Chicago, Illinois.

The purine nucleoside, adenosine, its nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine
monophosphate (AMP), and the pyrimidine nucleotide, uridine triphosphate (UTP) (Fig. 15.1 ), play a critical role in central and
peripheral nervous system homeostasis and function as extracellular messengers to regulate cell function. The effects of adenosine
and the nucleotides are mediated by activation of distinct P1 (adenosine) and P2 (ATP) cell-surface receptors present on neurons,
astrocytes, and microglia, as well as other cells that are present in the central nervous system (CNS) under different conditions (e.g.,
macrophage infiltration). These receptors are generically known as purinergic receptors (1 ).

FIGURE 15.1. Structures of P1 and P2 agonists and modulators of adenosine availability.

Adenosine, ADP, and ATP, and, to a lesser extent, UTP are well-known intracellular constituents, intimately involved in all aspects
of cell function acting as enzyme cofactors, sources of energy, and building blocks for DNA. Thus, the factors regulating their
availability in the extracellular space as chemical messengers have been an area of active research and considerable debate since
the late 1970s (2 ).

ATP can be released as a cotransmitter together with acetylcholine, norepinephrine, glutamate, γ-aminobutyric acid (GABA),
calcitonin gene–related peptide, vasoactive intestinal peptide, and neuropeptide Y (3 ). ATP is available on demand, and the body
can synthesize its own weight in ATP per day (4 ). Even though extracellular ATP levels can reach millimolar concentrations in the
extracellular local environment after release or cellular perturbation (1 ), these concentrations are miniscule compared with the
overall steady-state nucleotide content of the cell. Once released, in addition to interacting directly with P2 receptors, ATP can be
hydrolyzed by a family of approximately 11 ectonucleotidases that metabolize ATP, ADP, diadenosine polyphosphates such as Ap4A,
Ap5A (Fig. 15.2 ), and nicotinamide-adenine dinucleotide (5 ). Ecto-ATPases hydrolyze ATP to ADP, ectoapyrases convert both ATP
and ADP to AMP, and ecto-5′-nucleotidase converts AMP to adenosine. The activities of ectoapyrase and ecto-5′-nucleotidase can
change with cellular dynamics (6 ), and in guinea pig vas deferens, soluble nucleotidases are released together with ATP and
norepinephrine (7 ), a finding representing a potential mechanism to limit the actions of extracellular ATP by enhancing its
inactivation. The metabolic pathways linking ATP, ADP, AMP, and adenosine and the potential for each of these purines to elicit
distinct receptor-mediated effects on cell function form the basis of a complex, physiologically relevant, purinergic cascade
comparable to those involved blood clotting and complement activation (8 ) (Fig. 15.3 ).
 P.192

FIGURE 15.2. Structures of P2 receptor agonists.

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FIGURE 15.3. The purinergic cascade. ATP is released into the extracellular milieu from nerves or cells, where they can
interact to form a purinergic cascade. ATP acts at a variety of P2 receptors (see text) and is sequentially degraded to ADP and
AMP by ectonucleotidase activity. ADP interacts with P2T receptors. AMP gives rise to adenosine, which can interact with the
various P1 receptors (A1, A2A, A2B, A3). Adenosine can also be formed by intracellular 5′-nucleotidase activity. Adenylate charge
indicates the transfer of energy in the form of adenine nucleosides or nucleotides from one cell to another (see ref. 13). KATP
is an ATP-modulated potassium channel.
The extracellular effects of ATP on the various members of the P2 receptor family are terminated either by receptor desensitization
or by dephosphorylation of the nucleotide, leading to the formation of ADP, AMP, and adenosine. These latter compounds have their
own receptor-mediated functional activities, some of which are antagonistic to one another. For instance, ATP antagonizes ADP
actions on platelet aggregation, whereas adenosine-elicited CNS sedation contrasts with the excitatory actions of ATP on nerve cells
(9 ). In the broader framework of ATP-modulated proteins (or ATP-binding cassette proteins), ATP-sensitive potassium channels (KATP)
undergo activation when intracellular ATP levels are reduced (10 ,11 ). Thus, as P2 receptor–mediated responses decrease because
of ATP hydrolysis to adenosine, P1-mediated responses and KATP-mediated responses are enhanced. In addition to activating the as
yet uncloned platelet P2T receptor, ADP also enhances its own availability. Activation of A1 and A2A receptors can inhibit ATP
availability (1 ), and activation of hippocampal A2A and A3 receptors can desensitize A1 receptor–mediated inhibition of excitatory
neurotransmission (12 ). The transfer of purines transfer from one cell to another in the context of cellular adenylate charge (13 )
reflects another means by which purines can modulate cellular communication, in terms of both information transfer and alteration
of the target environment. ATP also functions as a substrate for synaptic ectokinases, which modulate the phosphorylation state of
the synaptic membrane (14 ) and, consequently, the intrinsic properties of the synapse. Once in the extracellular space, ATP thus
has the ability to function as a pluripotent modulator of synaptic function.
 P.194

The corresponding role of UTP in terms of functional synaptic signaling is less well understood (15 ), and although high
concentrations of exogenous uracil have been shown to modulate dopaminergic systems in the CNS (16 ), data on the existence of a
“uridine receptor” equivalent to the P1 receptor are limited (17 ).
Extracellular adenosine levels at rest are in the range of 30 to 300 nM (18 ), and they subserve a physiologic role in tissue
homeostasis as reflected by the CNS stimulant actions of caffeine, a natural methylxanthine that acts as an antagonist to counteract
the sedative actions of endogenous adenosine, and the role of the nucleoside as an endogenous hypnotic (19 ). Adenosine also acts
as an autocrine homeostatic agent or, as conceptualized by Newby (20 ), a “retaliatory metabolite,” to regulate the tissue energy
supply-and-demand balance in response to changes in blood flow and energy availability and thus to conserve tissue function under
adverse conditions. Reduced oxygen or glucose availability resulting from tissue trauma, such as during stroke, epileptogenic activity,
and reduced cerebral blood flow, leads to ATP breakdown and the formation of ADP, AMP, and adenosine.
Under basal conditions, extracellular levels of adenosine are tightly regulated by ongoing metabolic activity. Bidirectional
nucleoside transporters and the enzymes adenosine deaminase (ADA) and adenosine kinase (AK) regulate adenosine removal from
the extracellular space (21 ). Numerous studies have shown that inhibition of AK is physiologically more relevant in increasing
extracellular adenosine levels than inhibition of ADA or adenosine transport. AK inhibitors are also more effective in enhancing the
neuroprotective actions of endogenous adenosine than inhibitors of ADA or adenosine transport (8 ,21 ). Selective AK inhibitors, such
as GP 3269 and ABT-702 (Fig. 15.1 ), and ADA inhibitors, such as compound 1 (Fig. 15.1 ), are effective site- and event-specific
agents that can locally enhance levels of adenosine in areas of tissue trauma and thus have the potential to avoid the potential
cardiovascular side effects associated with a general elevation of extracellular levels of the purine (8 ). However, data have shown
that in vivo administration of AK inhibitors (22 ), even at single doses close to where these agents show efficacy in animal models of
epilepsy and pain, results in brain microhemorrhaging that can lead to minicerebral infarcts and cognitive impairment. Based on this
finding, the AK approach to selective modulation of endogenous adenosine function does not appear to have a sufficient therapeutic
window in CNS tissues to be a viable drug discovery target.
Adenosine has both presynaptic and postsynaptic effects on neurotransmission processes (12 ), whereas ATP has excitatory actions in
a variety of neuronal systems including rat trigeminal nucleus, nucleus tractus solitarius, dorsal horn, and locus ceruleus. The
nucleotide also functions as a fast transmitter in guinea pig celiac ganglion and rat medial habenula (9 ). Electrophysiologic studies
on P2X and neuronal nicotinic receptor (nAChR)–mediated responses suggested that these two ligand-gated ion channels (LGICs)
interacted with one another, with each receptor containing a inhibitory binding site for agonists active at the corresponding
receptor and resulting in functional cross-talk between the systems (23 ). Recombination of nAChR α-subunits with P2X receptor
subunits to form functional receptor constructs has also been reported (24 ), a finding further suggesting that heterooligimerization
between these two different classes of LGICs may occurs and represents a molecular basis for the cross-talk hypothesis. This finding
also adds a layer of further complexity to an already complex situation in understanding the precise subunit composition of
functional P2X receptors. Dimerization of the A1 subtype of the P1 (adenosine) G-protein–coupled receptor (GPCR) also occurs (25 ),
a finding consistent with the emerging view that functional homooligimerization and heterooligimerization of a variety of GPCRs is
the norm rather than the exception (26 ).

 P1 AND P2 RECEPTORS
 MITOCHONDRIAL PURINE RECEPTORS?
 THERAPEUTIC POTENTIAL OF PURINES IN NERVOUS TISSUE
 PURINERGIC THERAPEUTICS
 CHALLENGES IN THE DEVELOPMENT OF CNS-SELECTIVE THERAPEUTIC AGENTS
 ACKNOWLEDGMENTS

P1 AND P2 RECEPTORS
Part of "15 - Purinergic Neurotransmission "
Four distinct P1 receptors sensitive to adenosine and 12 P2 receptors sensitive to ADP, ATP, and UTP have been cloned and
characterized (1 ), thus providing a diversity of discrete cellular targets through which adenosine, ADP, ATP, and UTP can modulate
tissue function (Table 15.1 ). The four adenosine-sensitive P1 receptors are designated A1, A2A, A2B, and A3. Functional P2 receptors
are divided into ionotropic P2X receptors, a family of eight LGICs (P2X1 to P2X8), and the metabotropic P2Y family, which consists of
the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 GPCRs. The missing numbers in the P2Y family sequence are proposed receptors
that have been subsequently found to lack functional responses, are species variants, or have been inadvertently assigned to the P2
receptor family (1 ,8 ).

TABLE 15.1. P2X RECEPTORS

Adenosine (P1) Receptors

All four P1 GPCRs—A1, A2A, A2B, and A3 (Table 15.1 )—are heterogeneously distributed in a variety of mammalian tissues including
heart, smooth muscle, kidney, testis, platelets, leukocytes, and adipocytes. in addition to the CNS. The A1 receptor is widely
distributed in the CNS and is functionally coupled to inhibition of cyclic AMP (cAMP) formation, stimulation of potassium
conductance, inhibition of N-channel–mediated calcium conductance, stimulation of phospholipase C production, and modulation of
nitric oxide production (1 ,12 ). Selective agonists for the A1 receptor are all adenosine analogues and include cyclohexyl (CHA; A1 Ki
= 1 to 5 nM), cyclopentyl (CPA; A1 Ki = 0.6 nM), and 2-chlorocyclopentyl (CCPA; Ki = 0.6 nM) adenosine (27 ) (Fig. 15.1 ). Agonist
effects at the
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A1 receptor are selectively blocked by the 8-substituted xanthines, cyclopentylxanthine (Fig. 15.3 ) (CPX; Ki = 0.46 nM) and CPT (Ki =
24 nM), and by nonxanthines such as N-0861 (Ki = 10 nM). The A1 receptor shows distinct species pharmacology (8 ). Like other GPCRs,
it can be allosterically modulated (28 ) by compounds such as PD 81,723 (Fig. 15.1 ) that, although not directly interacting with the
agonist binding site of the receptor, stabilize an agonist-preferring conformation of the A1 receptor independent of G-protein
interactions (29 ).

The A2 receptor exists in two distinct molecular and pharmacologically subtypes, both of which are linked to activation of adenylate
cyclase (1 ). The A2A receptor has high affinity for adenosine, may also use N- and P-type Ca2+ channels as signal transduction
mechanisms, and is localized in the striatum, nucleus accumbens, and olfactory tubercle regions of mammalian brain. The lower-
affinity A2B receptor is more ubiquitously distributed throughout the CNS (1 ,30 ). The adenosine analogue, CGS 21680C (Fig. 15.1 )
(Ki A2A = 15 nM), is the present agonist of choice for the A2A receptor with the xanthine antagonists KF 17837 (Fig. 15.4 ) (Ki A2A = 24
nM) and CSC 8-(3-chlorylstyryl) caffeine (Ki A2A = 9 nM) and nonxanthines such as SCH 58261 (Ki A2A = 2.3 nM) and ZM 241385 (Ki A2A =
0.3 nM), being up to 6,800-fold selective for the A2A receptor (27 ). Like the A1 receptor, the A2A receptor also shows species-
dependent pharmacology (8 ). The A2B receptor has been cloned and is widely distributed in brain and peripheral tissues (1 ,30 ).
However, its functional characterization has proven difficult because of a paucity of selective ligands. Responses more potently
elicited by the nonselective adenosine agonist, NECA (Fig. 15.1 ) and not by selective A1-, A2A-, or A3-receptor agonists can be
attributed to A2B-receptor activation. Enprofylline (Fig. 15.3 ) is a selective, albeit weak, A2B-receptor antagonist (30 ).

FIGURE 15.4. Structures of P2 receptor antagonists.

The A3 receptor was the first P1 receptor identified by cloning rather than by pharmacologic characterization and is linked to
inhibition of adenylate cyclase and elevation of cellular inositol 1,4,5-triphosphate (IP3) levels and intracellular Ca2+. It also shows
distinct species-dependent pharmacology, especially in regard to xanthine antagonist blockade of the rat A3 receptor (1 ,31 ,32 ),
and it shows widespread distribution with low levels in brain. IB-MECA (Fig. 15.1 ) (Ki = 1nM) and its 2-chloro analogue (Cl- IB-MECA;
Ki = 0.3 - 0.7 nM) are potent and selective A3-receptor agonists (27 ). The human, but not the rat, A3 receptor is selectively blocked
by xanthines (31 ), such as DBXRM (Ki A3 = 229 nM), and by nonxanthines such as MRS 1191, MRS 1220, L-249313, L-268605, and MRE-
1008-21M (Fig. 15.4 ). A3 receptors are involved in mast cell function, eosinophil apoptosis, and the phenomenon known as
preconditioning that occurs during ischemic reperfusion of the heart that protects against myocardial infarction (1 ,33 ).

P2 (ATP and UTP) Receptors

P2 receptors were originally classified on the basis of the rank-order potency of agonists structurally related to ATP. Most of these
putative receptors (with the exception of the P2T receptor) have been subsequently cloned and functionally characterized in various
heterologous expression systems (1 ). However, their functional characterization in native tissues and in animals has been limited by
a paucity of potent, selective, and bioavailable ligands, both agonists and antagonists. All known P2-agonist ligands are analogues of
ATP, UTP, and ADP and, irrespective of their degree of chemical modification, show varying degrees of susceptibility to extracellular
degradation and differences in intrinsic activity (27 ,34 ). The selectivity and potency of these agonists are thus very much
dependent on the tissue preparation and species used and also on the experimental protocol. Indeed, few studies exist in which a
systematic evaluation of the relative selectivity of P2-receptor agonists has been determined. BzATP (Fig. 15.2 ), which is widely
used as a selective agonist for the P2X7 receptor (EC50 = 18 μM), is, however, far more potent at transfected rat and human P2X1 (EC50
= 1.9 nM) and P2X3 (EC50 = 98 nM) receptors (35 ) and thus cannot be used, a priori, in defining a P2X7-receptor–mediated response.

P2 receptors are present on excitable tissues, such as neurons,

 P.196

glia, and smooth muscle cells (1 ), and can be grouped into three classes based on agonist effects (36 ). Group 1, comprising the
P2X1 and P2X3 receptors, has high ATP affinity for ATP (EC50 = 1 μM) and is rapidly activated and desensitized. Group 2 includes the
P2X2, P2X4, P2X5, and P2X6 receptors that have lower ATP affinity (EC50 = 10 μM), have a slow desensitization profile, and exhibit
sustained depolarizing currents. The only receptor in Group 3 is the P2X7 LGIC, which has low ATP affinity (EC50 = 300 - 400 μM) and
shows little or no desensitization on agonist exposure.

P2X Receptors
P2X receptors are ATP-gated LGICs formed from various P2X subunits that share a common motif of two transmembrane-spanning
regions (2TM). Like the amiloride-sensitive epithelial Na+ channel, P2X receptor subunits have a large extracellular domain with both
the N- and C-termini located intracellularly (1 ,37 ). A functional P2-receptor channel consists of multimeric combinations of the
various P2X subunits to form a nonselective pore permeable to Ca2+, K+, and Na+ that mediates rapid (approximately 10-millisecond)
neurotransmission events. Available evidence indicates that functional P2X receptors are trimeric, in contrast to the typical
pentameric structure of other LGICs (38 ). In addition to putative P2X1 to P2X7 homomers, P2X1/5, P2X2/3, and P2X4/6 functional
heteromers have been identified (1 ,39 ). P2X5 and P2X6 receptors do not appear to exist in homomeric form, but rather as
heteromers with other P2X receptor subtypes. Unlike other LGICs, such as nAChRs, the 5-hydroxytryptamine (5-HT3) receptor, little
is known regarding the agonist (ATP) binding site on P2X receptor constructs or of ancillary sites that may modulate receptor
function.

The utility of current P2-receptor antagonists, such as PPADS, DIDS, reactive blue-2, and suramin (Fig. 15.5 ) (27 ), is limited by
their lack of selectivity for different P2
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 P.198

receptors and other proteins (34 ) or by their limited potency and bioavailability. These compounds can also inhibit the
ectonucleotidases responsible for ATP breakdown, thus confounding receptor characterization (40 ). Radioligand-binding assays for
P2 receptors are also far from robust; available ligands binding to cell lines lack any type of P2 receptor (41 ). The use of high
throughput screening techniques to identify novel ligands thus depends on functional fluorescence assays such as FLIPR
(fluorescence imaging plate redder) rather than binding.

FIGURE 15.5. Structures of P1 receptor antagonists.

Among the newer P2X-receptor antagonists (Fig. 15.5 ) are the following: TNP-ATP, a noncompetitive, reversible allosteric
antagonist at P2X1 and P2X3 receptors with nanomolar affinity (42 ) that also has weak activity at P2X4 and P2X7 receptors; Ip5I, a
potent, selective P2X1 antagonist (Ki < 100 nM) antagonist (43 ); KN-62, a potent (IC50 = 9 to 13 nM), noncompetitive antagonist of the
human P2X7 receptor that is inactive at the rat P2X7 receptor (44 ). The ATP analogue, A3P5PS (Fig. 15.5 ), is a partial agonist–
competitive antagonist at the turkey erythrocyte P2Y1 receptor (27 ), with the derivative, MRS 2179 being a full P2Y1-receptor
antagonist (IC50 =330 nM). AR-C 69931-MX (Fig. 15.5 ) is a potent, selective antagonist at the ADP-sensitive P2T/P2YAC receptor
involved in platelet aggregation that is currently in clinical trials as a novel antithrombotic agent (45 ).

P2X1 receptors are activated by 2MeSATP, ATP, and αβ-meATP (Fig. 15.2 ), and they exhibit rapid desensitization kinetics (Table
15.2 ) (1 ). P2X1 subunits are present in the dorsal root, in trigeminal and celiac ganglia, and in spinal cord and brain. The P2X2
receptor is activated by 2MeSATP and ATPγS but is insensitive to αβ-meATP and βγ-meATP (Fig. 15.2 ). It is present in brain, spinal
cord, superior cervical ganglia, and adrenal medulla. P2X2-1, P2X2-2, P2X2-3R, and P2X2-3 receptors are splice variants of the P2X2
receptor that have been localized, among other places, to the cochlear endothelium, an area in the ear associated with sound
transduction (46 ). P2X1 and P2X2 receptors can be blocked by PPADS and suramin (1 ). The P2X3 receptor has a rank order of
activation in which 2MeSATP >> ATP > αβ-meATP and is localized to a subset of sensory neurons that includes the dorsal root,
trigeminal, and nodose ganglia (1 ). It has similar properties to the P2X1 subtype including αβ-meATP sensitivity and rapid
desensitization kinetics. P2X2 and P2X3 subunits can form a functional heteromeric P2X2/3 receptor in vitro (39 ) that combines the
pharmacologic properties of P2X3 (αβ-meATP sensitivity) with the kinetic properties of P2X2 (slow desensitization). P2X4 receptors
are activated by 2MeSATP and are only weakly activated by αβ-meATP. The rat and human homologues of the P2X4 receptor differ in
their sensitivity to suramin and PPADS; the human P2X4 receptor is weakly sensitive, and the rat P2X4 receptor is insensitive to these
P2X-receptor antagonists (1 ). The P2X4 receptor is present in rat hippocampus, superior cervical ganglion, spinal cord, bronchial
epithelium, adrenal gland, and testis, as well as human brain. The agonist profile for the P2X5 receptor is ATP > 2MeSATP > ADP with
αβ-meATP being inactive. This receptor does not exhibit rapid desensitization kinetics but is blocked by suramin and PPADS. Message
for the P2X5 receptor is present in the central horn of the cervical spinal cord, in trigeminal and dorsal root ganglia neurons, and in
the brain in the mesencephalic nucleus of the trigeminal nerve. The P2X6 receptor is present in the superior cervical ganglion,
cerebellar Purkinje cells, spinal motoneurons of lamina IX of the spinal cord, superficial dorsal horn neurons of lamina II, and
trigeminal, dorsal root, and celiac ganglia (1 ). P2X4 and P2X6 subunits form functional heteromers in vitro (39 ).

TABLE 15.2. CLASSIFICATION

OF P2Y RECEPTORS

The P2X7 receptor, also known as the P2Z/P2Z receptor before it was cloned (47 ), is present in the superior cervical ganglion and
spinal cord, mast cells, and macrophages (48 ). Cerebral artery occlusion results in an increase in P2X7 immunoreactivity in the
stroke-associated penumbral region (49 ). The P2X7 receptor has a long (240 amino acid) intracellular C-terminal region that allows
the receptor to form
 P.199

a large nonselective cytolytic pore on prolonged or repeated agonist stimulation (1 ,48 ). Exposure of the P2X7 receptor to ATP for
brief periods (1 to 2 seconds) results in transient pore opening that mediates cell-to-cell communication. Prolonged receptor
activation triggers cytolytic pore formation with the initiation of an apoptotic cascade involving caspase-1 (interleukin 1β convertase)
and an associated release of IL-1β from macrophages (48 ). The P2X7 receptor is partially activated by saturating concentrations of
ATP and is fully activated by the ATP analogue BzATP (Fig. 15.2 ). The ability to form a cytolytic pore was considered unique for the
P2X7 receptor, but other P2X receptors such as P2X and P2X show the same phenomenon on prolonged exposure to ATP, a finding
indicating that the intracellular C-terminal tail is not a prerequisite for cytolytic pore formation (50 ,51 ).

P2Y Receptors
P2Y receptors are GPCRs activated by purine or pyrimidine nucleotides (1 ,52 ). The seven mammalian functional subtypes, P2Y1,
P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13, have been cloned and are coupled to Gq11. Receptor activation results in stimulation of
phospholipase C and IP3 activation and subsequent release of calcium from intracellular stores. The P2T receptor, present in platelets
and preferentially sensitive to ADP, has been cloned, as the P2Y12 receptor.

The P2Y1 receptor is preferentially activated by adenine nucleotides, with 2MeSATP the most potent. UTP and UDP are inactive at
this receptor. Suramin, PPADS, cibacron blue, A3P5PS, and MRS 2179 (Fig. 15.5 ) are competitive antagonists at this receptor (1 ,27 ).
The P2Y2 receptor is activated by both ATP and UTP; nucleotide diphosphates are inactive (1 ,52 ). Antagonists such as suramin are
less efficacious at the P2Y2 receptor. UTP is the preferred agonist for the P2Y4 receptor, with ATP and the nucleotide diphosphates
inactive. Diphosphates are more active at the P2Y6 receptor than triphosphates, and this has led to the classification of the P2Y6
receptor as a UDP-preferring receptor. The P2Y11 receptor is unique among other P2Y receptors in that only ATP serves as an agonist
for this receptor (53 ). The P2Y12 and P2Y13 receptors are ADP-selective receptors.

Diadenosine polyphosphates including Ap4A and Ap5A (Fig. 15.2 ) comprise another group of purine-signaling molecules that
modulate cell function by activation of cell-surface P2 receptors (1 ,54 ). Whereas an Ap4A receptor that modulates
neurotransmitter release and amphetamine-elicited Ap4A release has been pharmacologically characterized in nervous tissue, it is
unclear whether diadenosine polyphosphate actions involve distinct receptor subtypes or reflect activation of known P2 receptors.
Receptors for the diadenosine polyphosphates have not yet been cloned.

MITOCHONDRIAL PURINE RECEPTORS?

Part of "15 - Purinergic Neurotransmission "

In addition to functioning as the key source of ATP within the cell, mitochondria play a key role in the apoptotic cascade as the
source of the cytochrome C that is released after changes in mitochondrial transition pore function elicited by members of the bcl-2
family of cell death proteins (55 ). The ability of the P2X7 receptor to initiate an apoptotic cascade by activation of caspase-1 (48 )
and the key role of mitochondria in various degenerative diseases (56 ,57 ) raise the question whether intracellular P2 receptors are
present on the outer mitochondrial membrane and may provide a direct mechanism for ATP to influence mitochondrial function.

THERAPEUTIC POTENTIAL OF PURINES IN NERVOUS TISSUE

Part of "15 - Purinergic Neurotransmission "

Adenosine potently inhibits the release of the neurotransmitters dopamine, GABA, glutamate, acetylcholine, serotonin, and
norepinephrine and acts through presynaptic A1 receptors (12 ). Adenosine acts preferentially on excitatory versus inhibitory
neurotransmitter release, a finding suggesting a degree of physiologic specificity in modulating brain function. Adenosine also
directly modulates postsynaptic neuronal excitability by activating A1 and A2A receptors resulting in hyperpolarization of the
postsynaptic membrane.

Over the past 2 decades, many studies have provided evidence of involvement of purines in the actions of various CNS-active drugs
including antipsychotics, antidepressants, anxiolytics, and cognition enhancers. These studies have come from experiments in which
the effects of known CNS drugs representative of these therapeutic classes were examined for their ability to modulate adenosine-
mediated responses in the CNS, or, alternatively, they studied the effects of various P1 ligands, both agonists or antagonists, on the
effects of such prototypic CNS agents. In many instances, only single, somewhat high, concentrations of an isolated compound, or
limited numbers of compounds, were used to generalize to a complete class of psychotherapeutic agents, often with no negative
control data, thus limiting the value of the data (58 ).

For P2 receptors, the absence of ligands, agonists, and antagonists has limited the functional characterization of the various
receptor subtypes. The delineation of a role for P2 receptors in CNS disorders has been postulated largely on the basis of in situ
localization of the mRNAs encoding the different P2 receptor subtypes or of immunohistochemical studies.

Transgenic Models of P1 and P2 Receptor Function

For both P1 and P2 receptors, the use of mice either deficient in, or overexpressing, a targeted receptor can potentially
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provide a unique means to assess the role of the given receptor, the phenotype of which will provide information on the role of the
receptor. Although this approach is not always straightforward because some phenotypes are fatal and, in others, a knockout of one
receptor leads to compensation in associated receptor systems such that the resultant mouse phenotype is atypical, knockouts can
be helpful in the absence of selective antagonists or antisense probes.

P1 receptor knockouts show altered cardiovascular function (A1) and reduced exploratory activity, aggressiveness, hypoalgesia, and
high blood pressure (A2A) (59 ). P2 knockouts are associated with decreased male fertility (P2X1) (60 ), decreased nociception and
bladder hyporeflexia (P2X3) (61 ), decreased platelet aggregation and bleeding time (P2Y1) (62 ), and reduced chloride secretion
(P2Y2) (63 ). A preliminary report on a P2X7 knockout has appeared (64 ).

PURINERGIC THERAPEUTICS
Part of "15 - Purinergic Neurotransmission "

Three distinct classes of compound can modulate P1 and P2 receptor function: (a) conventional agonist, partial agonist and
antagonist ligands; (b) allosteric modulators of receptor function; and (c) modulators of the endogenous systems that regulate the
extracellular availability of ATP, adenosine, UTP, and their respective nucleotides. This last group includes the various ecto-ATPases
that catalyze the degradation of nucleotides (5 ), ADA, AK, and the bidirectional member transporter systems that remove
adenosine from the extracellular environment (21 ,65 ). From data on AK effects in brain tissue (22 ), it appears that modulation of
endogenous adenosine levels by inhibition of AK is not a viable drug discovery approach.

Efforts over the last 25 years to develop directly acting P1-receptor agonists and antagonists as therapeutic agents (8 ) have proven
less than successful because of a combination of the choice of disease states in which other therapeutic modalities are clearly
superior (58 ) and side effects are associated with global receptor modulation. Partial agonists, allosteric modulators, and novel
modulators of ATP metabolism may prove clinically useful agents with improved therapeutic indices (65 ).

Stroke and Ischemia

Extracellular adenosine levels are markedly increased after hypoxia and focal ischemia, a finding providing additional evidence that
the purine acts as a homeostatic neuroprotective agent (8 ). Adenosine-receptor agonists such as CHA reduce stroke-related cell
death and hippocampal neurodegeneration, whereas adenosine antagonists exacerbate ischemic brain damage by enhancing
glutamate release. The neuroprotective effects of adenosine are mediated by several P1 receptors: A1-receptor activation stabilizes
neuronal membrane potential, inhibits neuronal excitability and glutamate release (8 ,12 ), and thus prevents initiation of the
stroke cascade (66 ).

Adenosine also hyperpolarizes astrocyte membranes limiting extracellular glutamate and potassium accumulation and modulates
local cerebral blood flow and local inflammatory responses, such as platelet aggregation, neutrophil recruitment, and adhesion
acting through the A2A receptor (67 ). A3-receptor agonists have biphasic effects on cell survival. At nanomolar concentrations, they
are neuroprotective and inhibit apoptosis, but at micromolar concentrations they are neurotoxic (31 ).

mRNA for the P2X7 receptor is up-regulated on microglial cells in the ischemic penumbral region 24 hours after middle cerebral
artery occlusion in the rat (49 ), a finding indicating that cytolytic pore formation and inflammatory cytokine release are associated
with neural trauma and neurodegeneration. Antisense to the P2X7 receptor or selective receptor antagonists may represent a novel
approach to the treatment of stroke.

Epilepsy
Seizure activity is associated with rapid and marked increases in CNS adenosine concentrations in animals (68 ), as well as in
patients with epilepsy with spontaneous-onset seizures (69 ). Seizure activity induced by a variety of chemical and electrical stimuli
in animal models is reduced by adenosine and related agonists (68 ) acting through A1receptors. In electrically kindled seizure
models, adenosine agonists reduce seizure severity and duration without significantly altering seizure threshold. These
anticonvulsant effects are blocked by doses of methylxanthines that, when given alone, have no observable effect on seizure
activity (68 ), a finding leading to the hypothesis that adenosine functions as an endogenous anticonvulsant.

Neurodegeneration: Alzheimer’s Disease and Parkinson’s Disease

The nerve cell death that follows excessive glutamate release and changes in calcium homeostasis after ischemia and hypoxia may
reflect an acute manifestation of more subtle, long-term changes associated with apoptotic and necrotic cell death in Alzheimer’s
disease (AD) and Parkinson’s disease (PD). Adenosine antagonists including caffeine, theophylline, and BIIP 20 (Fig. 15.4 ) are potent
CNS stimulants (8 ,18 ), and they can enhance cognition in animal models by blocking the actions of endogenous adenosine. Certain
compounds acting by purinergic mechanisms, such as BIIP 20 and propentofylline, have been examined in the clinic for their efficacy
in cognitive disorders. Although provocative clinical data have been generated, neither compound showed sufficiently robust
efficacy in larger AD trials
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to warrant continuation. However, aged patients with rheumatoid arthritis who consume large quantities of antiinflammatory agents
such as indomethacin show an inverse correlation for the incidence of AD, a finding highlighting the pivotal role of inflammation in
disease origin. Adenosine agonists and AK inhibitors have marked antiinflammatory activity (67 ), inhibiting free radical production,
and thus they may be effective in maintaining cell function in AD, in addition to modulating cytotoxic events.
Trophic factors in nervous tissue act to ensure neuronal viability and regeneration. Withdrawal of nerve growth factor, which exerts
a tonic cell death–suppressing signal, leads to neuronal death. Polypeptide growth factors linked to receptor tyrosine kinases, such
as fibroblast growth factors, epidermal growth factor, and platelet-derived growth factor, are increased with neural injury (70 ).
ATP can act in combination with various growth factors to stimulate astrocyte proliferation and to contribute to the process of
reactive astrogliosis, a hypertrophic-hyperplastic response typically associated with brain trauma, stroke and ischemia, seizures, and
various neurodegenerative disorders. In reactive astrogliosis, astrocytes undergo process elongation and express glial fibrillary acidic
protein, an astrocyte-specific intermediate filament protein with an increase in astroglial cellular proliferation. ATP increases glial
fibrillary acidic protein and activator protein-1 (AP-1) complex formation in astrocytes and mimics the effects of basic fibroblast
growth factor (70 ). Both ATP and guanosine triphosphate induce trophic factor (nerve growth factor, neurotrophin-3, fibroblast
growth factor) synthesis in astrocytes and neurons. The effects of guanosine triphosphate are, however, not consistent with any
known P2-receptor profile. Nonetheless, these studies have focused research on the hypoxanthine analogue, neotrofin (AIT-082) (Fig.
15.1 ), which up-regulates neurotrophin production and enhances working memory and restores age-induced memory deficits in mice
(71 ). This compound has shown positive effects in early phase II trials for AD.
In 1974, Fuxe showed that methylxanthines such as caffeine could stimulate rotational behavior and could potentiate the effects of
dopamine agonists in rats with unilateral striatal lesions Conversely, adenosine agonists blocked the behavioral effects of dopamine
(72 ). Anatomic links between central dopamine and adenosine systems are well established; adenosine A2A receptors are highly
localized in striatum, nucleus accumbens, and olfactory tubercle, brain regions that also have high densities of dopamine D1 and D2
receptors. mRNAs for adenosine A2A receptors and dopamine D2 receptors are co-localized in GABAergic-enkephalin striatopallidal
neurons in the basal ganglia (Fig. 15.6 ) that form an “indirect” pathway from the striatum to the globus pallidus that originates
from striatal GABA-enkephalinergic neurons. Through GABAergic relays, this pathway interacts with a glutaminergic pathway from
the subthalamic nucleus that can activate the internal segment of the pars reticulata, which, turn, through a pars reticulata–
thalamic GABAergic pathway, inhibits the thalamic-cortical glutaminergic pathway. Dysfunction of this pathway may underlie the
movement disorders seen in Huntington chorea and PD. A direct pathway originating in striatal GABAergic–substance P–
dynorphinergic neurons inhibits the internal segment of the pars reticulata to disinhibit the ascending thalamic glutaminergic
pathway and to activate the cortex (Fig. 15.6 ). The balance between the direct (cortical activating) and indirect (cortical inhibiting)
striatal dopaminergic pathways provides a tonic regulation of normal motor activity. These studies indicate that striatal adenosine
A2A receptors may play a pivotal role in neurologic disorders involving basal ganglia dysfunction such as PD. The A2A agonist, CGS
21680, given intrastriatally, attenuates the rotational behavior produced by dopamine agonists in unilaterally lesioned rats.
Mechanistically, radioligand-binding studies have shown an increased efficacy of CGS 21680 in reducing the binding affinity of
supersensitive D2 receptors, a finding supporting the increased sensitivity of animals with supersensitive dopamine receptors to CGS
21680 treatment. Repeated administration of the dopamine antagonist, haloperidol can up-regulate the density of both D2 and A2A
receptors in rat striatum.

FIGURE 15.6. Dopamine–adenosine (ADO) interactions in the substantia nigra. An indirect pathway dopaminergic pathway
arises from the striatal GABA-enkephalinergic dopaminergic neurons on which both dopamine D1 and adenosine A2A receptors
are co-localized. Through a GABAergic interneuron originating in the external globus pallidus, the indirect pathway connects to
a glutaminergic pathway arising in the subthalamic nucleus. This, in turn, can activate the internal segment of the pars
reticulata and, through another GABA pathway, inhibit ascending glutaminegic neurons arising from the thalamus that
innervate the cortex. The direct pathway arises from striatal GABA–substance P–dynorphinergic neurons that, through a
GABAergic relay, inhibit the internal segment of the pars reticulata to disinhibit the ascending thalamic-cortical glutaminergic
pathway. The balance between the direct (activating) and indirect (inhibitory) striatal dopaminergic pathways can then
tonically regulate normal motor activity. Dopaminergic inputs arising from the substantia nigra pars compacta can facilitate
motor activity, inhibiting the indirect pathway by activation of D2 receptors and activating the direct pathway by D1 receptor
activation. (Adapted from Svenningsson P, Le Moine C, Fisone G, et al. Distribution, biochemistry and function of striatal
adenosine A2A receptors. Prog Neurobiol 1999;59:355–396; and Richardson PJ, Kase H, Jenner PG. Adenosine A2A receptor
antagonists as new agents for the treatment of Parkinson’s disease. Trends Pharmacol Sci 1997;18:338–344.)

Adenosine A1 receptor activation can reduce the high-affinity state of striatal dopamine D1 receptors, the A1 receptor agonist, and
CPA blocking D1-receptor–mediated locomotor activation in reserpinized mice (72 ). The nonselective adenosine agonist, NECA, can
attenuate the perioral dyskinesias induced by D1-receptor activation in rabbits. Acting through striatal A2A and A1 receptors,
adenosine directly modulates dopamine-receptor–mediated effects on striatal GABA-enkephalinergic neurons and striatal GABA–
substance P neurons (Fig. 15.6 ). These adenosine agonist–mediated effects are independent of G-protein coupling and may involve
an intramembrane modulatory mechanism involving receptor heterooligimerization (26 ).
The dynamic interactions between dopaminergic and purinergic systems in striatum suggest that dopaminergic dysfunction may be
indirectly ameliorated by adenosine receptor modulation. Selective adenosine A2A receptor antagonists such as KF 17837 and KW
6002 (Fig. 15.4 ) have shown positive effects in 1-methyl-4-[henyl-1,2,3,6-tetrahydropyridine–lesioned marmosets and cynomolgus
monkeys, well characterized animal models of PD, enhancing the effects of L-dopa (73 ,74 ). KW-6002 has successfully completed
human phase I trials. More recently, a 30-year longitudinal study of 8,004 Japanese-American man enrolled in the Honolulu Heart
Program showed an inverse association of the incidence of PD with caffeinated coffee consumption. In men who drank no coffee,
the incidence of PD was 10.4 per 10,000 person-years, and it was 1.9 per 10,000 person-years in men drinking at least 28 oz of
coffee per day (75 ).
Adenosine agonists can mimic the biochemical and behavioral
 P.202

actions of dopamine antagonists in animal models by activation of A2A receptors (9 ,72 ), a process that inhibits dopamine synthesis
and attenuates dopamine transductional processes. CGS 21680, like typical and atypical neuroleptics, can reverse apomorphine-
induced loss of prepulse inhibition (76 ). These actions involve a decrease in dopaminergic neurotransmission, with adenosine
receptor agonists acting as functional dopamine antagonists. Adenosine agonists have a behavioral profile similar to that of
dopamine antagonists in a conditioned avoidance response paradigm (77 ), in which they potently disrupting avoidance responding
without significantly impairing escape behavior. They also produce catalepsy at the same dose levels effective in attenuating
conditioned avoidance response, a property shared by typical neuroleptic agents such as haloperidol. CI-936, an A2A agonist (Fig.
15.1 ), entered clinical trials in the mid 1970s as a novel antipsychotic agent, but its development was discontinued for unstated
reasons.
 P.203

Sleep
The hypnotic and sedative effects of adenosine are well known, as are the central stimulant activities of the various xanthine
adenosine antagonists including caffeine (18 ). Direct adenosine administration into the brain elicits an EEG profile similar to that
observed in deep sleep, an increase in rapid eye movement (REM) sleep with a reduction in REM sleep latency resulting in an
increase in total sleep. In contrast, caffeine suppresses REM sleep and decreases total sleep time. Microdialysis studies have shown
that extracellular adenosine concentrations are increased in basal forebrain in direct proportion to periods of sustained wakefulness
and decline during sleep, a finding indicating that adenosine functions as a endogenous sleep regulator (19 ). Infusion of the A2A
agonist, CGS 21680, into the subarachnoid space associated with the ventral surface of the rostral basal forebrain, an area
designated the prostaglandin D2–sensitive sleep-promoting zone, increased slow-wave and paradoxical sleep, effects that were
blocked by the A2A antagonist, KF 17837 (78 ). The A1-selective agonist, CHA, suppressed slow-wave and paradoxical sleep before
eliciting an increase in low-wave sleep.

Pain
The role of purines in pain perception is well established (79 ,80 and 81 ), and both P1 agonists and P2X antagonists may represent
novel approaches to nociception. ATP application to sensory afferents results in neuronal hyperexcitability and the perception of
intense pain (79 ). These pronociceptive effects are mediated by P2X3 and P2X2/3 receptors present on sensory afferents and in the
spinal cord. The nucleotide also induces nociceptive responses at local sites of administration and can facilitate nociceptive
responses to other noxious stimuli, such as substance P. P2 receptor antagonists such as suramin and PPADS, even though they are
limited in their in vivo effects, reduce nociceptive responses in animal models of acute and persistent pain (1 ,79 ). ATP is released
from certain cell types (e.g., sympathetic nerves, endothelial cells, visceral smooth muscle) in response to trauma (1 ,8 ,79 ), and
P2X3-receptor expression is up-regulated in sensory afferents and spinal cord after damage to peripheral sensory fibers. P2X3-
receptor knockout mice have reduced nociceptive responses (61 ). The effects of adenosine are opposite effects to those of ATP
(80 ), a finding suggesting that the nociceptive effects of ATP can be autoregulated by adenosine production from the nucleotide.
Adenosine, adenosine-receptor agonists, and AK inhibitors inhibit nociceptive processes in the brain and spinal cord. When given
intrathecally, these agents have analgesic activity in a broad spectrum of animal models (e.g., mouse hot plate, mouse tail flick, rat
formalin, mouse abdominal constriction, rat neuropathic pain models), effects that are blocked by systemic or intrathecal
administration of adenosine antagonists. Adenosine A1-receptor agonists modulate acutely evoked and inflammation-evoked
responses of spinal cord dorsal horn nociceptive neurons and can also inhibit pain behaviors elicited by spinal injection of substance
P and the glutamate agonist, N-methyl-D-aspartate (NMDA). Glutamate is a key mediator of the abnormal hyperexcitability of spinal
cord dorsal horn neurons (central sensitization) associated with clinical pain states. A1 agonists can inhibit the spinal cord release of
glutamate and can also reduce cerebrospinal fluid levels of substance P in rat, another key mediator of nociceptive responses.
Adenosine has both presynaptic and postsynaptic effects on transmission from primary afferent fibers to neurons of the substantia
gelatinosa of the spinal dorsal horn (12 ,80 ,81 ), and it involves both peripheral and supraspinal mechanisms. Adenosine agonists
such as CHA and NECA, were 10- to 1,000-fold more potent in inhibiting acetylcholine-induced writhing in mice when these agents
were administered intracerebroventricularly than orally, a finding indicating a supraspinal site of action. The ability of adenosine to
inhibit peripheral neurotransmitter (12 ), and inflammatory processes (67 ), may block peripheral sensitization, a key feature of the
pain resulting from tissue injury and inflammation.

Adenosine agonists are also active in human pain states (81 ). Spinal administration of the A1 agonist, R-PIA, relieved allodynia in a
patient with neuropathic pain without affecting normal sensory perception, whereas adenosine infusion at doses without effect on
the cardiovascular system improved pain symptoms and reduced spontaneous pain and ongoing hyperalgesia and allodynia in patients
with neuropathic pain. Low-dose infusion of adenosine during surgical procedures reduced the requirement for volatile anesthetic
and also for postoperative opioid analgesia (82 ). AK inhibitors, such as CP 3269 and ABT-702 (Fig. 15.1 ), are effective analgesic
agents in animal pain models by effects that can be blocked by xanthine adenosine antagonists.

CHALLENGES IN THE DEVELOPMENT OF CNS-SELECTIVE THERAPEUTIC

AGENTS
Part of "15 - Purinergic Neurotransmission "

The field of purinergic molecular biology and pharmacology has exploded as more is learned about the cellular targets through which
ATP, ADP, AMP, and adenosine (and UTP) produce their effects on mammalian tissues. A clear historical delineation between the P1
and P2 fields is that in the former, more than 20 years of pharmacology and medicinal chemistry resulted in the identification of
receptor selective ligands before the receptors were cloned. In contrast, definitive evidence for the existence of the P2-receptor
family resulted from both pharmacologic and cloning studies. The latter have resulted in the identification of a remarkable diversity
of receptors responsive to ATP, unfortunately in the absence of selective, bioavailable ligands, especially antagonists, that will
allow a clearer understanding of P2-receptor
 P.204

function in normal and pathologic states. Evidence of the oligomerization of GPCRs and the emerging data on P2X heteromers both
within the P2-receptor family and with other LGICs, such as nAChRs, suggest that the dynamics and the actual composition of
systems targeted by purinergic receptors are potentially very complex (83 ).
Early efforts to develop therapeutics based on the modulation of P1-receptor–mediated processes met with limited success. Only
adenosine has been approved for use as a cardiac imaging agent and for the treatment of supraventricular tachycardia, acute
systemic uses that avoid some of the side effects seen with long-acting adenosine agonists. Similarly, the unexpected in vivo effects
of AK inhibitors suggest that this is not a viable approach to the discovery of new drugs. The use of the adenosine antagonist
theophylline for the treatment of asthma and the widespread use of caffeine as a CNS stimulant represent other P1-targeted
therapeutics. The evaluation of A2A antagonists as indirect dopamine agonists for use in PD (73 ,74 and 75 ) is an intriguing and novel
approach to treating this neurodegenerative disorder, although the side effect liabilities are unknown at present.
In contrast, the highly discrete localization of P2X3 receptors to sensory nociceptive neurons (79 ) has led to an intensive effort to
identify P2X3 antagonists as novel analgesic agents. Similarly, the discrete localization of other P2 receptors and evidence from
mouse knockout studies suggest that selective agonists and antagonists for these receptor subtypes may represent very novel
therapeutic agents as well as research tools to understand target function.
A caveat in the drug discovery process, as in all life’s endeavors, is that the less that is known regarding the functional liabilities of
a molecular target, the more attractive it is as drug target. In the area of purinergic medications, the identification of new ligands
in combination with a broader-based evaluation of compound efficacy and side effect liability will greatly assist in the prioritization
of therapeutic targets that are amenable to modulation by purinergic ligands (57 ). Finally, the renewed interest in mitochondria as
cellular organelles that have function beyond energy production (56 ) represents an additional level of molecular targeting for P1-
and P2-receptor ligands that may have benefit in treating human disease states, especially those involving apoptosis (55 ).

ACKNOWLEDGMENTS
Part of "15 - Purinergic Neurotransmission "
I would like to thank Mike Jarvis for his contributions to the previous CD-ROM version of this chapter. Because of space limitations, it
is not possible to cite primary literature sources exhaustively. The reader is referred to reference 1 for a more comprehensive
bibliography.

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78. Satoh S, Matsumura H, Suzuki F, et al. Promotion of sleep mediated by the A2a-adenosine receptor and possible involvement
of this receptor in the sleep induced by prostaglandin D2 in rats. Proc Natl Acad Sci USA 1996;93:5980–5984.

79. Burnstock G. A unifying purinergic hypothesis for the initiation of pain. Lancet 1996;347:1604–1605.

80. Sawynok J. Purines in pain management. Curr Opin CPNS Invest Drugs 1999;1:27–38.

81. Salter MW, Sollevi A. Roles of purines in nociception and pain. In: Abbracchio MP, Williams M, eds. Purinergic and
pyrimidinergic signaling. I. Molecular, nervous and urogenitary system function handbook of experimental pharmacology, vol
151/I. Heidelberg: Springer-Verlag, 2001:371–401.

82. Fukunaga AF. Purines in anesthesia. In: Jacobson KA, Jarvis MF, eds. Purinergic approaches in experimental therapeutics.
New York: Wiley Liss, 1997:471–494.

83. Williams M. Purines: from promise to premise. J Auton Nerv Syst 2000;81:285–288.
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16
Neurotrophic Factors and Intracellular Signal Transduction Pathways

David S. Russell

Ronald S. Duman

David S. Russell and Ronald S. Duman: Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale
University School of Medicine, Connecticut Mental Health Center, New Haven, Connecticut.

A clear limitation of treating diseases of the central nervous system arises from the loss of regenerative potential of the brain at a
very early age. Improper development of neural circuits or injury of neurons appears to be permanently fixed in the adult, with
seemingly little hope of restorative therapy. This is most clearly true of the spectrum of neurologic diseases, such as
neurodegenerative diseases (e.g., Alzheimer's or Parkinson's disease and stroke) and inflammatory disease (e.g., multiple sclerosis).
In addition, breakthrough disorders such as epilepsy or myoclonus reflect inappropriate “wiring” or lack of appropriate feedback
control of neuronal activity. It has also become apparent that some psychiatric diseases represent similar fixed deficits or lack of
appropriate functional adaptation. Neurochemical and neuroanatomic deficits have been documented in affective diseases,
schizophrenia, and anxiety disorders. Methods to recreate the early restorative potential of the brain would have great potential for
significantly, and possibly permanently, reversing the often debilitating features of these neurologic and psychiatric diseases.

Over the last several years, of body of literature has delineated the important roles of neurotrophic factors in guiding the
development of the nervous system. The first identified neurotrophic factor, nerve growth factor (NGF), was identified and
characterized in the pivotal studies of Levi-Montalcini (1 ). The general properties of NGF now essentially define what
neuroscientists consider a neurotrophic factor. A neurotrophic factor is capable of supporting the survival of at least one population
of neurons in culture. It is secreted by a target tissue (either neuronal or nonneuronal) and acts on the neurons that innervate that
tissue to support their survival or differentiation. Finally, a neurotrophic factor is expressed in the appropriate region and at the
appropriate time in development to support the survival of a particular neuronal population. Although several variations and
extensions of these principles have been delineated, the basics of defining a neurotrophic factor remain the same.

An exciting development in the neurosciences has been the realization that neurotrophic factors play important roles in the adult
brain. The time course of neurotrophic factor expression is intriguing and indicates important function in the adult nervous system,
as well as during development (2 ,3 and 4 ). The expression of these factors usually is very high during early development, a time of
substantial growth, differentiation, and modeling of the nervous system. Later the levels generally drop, but they do not subside
completely. In fact, in most cases in which it has been explored, the continued presence of these factors is substantial and is critical
throughout adulthood (e.g., see refs. 5 ,6 and 7 ). Neuronal populations continue to depend on these factors for survival and
optimal functioning.

More intriguing, although perhaps not surprising, studies have clearly shown that after development, these factors participate in the
ongoing remodeling of neuronal function that underlies the adaptability or plasticity of neurons. In some cases, specific neurotrophic
factors have been found to be necessary and sufficient for these changes to occur, from hippocampal plasticity and long-term
potentiation (8 ,9 ,10 and 11 ) to the acquisition of new songs by songbirds (12 ,13 and 14 ). Models of neuronal now often
incorporate components of neurotrophic factor signaling to explain synaptic alterations or strengthening. Contrary to the original
models, these signaling events have been found not only to be retrograde signals from target neurons or other tissues, but also to be
anterograde or autocrine signals. Furthermore, numerous studies have demonstrated that the expression of at least some of these
factors can be rapidly regulated in the adult, a finding supporting a dynamic role in mediating responses to the environment.

Ongoing work in the neurotrophic factor field has been

 P.208
devoted to characterizing the pathways that underlie the intracellular signaling of these factors. This information may then be used to treat many different neurologic and psychiatric
disorders, given the apparent critical roles of neurotrophic factors in the normal functioning and adaptability of the brain, as well as their potential to recapitulate early
developmental processes to restore damaged or maladaptive neural systems. This review focuses on the neurobiology of these factors, their interactions with classical neurotransmitter
systems, and their potential roles in the origin and possible treatment of psychiatric illnesses.

 NEUROTROPHINS
 TRK RECEPTORS
 TRK DOCKING PROTEINS
 NEUROTROPHIC FACTOR INTRACELLULAR SIGNALING PATHWAYS: RAS/ERK (MAPK) CASCADE
 PLC-γ CASCADE
 PI-3-K CASCADE
 INTERACTION OF NEUROTROPHIN SIGNALING CASCADES
 REGULATION OF NEUROTROPHIN SIGNALING BY ACTIVATION OF G-PROTEIN–COUPLED RECEPTORS
 SOME OTHER CLASSES OF NEUROTROPHIC FACTORS AND THEIR SIGNALING SYSTEMS
 ROLE OF NEUROTROPHIC FACTORS IN THE ACTIONS OF PSYCHOTROPIC DRUGS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 DISCLAIMER

NEUROTROPHINS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

The number of neurotrophic factors has now been expanded in to the dozens, each with unique a specificity in terms of biological activity, regional and temporal expression, and
target specificity. Some factors previously known for their effects in other systems, such as insulin-like growth factors or tumor-derived factors, have now clearly been found also to be
neurotrophic factors. These have been grouped into different families largely based on homology at the level of nucleotide sequence and therefore evolutionary relatedness. Perhaps
the best understood and most widely expressed in the brain of these families are the neurotrophins (NTs) (2 ,15 ). NGF is the prototype of the NT family, which also now includes
brain-derived neurotrophic factor (BDNF), NT-3, and NT-4 (or NT-4/5). NGF has the most restricted specificity among these. NGF in the brain acts specifically on cholinergic neurons. In
the rest of the nervous system, it also acts on sympathetic and sensory neurons. BDNF and NT-3 are widely and highly expressed, particularly in cortical and neocortical structures. NT-
4 is also widely expressed, although generally at lower levels in the adult than are the others. The NTs are small, secreted proteins of about 12 kd that contain characteristic
intramolecular disulfide bonds. These then form active noncovalent homodimers. They are found stored in vesicles clustered near the membrane. Each has been cloned and expressed
in active recombinant forms.

TRK RECEPTORS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

Generally better conserved than their ligands, the neurotrophic factor receptors also form families of related proteins (16 ,17 ). These receptors can be found in many different forms,
from single, active proteins to large heteromeric complexes. Common to these are an extracellular ligand-binding portion, a mechanism to transduce this signal across the membrane,
and at least one intracellular signaling apparatus. These may be contained in single proteins or distributed among several interacting proteins. Most, if not all, of the neurotrophic
factor receptor complexes include a protein tyrosine kinase. Although phosphorylation on tyrosine residues represents a relatively small proportion of all protein phosphorylation in the
cell, it seems to be a critical part of neurotrophic factor signal transduction and function.

The NTs act through receptors known as Trk receptors. Given their name from a troponin/receptor kinase gene fusion identified from colon carcinoma, it has now been found that in
their normal (protooncogene) forms, each Trk receptor contains a ligand-binding domain, a single transmembrane domain, and an intrinsic, intracellular tyrosine kinase domain
(16 ,17 ). Each receptor, when transfected into a cell line, is capable of transducing the appropriate NT signals independently of other receptor proteins (18 ). There is specificity
among the Trk receptors at physiologic NT concentrations. TrkA is a receptor for NGF, whereas TrkC is preferentially bound by NT-3. TrkB serves as a receptor for both BDNF and NT-4.
The expression patterns of these receptors correlate with known sensitivity of those neurons to specific NTs. Several studies, particularly in mice with engineered deletions of NTs or
their receptors, have shown significant complexity to these interactions. A review of this work falls beyond the scope of this review.

TRK DOCKING PROTEINS

Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

On binding of an NT, the Trk receptor tyrosine kinase becomes activated. The most critical substrate of this activity appears to be the receptor itself. The receptor becomes rapidly
autophosphorylated, and this is critical to receptor function. The receptor autophosphorylation sites form docking sites for the interaction of downstream signaling molecules (Fig.
16.1 ). Many signaling proteins contain domains that specifically bind to tyrosine residues when they are phosphorylated. Further binding specificity is mediated by the amino acids
surrounding the autophosphorylated tyrosine. The domains of the signaling molecules that are used to bind the tyrosine residues seem to fall into a small number of conserved motifs
(19 ).

FIGURE 16.1. Neurotrophin intracellular signal transduction pathways. This

schematic represents the three major signaling pathways emanating from a Trk-
like tyrosine kinase receptor. The ligand, here a neurotrophin dimer, binds to its
receptor and activates the tyrosine kinase activity. This results in
autophosphorylation and phosphorylation of substrates. A complex forms in which
docking proteins bind to the autophosphorylated receptor and are activated. These
docking proteins bind to the receptor phosphorylation sites by SH2 domains (PLC-γ)
or PTB domains (IRS and Shc). The three pathways shown here are as follows: (1)
The PLC-γ pathway leads to the production of diacylglycerol and intracellular
calcium. This results in activation of CAM kinases and PKC. (2) PI-3-Kinase (PI-3-K),
comprising an 85-kd regulatory subunit and a 100-kd catalytic subunit, generally
becomes activated by binding to an insulin receptor substrate (IRS)–like adaptor
protein, which interacts with the receptor and then activates signaling proteins.
IRS proteins also bind other signaling molecules such as Fyn and Syp. The PI-3-K
then activates AKT and p70 S6-kinase (p70 S6K). AKT has an antiapoptotic effect
through actions on Bad and caspaces. (3) Ras becomes activated through the
stimulation of the GDP/GTP exchange activity of SOS, which is in a complex with
Shc and Grb2. Activated Ras then turns on the cascade of kinases including Raf,
MEK, and erks (also known as MAP kinases). Erks stimulate many known effectors.
Each of these pathways then exerts a number of nuclear and nonnuclear actions
with short- and long-term consequences for the cells.

Most proteins that bind to phosphorylated tyrosines fall into one of two groups. The most common phosphotyrosine binding motif is the src-homology domain 2, or SH-2 domain. SH-2
domains are typically identified based on their homology to other SH-2 domain–containing proteins. Some of these have been shown directly to possess specificity for phosphorylated
tyrosines in the appropriate amino acid context. The SH-2 domain–containing proteins also often contain, or interact with proteins containing, an src-homology domain 3, or SH-3 (20 ).
The other, unrelated conserved motif that directs a separate type of specific protein–protein interaction has been termed, appropriately, a phosphotyrosine binding domain, or PTB
domain. Proteins containing a PTB domain bind to a distinct set of phosphorylated tyrosine residues from those with SH-2 domains (21 ).

After these signaling proteins bind to the activated, autophosphorylated

 P.209

receptor, they become activated. The mechanisms by which they are activated are not entirely clear. Often they, too, become
phosphorylated on tyrosine residues. In addition, their recruitment to the membrane or into signaling complexes plays a role in the
initiation of their activity (22 ,23 ). Activation of each of these NT-signaling proteins triggers distinct downstream cascades of target
enzymes and other biological effects. Although there is great diversity of neurotrophic factor receptors, they seem to trigger only a
few well-conserved types of downstream signaling pathways. Among the best characterized of the pathways include the
Ras/extracellular signal regulated kinase (ERK) pathway, the phosphotidylinositol-3′-OH-kinase (PI-3-K) pathway, and the
phospholipase C-γ (PLC-γ) pathway (24 ). In addition, specific tyrosine phosphatases are activated that modulate these responses
and may contain pathway-activating properties of their own.

NEUROTROPHIC FACTOR INTRACELLULAR SIGNALING PATHWAYS:

RAS/ERK (MAPK) CASCADE
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

The Ras/ERK pathway is regulated by the activity of the Ras proteins. Ras is a small, membrane-associated protein that serves as a
transducer of signal from tyrosine kinase activity to ERK proteins, among other activities (25 ). The activity of Ras depends on the
type of the guanine nucleotide it is bound to. Hence, Ras is a G protein, although distinct from the heterotrimeric G proteins
coupled to many neurotransmitter receptors. Ras is active when binding guanosine triphosphate (GTP), but at rest it is inactive and
bound to guanosine diphosphate (GDP). Activation of Trk receptor tyrosine kinases leads to the binding of an adapter protein in the
Shc family. Shc becomes tyrosine phosphorylated and binds a Grb protein, such as Grb2. Shc contains a PTB domain and an SH-2
domain, whereas Grb2 contains two SH-2 domains and a SH-3 domain. This complex then activates a GDP-GTP exchange factor, such
as SOS, which, in turn, activates Ras through GTP binding.

Once activated, Ras recruits a serine kinase of the Raf family to the membrane, where it is activated. This initiates a cascade in
which Raf activates MEK (from MAPK or ERK kinase), and then MEK phosphorylates and activates ERKs (26 ). ERKs, also known as
mitogen-activated protein kinase (MAPKs), are abundant, multifunctional, intracellular kinases with many different cellular
activities. ERKs have been shown to phosphorylate such diverse proteins as tyrosine hydroxylase, transcription factors, regulators of
protein translation, microtubule proteins, and many others (27 ,28 ). In cells in vitro, ERKs have been shown to mediate neuron
survival, neuritic process elongation (29 ), and levels of specific neuronal enzymes and ion channels, among other effects (30 ). ERKs
have been shown to be important in hippocampal long-term potentiation in brain slices (31 ). Some effects of ERK activation are
very rapid, whereas others are delayed and persistent.

PLC-γ CASCADE
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

A second NT-signaling pathway involves PLC-γ activation (32 ,33 ). Like the better-understood PLC-β, PLC-γ cleaves
phosphatidylinositol phosphates into diacylglycerol and inositol phosphates. Diacylglycerol can activate protein kinase C (PKC),
whereas inositol-1,4,5-tris phosphate releases intracellular stores of calcium. Intracellular calcium can exert numerous effects from
the activation of Ca2+/calmodulin–dependent
 P.210

protein kinases to the production of cyclic adenosine monophosphate through some adenylyl cyclases. All these are known to have
powerful effects on neurons. Unlike PLC-β, which is regulated by heterotrimeric G-protein–coupled receptors, PLC-γ is regulated by
tyrosine phosphorylation (34 ). PLC-γ contains SH-2 and SH-3 domains. When bound to tyrosine phosphorylated receptors, it is
recruited to the membrane and becomes phosphorylated, which activates its PLC activity. Virtually nothing is known about the role
of PLC-γ in the intact brain, although it is likely to exert important effects on neuronal function.

PI-3-K CASCADE
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

Somewhat less well understood is the PI-3-K pathway (35 ,36 and 37 ). Several types of PI-3-Ks have been identified. Type 1 PI-3-Ks
are regulated by tyrosine kinase activity. They are heterodimers of a catalytic α subunit and a regulatory β subunit. The β subunit
contains SH-2 and SH-3 domains. When bound to phosphorylated tyrosines, the β subunit activates the catalytic activity of the α
subunit. This phosphorylates phosphatidylinositol on the 3′-hydroxl group (distinct from the 4′,5′ phosphorylated forms mentioned
earlier). Furthermore, PI-3-K has been shown to possess protein kinase activity and can bind Ras (38 ). The PI-3-K lipid product,
phosphotidylinositol-3′-phosphate, activates at least two protein kinases, AKT and S6-kinase. AKT is best known for its powerful
ability to oppose programmed cell death (i.e., antiapoptotic effects), although it has other metabolic actions as well. S6-kinase is
named for its ability to phosphorylate the ribosomal subunit S6 (although not to be confused with ribosomal S6-kinase RSK), and it
has numerous other cellular effects as well. Currently, these effects are less well elucidated than those of the ERKs. Within neurons,
PI-3-K has been shown to mediate cell survival, initiation of neuritic process outgrowth, and acquisition of sensitivity to glutamate
excitotoxicity (39 ), among other actions. Again, little is known about its role in intact brain.

Although PI-3-K possesses some ability to bind phosphorylated receptors itself, it seems largely to be activated by receptors through
docking proteins. Important PI-3-K docking proteins are the insulin receptor substrate (IRS) family of proteins (40 ). IRS1, IRS2, and
IRS4 are expressed in brain (41 ). More distantly related PI-3-K docking proteins are the GAB family of proteins. All these bind to the
receptors through PTB domains and become phosphorylated on numerous tyrosines. Most of these tyrosines are then bound by PI-3-K,
leading to a substantial amplification of PI-3-K signaling. IRS proteins can be bound by other signaling molecules such as protein
tyrosine phosphatases and also possess numerous serine and threonine phosphorylation sites. Therefore, it is likely that the IRS
proteins are important sites of convergence of numerous types of signaling pathways.

INTERACTION OF NEUROTROPHIN SIGNALING CASCADES

Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

Numerous levels of complexity have been found in the downstream signaling pathways of the NT receptors. PI-3-K has been shown to
contribute to ERK activation by Ras-dependent and Ras-independent process pathways (36 ,38 ,42 ). Ras can contribute to activation
of AKT, and both SHC and IRS can bind to the same phosphorylated tyrosine site, although with differing affinities (43 ). PLC-γ can
activate ERK in neurons and can theoretically terminate PI-3-K signaling by cleaving phosphotidylinositol-3′-phosphate. In addition,
most of these proteins exist in multiple isoforms arising either from different genes or differential splicing of the same gene. These
isoforms are differentially expressed during development and in different brain regions, although there is also a great deal of
overlap. The complement of signaling proteins and adaptors would be expected to determine the effects of the NT on particular
populations of neurons. Regulation of these proteins may influence plasticity or other neuronal responses. Furthermore, this
complexity of expression and cross-talk allows tremendous opportunity for potential sites of therapeutic intervention.

REGULATION OF NEUROTROPHIN SIGNALING BY ACTIVATION OF G-

PROTEIN–COUPLED RECEPTORS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

There is also evidence of cross-talk between G-protein–coupled receptor signaling pathways and the NT cascades. Many different
types of interactions occur between the G-protein receptor–coupled second messenger-dependent pathways and the Trk signaling
pathways. Only a few are discussed here, to demonstrate the complexity and possible types of interactions.

Activation of Trk Docking Proteins and the Ras/ERK Pathway by G-Protein–Coupled

Receptors
G-protein–coupled receptors are reported to activate the Ras/ERK by a pathway that is independent of their respective second
messenger systems (44 ). This alternate pathway is dependent on internalization of the receptor and recruitment of a soluble
tryosine kinase that directly phosphorylates the adaptor proteins (Shc and Gab) that lead to activation of Ras and subsequently the
Ras/ERK pathway. For example, internalization of the β-adrenergic receptor (βAR) leads to binding of β-arrestin, which inhibits
activation of the receptor. Studies demonstrated that β-arrestin also functions as an adaptor protein that binds both βAR and a
soluble tyrosine kinase, Src. Studies demonstrated that 5-hydroxytryptamine (5-HT1A) receptors activate the Ras/ERK pathway,
possibly through this mechanism (45 ). Regulation
 P.211

of the Ras/ERK pathway by internalization of G-protein–coupled receptors is not observed in all cases (e.g., 5-HT2A receptor), a
finding indicating receptor or cellular specificity in the control of this pathway.

Numerous other potential mechanisms of cross-talk between G protein and neurotrophic factor signaling pathways have been
identified. For instance, several other forms of PI-3-Ks have been elucidated, some of which are activated by G-protein–coupled
receptors, but presumably they lead to at least some similar downstream signaling effects through the production of similar
phosphorylated inositol lipids (35 ,36 and 37 ). Similarly, PLC-β, the G-protein–coupled form of PLC, would be expected to produce
at least some of the same effects as PLC-γ by activation of PKCs and mobilization of calcium. Furthermore, both PKC and levels of
cyclic adenosine monophosphate modulate the activity of Raf and hence the ERK pathway (46 ,47 ,48 and 49 ). Calcium mobilization
can activate ERKs through the intracellular tyrosine kinase, Pyk2 (50 ). Obviously, the potential for cross-talk with G-protein–coupled
systems is great, and sorting out the mechanisms relevant in various brain regions and mechanisms of plasticity is an important area
for investigation.

SOME OTHER CLASSES OF NEUROTROPHIC FACTORS AND THEIR

SIGNALING SYSTEMS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

Although this review focuses on the NT family of neurotrophic factors, several others have generated significant interest (Fig. 16.2 ).
For example, the glial cell line–derived neurotrophic factor (GDNF) family of proteins has been found to have profound effects on
dopaminergic and motor neurons, as well as enteric neurons (51 ,52 and 53 ). Thus far, this family includes GDNF, artemin,
persephin, and neurturin. This family, distantly related to the transforming growth factor-β (TGF-β) family, signals through a
heteromeric signaling complex (54 ). A common component of all the GDNF receptor complexes identified is the Ret protein. Ret
spans the membrane and contains an intracellular tyrosine kinase domain. This domain, which is less well characterized than for the
Trk receptor, can nonetheless signal at least some of the same intracellular signaling pathways, including the Ras/ERK pathway and
PI-3-K (55 ). Ret associates with various receptor-binding proteins (GFR-α subunits 1 through 4). These subunits do not span the
membrane, but are linked to the extracellular surface through a glycosyl-phosphatidylinositol moiety. The GFR-α subunits provide
specificity for ligand binding and participate in the activation of the Ret tyrosine kinase. Furthermore, evidence indicates that they
can stimulate activation of src-like tyrosine kinases independent of Ret. The known activities of the GDNF family of proteins has
spurred interest in its role in the pathogenesis and possible treatment of diseases such as Parkinson's disease, addiction, and
amyotrophic lateral sclerosis (52 ,53 ).

FIGURE 16.2. Other neurotrophic factor signaling cascades. This cartoon illustrates the general motifs used by neurotrophic
factor receptors to transduce a signal across the membrane and to couple with the intracellular signaling pathways. The light
gray ovals represent extracellular (Out) ligand-binding domains. The vertical striped intracellular (In) ovals are tyrosine kinase
domains. The Trk receptors, like EGF receptors, are single polypeptide chains that span the membrane. These contain intrinsic
extracellular ligand binding domains and intracellular tyrosine kinase activity. The insulin and IGF receptors are derived from
the cleavage and subsequent disulfide linking of two identical chains. These receptors also contain intrinsic binding and kinase
activities. The GDNF receptor is a heteromeric complex in which the ligand-binding specificity resides within a separate
phosphatidylinositolglycan-linked subunit (GFR-α). The signal from the ligand–GFR-α complex is then transduced across the
membrane by Ret, which contains an intracellular tyrosine kinase domain. The CNTF receptor is typical of the cytokine
receptors, which contain separate binding and transmembrane subunits, and then couple to a soluble intracellular tyrosine
kinase (a JAK). Other systems couple to the tyrosine kinase intracellular signaling pathway by interacting with one of several
membrane-associated intracellular tyrosine kinases. Some G proteins, for instance, act through src-like proteins, whereas
intracellular calcium can activate Pyk2, an intracellular tyrosine kinase in the focal adhesion kinase family.

Another large class of receptors couples to an intracellular tyrosine kinase known as the Janus kinase, or JAK (56 ,57 ). These
receptor kinases include extracellular binding components, one of which spans the membrane but does not have intrinsic kinase
activity. Instead, they bind to and activate specific members of the JAK family of kinases. The JAKs then activate certain
intracellular effector molecules, including IRS proteins, SHC, and others. Moreover, they interact with a unique group of proteins
called STATs. STAT proteins bind to JAK. After tyrosine phosphorylation, they are released and translocated to the nucleus, where
they function directly as DNA-binding transcriptional activators. Many neurotrophic factors activate the JAK/STAT pathway,
including ciliary neurotrophic factor, growth hormone, leptin, and many cytokines. Characterization of the role of STAT-mediated
transcription in brain lags behind that of other earlier identified transcription factors, but it is an area of intensive study.

Even factors known for their hormone functions or peripheral

 P.212

effects have been found to have substantial activity in the central nervous system. Insulin and insulin-like growth factor 1 (IGF-1),
and their respective transmembrane receptor tyrosine kinases, are expressed widely in brain and play roles in development and
behavior (58 ,59 ). Furthermore, epidermal growth factor (EGF) and EGF-like ligands in the TGF-α family are expressed in brain
along with their receptor, the EGF receptor. Evidence is accumulating for roles for these factors in the adult central nervous system
as well (60 ). Although not discussed in detail, these serve as further examples of the complexity of neurotrophic factor signaling at
many levels in the brain. These receptors also share coupling to the same modules of signaling pathway proteins as the Trks and Ret.
Apparently, there is a tremendous diversity of neurotrophic ligands and ligand-binding domains within their receptors that allows
fine anatomic and temporal specificity of action, along with the potential for synergistic or counterregulatory mechanisms. However,
the relatively smaller number of conserved signaling pathways to which they couple suggests that they share common mechanisms of
action to shape neuronal responses.

ROLE OF NEUROTROPHIC FACTORS IN THE ACTIONS OF

PSYCHOTROPIC DRUGS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

As investigations of the psychotropic drugs have been extended, it has become clear that these agents also influence the expression
of neurotrophic factors and their signal transduction systems. Regulation of neurotrophic factor signaling could thereby contribute
to the desired actions of therapeutic of agents, as well as the negative effects of other drugs. These possibilities are illustrated
briefly in this section.

Regulation of Neurotrophin Systems by Stress and Antidepressant Treatment

Preclinical and clinical studies have reported an atrophy or loss of neurons in limbic brain structures that could be related to
dysfunction of neurotrophic factor systems. Chronic physical or social stress can result in atrophy or death of stress-vulnerable
neurons in the hippocampus of rodents and nonhuman primates (61 ,62 ). More recently, investigators conducting brain imaging
studies reported that the volume of the hippocampus is reduced in patients suffering from depression of posttraumatic stress
disorder (63 ). Postmortem studies also demonstrate that the numbers of neurons and glia in prefrontal cortex are reduced in
patients with depression (63 ). The expression of BDNF in hippocampus is decreased by exposure of animals to stress (64 ). This
effect could contribute to the atrophy and death of hippocampal neurons, although it is also likely that other pathways are involved
in this effect (61 ,62 ).

In contrast to the actions of stress, antidepressant treatment increases the expression of BDNF, as well as its receptor TrkB, in
hippocampus (65 ,66 ). Up-regulation of BDNF is dependent on long-term antidepressant treatment, consistent with the time course
for the therapeutic action of these agents. Both norepinephrine and serotonin-selective reuptake inhibitor antidepressants increase
BDNF expression, a finding suggesting that this NT system may be a common postreceptor target of these monoamines and
antidepressant treatment. In addition, nonantidepressant psychotropic drugs do not increase BDNF expression in hippocampus, a
finding indicating that this effect is specific to antidepressants. The possibility that BDNF contributes to the therapeutic actions of
antidepressants is supported by behavioral studies. Infusion of BDNF into midbrain or hippocampus produces antidepressant-like
effects in behavioral models of depression, the forced swim test, and learned helplessness paradigms (67 ,68 ). Additional studies
will be required to elucidate further the role of BDNF, as well as other neurotrophic factors, in the pathogenesis and treatment of
depression. However, these findings have contributed to an exciting new hypothesis of depression.

Role of Neurotrophic Factors in the Actions of Drugs of Abuse

A picture is emerging that neurotrophic factors and their signaling pathways play important roles in mediating acute and chronic
changes in synaptic connectivity, neuronal physiology, and gene expression. A powerful and important model of environmentally
induced acute and persistent alterations in brain function is the effect of chronic exposure to drugs of abuse (69 ,70 ). Within
laboratory animals, exposure to any of several diverse addicting drugs leads a set of alterations in neuronal biochemistry,
electrophysiology, and morphology in specific brain regions implicated in addictive behaviors. Concomitantly, these animals display
alterations in behavior including tolerance, dependence, sensitization, craving, and drug-seeking behaviors reminiscent of the
behaviors seen in humans suffering from drug addiction. Specifically, alterations in the dopaminergic nucleus, the ventral tegmental
area (VTA), are reminiscent of the changes seen with neurotrophic factor withdrawal in cell culture: the cells become smaller with
less prominent neuritic processes, they have decreased neurofilament expression and axoplasmic transport, and they have
decreased expression and accumulation of tyrosine hydroxylase, the rate-limiting enzyme in dopamine syntheses and a critical
neuron type-specific protein. Infusing NTs such as BDNF or GDNF into the VTA can restore most, or all, of these features to normal
(71 ,72 ). Additional studies have also implicated endogenous NT-3 in the drug-induced changes (73 ). Furthermore, the role of ERK
signaling in this system has been established. ERK activity is increased in the VTA by chronic morphine exposure (74 ). Infusion of a
specific antisense oligonucleotide against ERK1 into the VTA again blocks the morphine-induced biochemical changes (75 ).
 P.213

Dissecting the mechanisms of signaling protein regulation within specific brain nuclei in the intact animal poses special challenges. However, using tools from in vitro studies, headway
is now starting to be made. For instance, the mechanism of this ERK up-regulation is unclear, but it has been shown that PLC-γ, which is capable of activating ERK, is up-regulated in
VTA after chronic morphine exposure (76 ). Although levels of the neurotrophic factors themselves have not been found to be significantly altered in VTA by chronic drug exposure,
they may be regulated indirectly by modulation of their signaling systems.

CONCLUSIONS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

The neurotrophic factors and their signal transduction cascades represent a complex array of pathways that influence many aspects of neuronal function and survival during
development as well as in the adult central nervous system. The characterization of these pathways has provided many new target sites for the development of novel agents that could
be used to treat a variety of neurologic and psychiatric illnesses. There is currently a tremendous amount of interest in this area, and agents are already available for selective
blockade of certain components of the Ras/ERK pathway. Moreover, characterization of the roles these pathways play in the normal nervous system may lead to identification of
abnormal conditions that underlie pathologic states. The opening of the field of growth factor action into the neurosciences opens opportunities that may be as rich or as powerful, if
not more, as those that have been presented with the more traditional neurotransmitter systems.

ACKNOWLEDGMENTS
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

We would like to acknowledge the support of United States Public Health Service grants DA00302, MH45481, MH53199, and 2 PO1 MH25642, the Veterans Affairs National Center Grant
for Posttraumatic Stress Disorder, the Veterans Affairs Medical Center, and the National Alliance for Research on Schizophrenia and Depression (NARSAD).

DISCLAIMER
Part of "16 - Neurotrophic Factors and Intracellular Signal Transduction Pathways "

Dr. Duman serves as a consultant to Pfizer, Psychogenics, Janssen, Lilly, and Pharmacia-Upjohn. In addition, he currently receives research support from Pfizer and serves as a member
of the scientific advisory board for Psychogenics.

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17
Regulation of Gene Expression

Eric J. Nestler

Steven E. Hyman

Eric J. Nestler: Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, Texas.

Steven E. Hyman: National Institute of Mental Health, Bethesda, Maryland.

For all living cells, regulation of gene expression by extracellular signals is a fundamental mechanism of development, homeostasis,
and adaptation to the environment. Indeed, the ultimate step in many signal transduction pathways is the modification of
transcription factors that can alter the expression of specific genes. Thus, neurotransmitters, growth factors, and drugs are all
capable of altering the patterns of gene expression in a cell. Such transcriptional regulation plays many important roles in nervous
system functioning, including the formation of long-term memories. For many drugs, which require prolonged administration for
their clinical effects (e.g., antidepressants, antipsychotics), the altered pattern of gene expression represents therapeutic
adaptations to the initial acute action of the drug.

Mechanisms that underlie the control of gene expression are becoming increasingly well understood. Every conceivable step in the
process is subject to dynamic regulation in the cell. This includes structural changes in the chromatin to make a particular gene
accessible for transcription, transcription of DNA into RNA, splicing of RNA into mRNA, editing and other covalent modifications of
the mRNA, translation of mRNA into protein, and, finally, post-translational modification of the protein into its mature, functional
form.

Molecular details of each of these regulatory steps are becoming increasingly available. In this chapter, we focus on the regulation
of gene expression by transcription factors because their role in mediating the ability of extracellular signals to alter gene
expression remains the best characterized.

 OVERVIEW OF TRANSCRIPTIONAL CONTROL MECHANISMS

 REGULATION OF GENE EXPRESSION BY EXTRACELLULAR SIGNALS
 CONCLUSIONS
 ACKNOWLEDGMENTS

OVERVIEW OF TRANSCRIPTIONAL CONTROL MECHANISMS

Part of "17 - Regulation of Gene Expression "

Regulation of Gene Expression by the Structure of Chromatin

In eukaryotic cells, DNA is contained within a discrete organelle called the nucleus, which is the site of DNA replication and
transcription. Within the nucleus, chromosomes—which are extremely long molecules of DNA—are wrapped around histone proteins
to form nucleosomes, the major subunits of chromatin (1 ,2 and 3 ). To fit within the nucleus, much of the DNA is tightly packed
into a “coiled coil.” Compared with transcriptionally quiescent regions, actively transcribed regions of DNA may be more than 1,000-
fold further extended. Chromatin does not just serve a structural role, however; in eukaryotes, chromatin plays a critical role in
transcriptional regulation. Chromatin can inhibit access of transcription factors to the DNA and can thereby repress gene expression.
In eukaryotic organisms, with their very large number of genes (approximately 40 × 103 in mammals), this means that the ground
state of gene expression is for genes to be turned off. Activation of gene expression requires that cells alleviate nucleosome-
mediated repression of an appropriate subset of genes. This is accomplished by means of activator proteins that modify chromatin
structure. The activation process, which involves transcription factors, along with histones and cofactors, displaces or remodels
chromatin, and opens up regions of the DNA, including the core promoters (see later) of genes, for the binding of regulatory proteins.

Transcription occurs when particular activator proteins displace nucleosomes. This permits a complex of proteins (described later)
called general transcription factors, to bind DNA at a particular type of element, called a core promoter, and to recruit RNA
polymerase. The construction of this protein complex at the transcription start site and the synthesis of the first phosphodiester
bond between nucleotides are referred to as transcription initiation (3 ). The RNA polymerase must successfully transcribe an
appropriate length of RNA without premature termination (elongation). Premature
 P.218

termination appears to be a regulated mechanism that controls expression of a small number of genes. Transcription of the RNA
must also terminate appropriately (termination).

Transcription Initiation: A Critical Biological Control Point

As described in the preceding section, transcription can be divided into three discrete steps: initiation, mRNA chain elongation, and
chain termination. Although biologically significant regulation may occur at any step in the process, transcription initiation appears
to be one of the most significant control points that gates the flow of information out of the genome. Certainly, as far as we know
now, transcription initiation is the step in gene expression that is most highly regulated by extracellular signals (3 ).

Transcription initiation involves two critical processes: positioning of the appropriate RNA polymerase at the correct start sites of
transcription and controlling the efficiency of initiations to produce the appropriate transcriptional rate for the circumstances of the
cell. These control functions depend on regulatory elements that recruit appropriate transcription factors to the DNA (Fig. 17.1 ).
Many transcription factors bind DNA directly; others interact indirectly through protein–protein interactions with factors that do bind
DNA themselves. Those regulatory elements that set the transcription start sites of a gene are called the basal or core promoters.
Other regulatory elements tether additional activator and repressor proteins to the DNA.

FIGURE 17.1. Scheme of a generalized polymerase II promoter. The figure shows two regulatory elements (open rectangles)
along the stretch of DNA (thin black line). These include the TATA element and a hypothetic activator or response element.
The TATA element is shown binding the TATA-binding protein, TBP. Multiple general transcription factors and RNA polymerase
II (pol II) associate with TBP. The general transcription factors are referred to with the nomenclature of TFII(x), for
transcription factors of a pol II promoter. Shown are general factors, TFIIA, B, E, F, and H. Each of these transcription
“factors” is actually composed of multiple individual proteins complexed together. TBP and its associated proteins are
collectively called TFIID. This basal transcription apparatus recruits RNA polymerase II. It also forms the substrate for
interactions with various activator proteins that bind to activator elements such as the one shown. Typical activator proteins
contain DNA-binding domains, dimerization domains, and transcription activation domains. The latter interact with the basal
transcription apparatus and may be modified by phosphorylation. Adapted from reference 14.

Core Promoters: Setting the Start Site and Direction of Transcription

In eukaryotes, transcription is carried out by three distinct RNA polymerases: RNA polymerases I, II, and III (4 ). These three
polymerases interact with different classes of genes, each of which contains distinct promoter elements. Polymerase I (pol I)
promoters are used by genes that encode large rRNAs (ribosomal RNAs). Polymerase II (pol II) promoters are used by genes that are
transcribed to yield mRNAs and hence proteins. Pol II promoters are also used by a subset of the genes that encode snRNAs that are
involved in RNA splicing. Polymerase III (pol III) promoters are used by genes that encode other small RNAs, including the remaining
snRNAs, small rRNAs, and tRNAs (transfer RNAs).

None of the RNA polymerases bind DNA directly; rather, the polymerases are recruited to the DNA by other proteins. The core
promoters for each of the three polymerases contain distinct elements on which different types of basal transcription complexes are
assembled, each using different transcription factors. Because the main focus of this chapter is regulated expression of protein-
encoding genes, only transcription by pol II is described.

The core promoters of genes transcribed by pol II are surprisingly diverse, but they share certain key features. By far the most
common core promoter element for pol II promoters is the TATA box (Fig. 17.1 ), a sequence rich in the nucleotides A and T located
between 25 and 30 bases upstream of the transcription start site. In TATA box–containing genes, mutation of this sequence can
inhibit transcription
 P.219

initiation or make it inaccurate. In addition to setting the start site of transcription, the TATA box sets the orientation of the basal
transcription complex and therefore the 5′ to 3′ direction in which pol II synthesizes the RNA. Many pol II promoters (including those
for many neurally expressed genes) lack a TATA box; in these cases, a poorly conserved core promoter element called an initiator is
found.

The TATA-binding protein (TBP) initiates the formation of the basal transcription complex along with multiple TBP-associated
proteins (TAFs) and multiple additional general transcription factors (Fig. 17.1 ). Each of the transcription factors represented in Fig.
17.1 was originally identified as a chromatographic fraction derived from cell nuclei, and it is a mixture of proteins. Thus, TBP
together with its TAFs was originally identified as a fraction called TFIID, where TFII is a nomenclature identifying general
transcription factors associated with pol II, and the final letter designates the fraction. TFIID, but not TBP by itself, is required to
build a basal transcription complex from TATA-less promoters.

Transcription Factors: Key Regulators of Gene Expression

The basal transcription apparatus is not adequate to initiate more than low levels of transcription. To achieve significant levels of
transcription, this multiprotein assembly requires help from additional transcriptional activators that recognize and bind to
regulatory elements found elsewhere within the gene. Because they are tethered to DNA—by their binding to specific recognition
sequences in the DNA—such proteins can be described as sequence-specific transcription factors (5 ,6 and 7 ).

Functional regulatory elements are generally found within several hundred bases of the start site of the gene to which they are
linked, but they can occasionally be found many thousands of base pairs (bp) away, either upstream or downstream of the start site.
Regulatory elements that exert control near the core promoter itself have been called promoter elements, and those that act at a
distance have been called enhancer elements, but the distinction between promoter and enhancer elements is artificial from a
mechanistic point of view. Both are generally composed of small, modular elements (generally 7 to 12 bp in length), each of which
is a specific binding site for one or more transcription factors. The fundamental logic of transcriptional regulation in eukaryotes is
that it is combinatorial: each gene has a particular combination of regulatory elements, the nature, number, and spatial
arrangement of which determines the gene's unique pattern of expression. These promoter or enhancer elements control the cell
types in which the gene is expressed, the times during development in which it is expressed, and the level at which it is expressed in
adults both basally and in response to physiologic and environmental signals (7 ).

Sequence-specific transcription factors typically contain several physically distinct functional domains (these are shown in Fig. 17.1 ):
(a) the DNA-binding domain recognizes and binds to a specific nucleotide sequence (i.e., response element); (b) the transcription
activation domain interacts with coactivators or with general transcription factors (i.e., components of the pol II complex) to form a
mature or fully active transcription complex; and (c) the multimerization domain permits the formation of homomultimers and
heteromultimers with other transcription factors. The modularity of these proteins is emphasized by the finding that particular
binding domains, activation domains, and interaction domains are used in different combinations in many naturally occurring
proteins. Experimentally, domains can be swapped from different activators to produce novel hybrid proteins that are functionally
active.

Many transcription factors are active only as dimers or higher-order complexes. Multimerization domains are diverse and include so-
called leucine zippers (described later), Src homology (SH-2) domains, and certain helical motifs (8 ,9 and 10 ). Within transcription
factor dimers, whether they are homodimers or heterodimers, both partners commonly contribute jointly to both the DNA binding
domain and to the activation domain. In some cases, dimerization can be a mechanism of negative control of transcription. This is
illustrated by the CREB (cyclic adenosine monophosphate [cAMP]–response element binding protein) family of transcription factors
discussed later.

Regulation of transcription factors by the formation of heterodimers is not an “all or none” proposition, however. Within the Fos
family of transcription factors (described later), some family members, such as c-Fos, are strong activators when dimerized with a
partner from the Jun family, such as c-Jun. Other Fos-related proteins, such as Fra1 (Fos-related antigen–1), bind DNA as
heterodimers with c-Jun, and they may still activate transcription, but at lower levels than c-Fos (11 ). Overall, the ability of
transcription factors to form heterodimers and other multimers increases the diversity of transcription factor complexes that can
form in cells and, as a result, increases the types of specific regulatory information that can be exerted on gene expression.

Sequence-specific transcriptional activator and repressor proteins may contact several proteins within the basal transcription
complex directly. In other cases, they interact with the basal transcription apparatus through the mediation of coactivator or
adapter proteins. In either of these situations, transcription factors that bind at a distance from the core promoter can still interact
with the basal transcription apparatus, because the DNA forms loops that bring distant regions in contact with each other.

Many activator proteins become involved only in the assembly of the mature transcription apparatus after modification, most
commonly phosphorylation, that occurs in response
 P.220

to extracellular signals. An important effect of many phosphorylation events is to alter the ability of the phosphoprotein to interact with other proteins. This is illustrated by CREB,
which can activate transcription only when phosphorylated on a particular serine residue (ser133) (12 ). As seen later, phosphorylation of ser133 permits CREB to interact with an
adapter protein, CBP (CREB-binding protein), which, in turn, contacts and activates the basal transcription apparatus (13 ).

REGULATION OF GENE EXPRESSION BY EXTRACELLULAR SIGNALS

Part of "17 - Regulation of Gene Expression "

Transcription Factors: Targets of Signaling Pathways

Most genes probably contain response elements that confer responsiveness to physiologic signals. Response elements work by binding transcription factors that are activated (or
inhibited) by specific physiologic signals, of which the most common is phosphorylation. Two general mechanisms of transcriptional regulation by extracellular signals are illustrated
Fig. 17.2 (14 ). In one mechanism, transcription factors that are present at significant levels in cells under basal conditions are rapidly activated by signaling cascades to activate or
repress transcription of responsive target genes. In the other major mechanism, transcription factors that are expressed at very low levels under basal conditions are themselves
induced by a physiologic signal, after which they can regulate expression of a series of additional genes.

FIGURE 17.2. Scheme of intracellular pathways underlying regulation of gene expression.

Activation of neurotransmitter, hormone, or neurotrophic factor receptors leads to the activation
of specific second messenger and protein phosphorylation pathways, which produce multiple
effects on neuronal function through the phosphorylation of numerous proteins. Among the
effects of these intracellular pathways on neuronal function is the regulation of gene expression.
This can be accomplished by two basic types of mechanisms. In one case, transcription factors,
already in the nucleus, are phosphorylated by protein kinases; this alters their transcriptional
activity and leads to alterations in the expression of specific target genes. CREB is an example of
a transcription factor that functions in this manner. Among the target genes for CREB family
transcription factors are those for other transcription factors, for example, Fos and Jun family
proteins. Increased expression of Fos and Jun then leads to alterations in the expression of
additional target genes.

A critical step in extracellular regulation of gene expression is the transduction of signals from the cell membrane to the nucleus; this can be accomplished by several different types
of mechanisms. Some transcription factors themselves translocate to the nucleus on activation. One example is provided by the steroid hormone receptor transcription factors,
discussed at length later. Another example is the transcription factor nuclear factor-κB (NF-κB) (15 ). This transcription factor is retained in the cytoplasm by its binding protein, IκB,
which masks the nuclear localization signal within NF-κB. Signal-regulated phosphorylation of IκB by protein kinase C leads to dissociation of NF-κB, which permits it to enter the
nucleus, where it can bind DNA; IκB is then proteolyzed within the cytoplasm.

Other transcription factors must be directly phosphorylated or dephosphorylated to bind DNA. For example, phosphorylation of STATs (signal transducers and activators of transcription)
by protein tyrosine kinases in the cytoplasm permits their multimerization, which, in turn, permits nuclear translocation and construction of an effective DNA binding site within the
multimer (16 ).

Still other transcription factors are already bound to their cognate cis-regulatory elements in the nucleus under basal conditions and are converted into transcriptional activators by
phosphorylation. CREB, for example, is bound to DNA elements termed cAMP-response elements (CREs) (Fig. 17.3 ) before cell stimulation. The critical nuclear translocation step in
CREB activation involves not the transcription factor itself, but activated protein kinases (cAMP-dependent protein kinase; also called protein kinase A) that, on entering the nucleus,
phosphorylate CREB. Alternatively, CREB activation can involve the nuclear translocation of second messengers, such as Ca2+ bound to calmodulin, which, on entering the nucleus
activate protein kinases that then phosphorylate CREB (Fig. 17.3 ). As stated earlier, phosphorylation converts CREB into a transcriptional activator by permitting it to recruit CBP into
the transcription complex.

FIGURE 17.3. Scheme of the regulation of CREB phosphorylation by several signaling pathways. The
figure illustrates how several signaling pathways converge on the phosphorylation of CREB at a single
serine residue, ser133. Neurotransmitters that stimulate adenylyl cyclase would increase CREB
phosphorylation by the activation of protein kinase A (PKA). On activation, free PKA catalytic
subunits would translocate to the nucleus, where they would phosphorylate ser133 of CREB.
Neurotransmitters that inhibit adenylyl cyclase would cause the opposite cascade and inhibit CREB
phosphorylation. Any of several signals that increase cellular Ca2+ levels (e.g., certain inotropic or G-
protein–coupled receptors, voltage-gated Ca2+ channels) would also increase CREB phosphorylation.
Here, it appears that a wave of increased Ca2+ would permeate the nucleus, where it would activate
certain Ca2+/calmodulin–dependent protein kinases (CaM kinases), particularly CaM-K IV, which
phosphorylates ser133 of CREB. In addition, growth factor regulated pathways lead to CREB
phosphorylation, although the details are not as well established. One possibility, shown in the
figure, is that activation of Ras-Raf-MEK pathways would lead to activation of ERK (a type of MAP
kinase), which would translocate to the nucleus and phosphorylate and activate RSK (ribosomal S6
kinase). RSK would then phosphorylate ser133 of CREB. MEK, MAP kinase and ERK kinase; ERK,
extracellular signal regulated kinase; RSK, ribosomal S6 kinase.

The remainder of this chapter provides a more in-depth discussion of several transcription factor families that have received a great deal of attention as mediators of neural and
behavioral plasticity in the adult.
 P.221

CREB Family of Transcription Factors

CREs were the first second messenger response element to be well characterized (12 ,17 ,18 ). As the name suggests, CREs confer
activation by cAMP on genes to which it is linked. Subsequently, it was found that the same element confers response to Ca2+ and to
the Ras pathway as well. CREs have been identified in many genes expressed in the nervous system, including those encoding
neuropeptides (e.g., somatostatin, proenkephalin, vasoactive intestinal polypeptide), neurotransmitter synthetic enzymes (e.g.,
tyrosine hydroxylase), signaling proteins (e.g., adenylyl cyclase type VIII), and transcription factors (e.g., c-Fos and CREB itself)
(12 ,17 ,18 ).

The idealized or “consensus” CRE sequence is TGACGTCA, although the actual CREs present in various genes differ slightly. The
consensus CRE sequence illustrates an important principle, the palindromic nature of many transcription factor–binding sites.
Examining the sequence TGACGTCA, it can be readily observed that the sequence on the two complementary DNA strands, which run
in opposite directions, is identical. Many regulatory elements are perfect or approximate palindromes because many transcription
factors bind DNA as dimers, with each member of the dimer recognizing one of the half-sites. The major protein that binds to CREs
is CREB. CREB binds to a CRE as a homodimer, with a higher affinity for palindromic than for asymmetric CREs.

When bound to a CRE, CREB activates transcription only when it is phosphorylated on its critical ser133. It does so, as described
earlier, because phosphorylated CREB, but not dephosphorylated CREB, can recruit the adapter protein, CBP, into the transcription
complex. CBP, in turn, interacts with the basal transcription complex.

Regulation by cAMP, Ca , and Growth Factors

2+

As discussed in previous sections, the regulation of CREB activation by phosphorylation illustrates the requirement for nuclear
translocation of protein kinases or second messengers when transcription factors are already found in the nucleus under basal
conditions and the role of phosphorylation in regulating protein–protein interactions. An additional important principle illustrated by
CREB is the convergence of signaling pathways. CREB is activated in response to activation of the cAMP or Ca2+ pathways. This occurs
because the same serine residue (ser133) is phosphorylated both by protein kinase A and by Ca2+/calmodulin–dependent protein
kinases (CaM kinases) (Fig. 17.3 ). CaM kinase IV appears to be the most important form of the enzyme that mediates this
phosphorylation (19 ,20 ). CREB also appears to be phosphorylated on ser133 by a growth factor–activated kinase, RSK—ribosomal S6
kinase—that is phosphorylated and activated by mitogenactivated protein (MAP) kinases (21 ).

Thus, diverse types of signaling pathways converge on the phosphorylation and activation of CREB. If each individual signal is
relatively weak, convergence may be a critical mechanism for achieving significant gene regulation, with some genes activated only
when multiple pathways are stimulated. Furthermore, some genes that contain CREs are known to be induced in a synergistic
fashion by the interaction of cAMP and Ca2+ signaling pathways. In addition
 P.222

to ser133, CREB contains other sites for phosphorylation by a variety of protein kinases, which may fine tune the regulation of CREB-
mediated transcription. For example, CaM kinase II phosphorylates a distinct serine residue in CREB, which diminishes the ability of
other kinases to phosphorylate ser133. Activation of CaM kinase II would therefore appear to mediate a dampening of the CREB
signal (19 ,20 ).

Role in Neural Plasticity

The convergent activation of a single transcription factor by multiple signaling pathways is particularly important in the nervous
system, because this is an important candidate mechanism for long-term neural adaptations, including those underlying long-term
memory, drug addiction, and fear conditioning. As discussed elsewhere in this volume, it is reasonably well established that some
forms of long-term memory require new gene expression. Furthermore, associative memory depends on the temporally coordinated
arrival of two different signals, which must then be integrated within target neurons and their circuits. Activation of CREB is
therefore a plausible candidate for playing an important role in long-term memory and related phenomena. Consistent with this
prediction, Drosophila in which CREB was inactivated by a dominant negative transgene, and mice in which CREB was inactivated by
homologous recombination (i.e., knockout), show deficits in long-term memory (22 ,23 ,24 and 25 ). Manipulation of CREB also
influences long-term potentiation in the hippocampus (23 ,24 and 25 ) and aspects of drug addiction (26 ,27 ,28 and 29 ). Although
much work remains to understand the precise role of CREB in these various phenomena, it does appear that CREB, which is regulated
by several major neural signaling pathways, is a critical mediator of many types of plasticity.

CREB-like Proteins
CREB illustrates yet another important principle of transcriptional regulation: CREB is a member of a larger family of related
proteins. Many transcription factors are members of families. CREB is closely related to proteins called the activating transcription
factors (ATFs) and the CRE modulators (CREMs), each generated by distinct genes. In addition, several alternative splice forms are
known for CREB, certain of the ATFs, and CREMs (30 ,31 ).

All these proteins bind CREs as dimers, and many can heterodimerize with CREB itself. ATF1 appears to be very similar to CREB; it
can be activated by both the cAMP and Ca2+ signaling pathways (30 ,31 ). Many of the other ATF proteins and CREM isoforms also
appear to activate transcription. However, certain CREMs (e.g., ICER—inducible cAMP element repressor) act to repress it (30 ).
These CREM isoforms lack the glutamine-rich transcriptional activation domain found in CREB-ATF family proteins that are
transcriptional activators. Thus, CREB-ICER heterodimers may occupy CREs, but fail to activate transcription. Like CREB, many of the
ATF proteins are constitutively made in cells, but ATF3 and certain CREM isoforms (e.g., ICER) are inducible.

Leucine Zipper Dimerization Motif

The dimerization domain used by the CREB-ATF proteins and several other families of transcription factors is called a leucine zipper
(8 ,9 ). This domain was first identified in the transcription factor C/EBP (CAATT-enhancing binding protein) (32 ), and it is also used
by the AP-1 proteins, as described in detail later. The so-called leucine zipper actually forms a coiled coil. The dimerization motif is
an α helix in which every seventh residue is a leucine; based on the periodicity of α helices, the leucines line up along one face of
the α helix two turns apart. The aligned leucines of the two dimerization partners interact hydrophobically to stabilize the dimer. In
CREB, C/EBP, and many AP-1 proteins, the leucine zipper is at the C-terminus of the protein. There is a region of highly basic amino
acid residues just upstream of the leucine zipper that forms the DNA binding domain. Dimerization by the leucine zipper juxtaposes
the adjacent basic regions of each of the partners; these juxtaposed basic regions undergo a conformational change when they bind
DNA, which results in what has been described as a “scissors grip.” This combination of motifs has led this superfamily of proteins to
be described as the basic-leucine zipper proteins or the bZIP proteins.

AP-1 Family of Transcription Factors

Another group of bZIP transcription factors that plays an important role in the regulation of neural gene expression by extracellular
signals comprises the activator protein-1 (AP-1) proteins. The name AP-1 was originally applied to a transcriptional activity
stimulated by protein kinase C activation (33 ). This activity was found to be composed of multiple proteins, which bind as
heterodimers (and a few as homodimers) to the DNA sequence TGACTCA, the AP-1 sequence. The consensus AP-1 sequence is a
heptamer that forms a palindrome flanking a central C or G, and differs from the CRE sequence by only a single base. Yet this one
base difference strongly biases protein binding away from CREB (which requires an intact CGTCA motif) to the AP-1 family of
proteins and means that, under most circumstances, this sequence will not confer cAMP responsiveness on a gene.

Many genes expressed in the nervous system contain AP-1 sites within their regulatory regions (34 ,35 and 36 ). Examples include
genes encoding neuropeptides (neurotensin and substance P), neurotransmitter receptors (D1 dopamine, NR1 NMDA, and GluR2
AMPA glutamate receptor subunits), neurotransmitter synthetic enzymes (tyrosine hydroxylase),
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and cytoskeletal proteins (neurofilament proteins), to name a few. In some cases, it has been possible to demonstrate a role for
these sites in regulation of gene promoter activity in vitro, although it has been very difficult to identify with certainty those genes
that are regulated by AP-1 transcription factors in the brain in vivo (35 ).
As alluded to earlier, the AP-1 sequence was described initially as a TPA-response element (TRE) because the phorbol ester, 12-O-
tetradecanoyl-phorbol-13-acetate (TPA), which activates protein kinase C, can induce gene expression through AP-1 proteins (33 ).
In addition, it is now thought that a major role of the AP-1 sequence is to confer responsiveness to growth factor–stimulated
signaling pathways such as the Ras/MAP-kinase pathways. This occurs by phosphorylation of specific AP-1 proteins by certain MAP
kinases.
AP-1 proteins generally bind DNA as heterodimers composed of one Fos family member and one Jun family member (34 ). Both
families are bZIP proteins: they form dimers through their leucine zipper domains. The known members of the Fos family are c-Fos,
Fra1, Fra2, and FosB and its alternatively spliced variant ΔFosB. The known members of the Jun family are c-Jun, JunB, and JunD.
Unlike Fos proteins, c-Jun and JunD (but not JunB) can form homodimers that bind to AP-1 sites, albeit with far lower affinity than
Fos-Jun heterodimers. The potential complexity of transcriptional regulation is greater still because some AP-1 proteins can
heterodimerize through the leucine zipper with members of the CREB-ATF family, such as ATF2 with c-Jun. AP-1 proteins can also
form higher-order complexes with apparently unrelated families of transcription factors. For example, AP-1 proteins can complex
with and thereby apparently inhibit the transcriptional activity of steroid hormone receptors (see later).
Among Fos and Jun proteins, only JunD is expressed constitutively at high levels in many cell types. The other AP-1 proteins tend to
be expressed at low or even undetectable levels under basal conditions, but, with stimulation, they may be induced to high levels of
expression. Thus, unlike regulation by constitutively expressed transcription factors such as CREB, regulation by Fos-Jun
heterodimers requires new transcription and translation of the transcription factors themselves (Fig. 17.2 ).

Cellular Immediate Early Genes

Genes that are transcriptionally activated by synaptic activity, drugs, or growth factors have often been classified roughly into two
groups. Immediate early genes (IEGs), such as the c-fos gene itself, are activated rapidly (within minutes) and transiently and do not
require new protein synthesis. Late-response genes, in contrast, are induced or repressed more slowly (over hours) and are
dependent on new protein synthesis. The term IEG was applied initially to describe viral genes that are activated “immediately” on
infection of eukaryotic cells, by commandeering host cell transcription factors for their expression. Viral IEGs generally encode
transcription factors needed to activate viral “late” gene expression. This terminology has been extended to cellular (i.e., nonviral)
genes, although this has created some confusion.
The terminology is problematic because there are many cellular genes induced independently of protein synthesis, but with a time
course intermediate between “classic” IEGs and late-response genes. In fact, some genes may be regulated with different time
courses or requirements for protein synthesis in response to different extracellular signals. Moreover, many cellular genes regulated
as IEGs encode proteins that are not transcription factors: for example, any gene induced by CREB could potentially show temporal
features of induction of an IEG. Despite these caveats, the concept of IEG-encoded transcription factors in the nervous system has
proved useful in thinking about the complexities of gene regulation. In addition, because of their rapid induction from low basal
levels in response to neuronal depolarization (the critical signal being Ca2+ entry) as well as various second messenger and growth
factor pathways, several IEGs have been used as cellular markers of neural activation, and this has permitted novel approaches to
functional neuroanatomy (37 ).
The protein products of those cellular IEGs that function as transcription factors bind to regulatory elements contained within a
subset of late response genes to activate or repress them. IEGs such as c-fos have therefore been termed “third messengers” in
signal transduction cascades, with neurotransmitters designated intercellular first messengers and small intracellular molecules,
such as cAMP and Ca2+, second messengers (34 ). There have, however, been misunderstandings among some neurobiologists that
IEGs are a necessary step in the signal-induced expression of most neural genes involved in the differentiated function of neurons. In
fact, as stated earlier, many genes involved in differentiated neural functions, including genes encoding certain neuropeptides and
neurotrophic factors, to name a few, are activated in response to neuronal depolarization or cAMP through phosphorylation of CREB
rather than through IEG third messengers.

Activation by Multiple Signaling Pathways

The most studied cellular immediate early gene is c-fos. The c-fos gene contains three binding sites for CREB (the strongest of which
is shown in Fig. 17.4 ). As a result, it is not surprising that the gene can be activated rapidly by neurotransmitters or drugs that
stimulate the cAMP or Ca2+ pathways (38 ).

FIGURE 17.4. Scheme of the regulatory region of the c-fos gene. The figure shows only three of the many known transcription
factor binding sites within this gene. These sites are as follows: a CRE (cAMP-response element), which binds CREB; a serum-
response element (SRE), which binds serum response factor (SRF) and Elk-1 (also called the ternary complex factor or TCF);
and an SIF-inducible element (SIE), which binds STAT proteins (signal transducers and activators of transcription). Proteins
binding at each of these sites are constitutively present in cells and are activated by phosphorylation. CREB can be activated
by protein kinase A, CaM kinases (CaM-Ks), or RSKs (ribosomal S6 kinases) (Fig. 17.3); Elk-1 can be activated by the MAP kinases
ERK1 and ERK2 (extracellular signal regulated kinases 1 and 2); and the STAT proteins can be activated by the JAK protein
tyrosine kinases. Thus, activation of the c-fos gene—by any of multiple signaling pathways—depends only on signal-induced
phosphorylation rather than on new protein synthesis. This explains the rapidity of its induction by a wide array of stimuli.
MEK, MAP kinase and ERK kinase; PKA, protein kinase A; RTKs, receptor tyrosine kinases. Adapted from reference 14.

The c-fos gene also can be induced by the Ras/MAP-kinase pathway (39 ,40 ). Neurotrophins, such as nerve growth factor, bind a
family of receptor tyrosine kinases (called Trks) that activate Ras. Ras then triggers a cascade
 P.224

of protein kinases, which results in the phosphorylation and activation of certain MAP kinases called extracellular signal regulated
kinases (ERKs). These ERKs can phosphorylate and activate additional protein kinases, such as RSK, which, among its other
substrates, can phosphorylate ser133 of CREB, as described earlier. However, an additional mechanism exists whereby ERK can
induce the c-fos gene, and this mechanism appears to predominate in many cell types (41 ). Here, the ERKs translocate into the
nucleus where they phosphorylate the transcription factor Elk-1 (also called the ternary complex factor or TCF). Elk-1 then
complexes with the serum response factor (SRF) to bind to and activate the serum response element (SRE) within the c-fos gene (Fig.
17.4 ). SREs are present within many other growth factor-inducible genes as well. In comparison with cAMP- or Ca2+-dependent
phosphorylation of CREB, the Ras/MAP kinase pathway depends on a complex chain of phosphorylation events. Nonetheless, these
events can occur very rapidly to induce gene expression.

Still another mechanism exists for c-fos induction: cytokine-activated signaling pathways that act through STATs (42 ). As stated
earlier, STATs are activated on their phosphorylation by certain protein tyrosine kinases. This permits STATs to form multimeric
complexes, translocate to the nucleus, and bind to their specific DNA response elements, generally now described at STAT sites.
However, the STAT site in c-fos had already been named the SIE or SIF-inducible element (SIF itself is an acronym for sis-inducible
factor, i.e., a factor induced by the oncogene v-sis, which activated c-fos though this site). STATs are activated by the class of
cytokines that interact with gp130-linked receptors, which include ciliary neurotrophic factor, LIF (leukemia inhibitory factor),
interleukin-6, leptin, and prolactin, to name a few (16 ,43 ). These receptors activate a cytoplasmic protein tyrosine kinase called
JAK (Janus kinase), which then phosphorylates the STATs. As shown in Fig. 17.4 , the c-fos gene contains an SIE, which binds STAT
proteins and mediates the induction of c-Fos by cytokines.

Most other Fos and Jun family proteins are also induced rapidly in response to diverse acute stimuli and, presumably, many of the
same mechanisms operate for the genes encoding these proteins. However, the response elements within these genes are not as
well characterized as are those for c-fos, and further research is needed to understand their regulation.

Regulation by Phosphorylation
Several AP-1 proteins are regulated at the post-translational level by phosphorylation. The best-established example is
 P.225

the phosphorylation of c-Jun, which provides a critical mechanism of regulation of AP-1-mediated signaling (44 ,45 ). c-Jun
phosphorylation occurs in response to activation of a MAP kinase–related signaling pathway that is activated by many forms of
cellular stress. In this pathway, a Ras-like G protein is activated by any of several insults (e.g., ultraviolet radiation, osmotic stress,
toxins, certain cytokines); this triggers a cascade of protein kinases analogous to that triggered by Ras and the neurotrophins
outlined earlier. The culmination of this pathway is the phosphorylation and activation of certain MAP kinases called SAP kinases
(stress-activated protein kinases) or alternatively JNKs (for Jun N-terminal kinases). JNKs phosphorylate c-Jun on serines 63 and 73
in its transcriptional activation domain and increase the ability of c-Jun to activate transcription. Phosphorylation and activation of
c-Jun have been implicated in the regulation of apoptosis (programmed cell death) pathways (45 ).

Generation of Unique AP-1 Complexes by Repeated Stimulation

After acute stimulation of cells, different members of the Fos family are induced with varying time courses of expression, which
leads to a progression of distinct AP-1 complexes over time (46 ). Thus, under resting conditions, c-fos mRNA and protein are barely
detectable in most neurons, but c-fos expression can be induced dramatically in response to numerous stimuli. As just one example,
experimental induction of a grand mal seizure causes marked increases in levels of c-fos mRNA in brain within 30 minutes and
induces substantial levels of c-Fos protein within 2 hours. c-Fos is highly unstable and is degraded back to low, basal levels within 4
to 6 hours (46 ). Administration of cocaine or amphetamine causes a similar pattern of c-fos expression in striatum (47 ,48 ). In
either of these stimulus paradigms, other Fos-like proteins are also induced, but with a longer temporal latency than c-Fos; their
peak levels of expression lag behind c-Fos by approximately 1 to 2 hours. Moreover, expression of these proteins persists a bit longer
than c-Fos, but it still returns to basal levels within 8 to 12 hours.

With repeated stimulation, however, the c-fos gene, and to an extent the genes for other Fos-like proteins, become refractory to
further activation (i.e., their expression becomes desensitized) (49 ,50 ). Instead, other Fos-like proteins continue to be expressed.
These proteins, originally termed chronic Fras (51 ,52 and 53 ), are now known to be biochemically modified isoforms of ΔFosB,
which exhibit very long half-lives in brain (54 ,55 and 56 ). As a result, these proteins accumulate in specific neurons in response to
repeated perturbations and persist long after cessation of these perturbations (Fig. 17.5 ). Although the precise physiologic
significance of these stable ΔFosB isoforms remains unknown, there is now direct evidence that ΔFosB plays an important role in
aspects of drug addiction (57 ) and in mediating various types of striatal-based movement disorders (e.g., see refs. 58 ,59 and 60 ).
More generally, ΔFosB may function as a sustained molecular switch that gradually converts an acute response to a long-lived
adaptation in the brain (61 ).

FIGURE 17.5. Scheme showing the composition of AP-1 complexes changing over time. Top: There are several waves of Fras
(Fos-related antigens) induced by many acute stimuli in neurons. C-Fos is induced rapidly and degraded within several hours of
the acute stimulus, whereas other “acute Fras” (e.g., FosB, ΔFosB, Fra1, Fra2) are induced somewhat later and persist
somewhat longer than c-Fos. The “chronic Fras” are biochemically modified isoforms of ΔFosB; they, too, are induced
(although at low levels) after a single acute stimulus but persist in brain because of their enhanced stability. In complexes with
Jun family proteins, these waves of Fras form AP-1 binding complexes with shifting composition over time. Bottom: With
repeated stimulation, each acute stimulus induces a low level of ΔFosB isoforms. This is indicated by the lower set of
overlapping lines, which indicate ΔFosB-induced by each acute stimulus. The result is a gradual increase in the total levels of
ΔFosB with repeated stimuli during a course of long-term treatment. This is indicated by the increasing stepped line in the
graph. The increasing levels of ΔFosB with repeated stimulation would result in the gradual induction of significant levels of a
long-lasting AP-1 complex, which could underlie persisting forms of neural plasticity in the brain. (Adapted from reference 53.)

Steroid Hormone Receptor (or Nuclear Receptor) Superfamily

The steroid hormones (e.g., glucocorticoids, gonadal steroids such as estrogen and testosterone, and mineralocorticoids), retinoids,
thyroid hormones, and vitamin D3 are small, lipid-soluble ligands that diffuse readily across cell
 P.226

membranes. Unlike amino acid–derived neurotransmitters and neuropeptides, their receptors are cytoplasmic, rather than localized
to the cell membrane. On ligand binding, these receptors translocate to the nucleus, whereupon they bind to specific hormone-
response elements (HREs) located in the regulatory regions of specific genes and thereby regulate expression of those genes. These
receptors are referred to as the steroid hormone receptor, or nuclear receptor, superfamily (62 ,63 and 64 ).

The glucocorticoid receptor (GR) exemplifies general mechanisms utilized by this superfamily (62 ,63 ,64 and 65 ). Under basal
conditions, the GR is retained in the cytoplasm by a large multiprotein complex of chaperone proteins, including the heat shock
protein Hsp90 and the immunophilin Hsp56. When bound by glucocorticoid, the GR dissociates from its chaperones and translocates
to the nucleus. The first activity of the GR to be identified was its function as a ligand-regulated transcription factor, as stated
earlier, by binding to glucocorticoid response elements (GREs). GREs are typically 15 bases in length; they consist of two palindromic
half-sites of six bases each separated by a 3-bp spacer. As described earlier for other transcription factors, this palindromic
organization of the GRE suggests that the GR binds as a dimer. Like many other transcription factors, the nuclear receptor
superfamily has a modular structure. The GR has three domains: an N-terminal transcriptional activation domain, a C-terminal
ligand binding domain, and an intervening DNA binding domain. The DNA-binding domain of the GR is characterized by a zinc finger
motif, in which multiple cysteines are organized around a central zinc ion. This type of DNA binding domain is used by many other
transcription factors, including the immediate early gene zif268/egr1 (see later). The DNA binding domain also contains a region
that permits dimer formation after GR monomers bind GRE half-sites.

GREs can confer either positive or negative regulation on genes to which they are linked, depending on the particular GRE involved
(62 ,63 ,64 and 65 ). One of the first positive GREs characterized is that within the metallothionein IIA gene, which encodes a
protein that chelates heavy metals. A well-characterized negative GRE is found within the proopiomelanocortin (POMC) gene. This
negative GRE permits glucocorticoids to repress the gene that encodes adrenocorticotropic hormone and is therefore an important
mechanism of feedback inhibition on further glucocorticoid synthesis.

GRs have many important physiologic actions that do not appear to be mediated by DNA binding. GRs can interfere with
transcriptional activity mediated by other transcription factors, particularly AP-1 and NF-κB. Although the mechanisms are not fully
understood, GRs appear to interact directly with AP-1 and NF-κB proteins to block their ability to activate transcription (62 ,63 ,64
and 65 ). An alternative mechanism by which glucocorticoids may interfere with NF-κB activity is by inducing expression of IκB, the
protein that holds NF-κB in the cytoplasm.

Other Transcription Factors in Neural Signaling

The CREB, AP-1, STATs, and steroid hormone receptor families are just a few of the literally hundreds, perhaps thousands, of
transcription factors that are expressed in neurons and glia. Most of these other factors are best understood with respect to their
roles in nonnervous tissues, although more recent work implicates them in neural signaling as well. Examples of these other
transcription factors have already been mentioned. NF-κB is activated by protein kinase C and immunologic signals and likely plays
an important role in the regulation of neural gene expression (15 ). C/EBP and its several family members are known to mediate
some of the effects of the cAMP pathway on gene expression and have been implicated in neural plasticity (32 ,66 ). Specific
protein-1 (SP-1), which binds to GC rich regions of promoters, is often thought of as a general transcription factor, that is, a
regulator of the basal rate of transcription. However, more recent research has shown that certain SP-1 family members are subject
to dynamic regulation and could mediate transcriptional changes induced by extracellular signals (5 ). AP-2 (activator protein-2)
binding sites are present in many neural-expressed genes, although we still know very little about its physiologic role (5 ). Zif268
(also called Egr-1) and related Egr family members are zinc finger transcription factors that, like c-Fos, are induced rapidly and
transiently in brain by many stimuli with temporal features of IEGs. Induction of Egr family proteins has been correlated with
induction of hippocampal long-term potentiation; however, their specific target genes remain poorly characterized (67 ).

CONCLUSIONS
Part of "17 - Regulation of Gene Expression "

Our discussion of nuclear signaling mechanisms highlights several important points. The first is that the potential number of
mechanisms by which the expression of a gene can be controlled is vast. This highlights the exquisite control over gene expression
that is required both for the generation of a diversity of cell types during development and for adaptation of cells to the
environment throughout life.

We devoted most attention to nuclear transcription factors, because these provide the best-understood mechanisms of how cells
adapt to external cues with alterations in gene expression. However, even the complexity of mechanisms discussed represents the
tip of the iceberg. Regulatory regions of genes are often far longer than the coding regions of genes. Regulatory information is
contained not only within the 5′ promoter regions of genes, but throughout intronic (and sometimes exonic) sequences as well as 3′
untranscribed regions. Within the 5′ regions, we focused on relatively small response elements, such as CRE and AP-1 sites. It is
extraordinary, indeed, that the difference of 1
 P.227

nucleotide (e.g., from a CRE to an AP-1 site) in a sequence of literally thousands can confer specificity on a gene with respect to its regulation. Nonetheless, we know that any given
gene likely contains many regulatory sites. Moreover, these sites do not function in isolation, but they influence one another. As a result, the rate of expression of a given gene
represents the temporal and spatial synthesis of multiple signaling pathways. Unraveling these layers of complexity is a daunting task, particularly in vivo, but it could hold important
clues for understanding neural and behavioral plasticity.

ACKNOWLEDGMENTS
Part of "17 - Regulation of Gene Expression "

This chapter is based on a more extensive, earlier chapter (see reference 14 ).

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Section II
Emerging Methods in Molecular Biology and Genetics

Samuel H. Barondes

Samuel H. Barondes: Center for Neurobiology and Psychiatry, Department of Psychiatry, University of California-San Francisco, San Francisco,

California 94143-0984.

Emerging Methods in Molecular Biology and Genetics - Introduction

When the American College of Neuropsychopharmacology was founded in the mid-1950s, molecular biology and genetics were in their infancy and

had little to offer neuropsychopharmacology. By 1967, when the first volume in this series was published, it still had not become apparent how

greatly our field would be influenced by research on genes and on DNA. Of more than a hundred papers in that volume--Psychopharmacology: A

Review of Progress 1957-1967--only a few used tools of molecular biology. None of those papers, including my own, envisioned the central role that

such tools have now come to play.

This central role, which is documented in this section, is the result of the development of two types of new technologies. One of them, the automated

sequencing of the nucleotides in DNA, facilitated the decoding of the structure of all genes, including those that make up the human genome. The

other consists of ways to manipulate the structure of individual genes in isolated cells or in intact organisms, and to measure their levels of expression.

This made it possible to directly study the biological actions of particular genes, and the effects of changes in their regulatory regions and coding

regions.

These technologic advances are now being applied to a variety of problems in neuropsychopharmacology. For example, measurements of the levels

of expression of large numbers of genes in various brain regions and nerve cells are providing information about the molecular basis of normal brain

functions, and the effects of drugs on these functions. The same technologies are also being used to learn about the molecular pathogenesis of mental

disorders. As work on the human genome continues it will lead to the identification of gene variants that influence susceptibility to mental disorders,

as well as of gene variants that influence the alternative ways that individuals respond to particular psychiatric drugs. It will also provide new targets

for the creation of better drugs, with greater efficacy and specificity.

The six chapters in this section provide a sampling of the molecular and genetic tools that are being used to advance neuropsychopharmacology.

Because these tools are changing so rapidly, the authors provide overviews rather than extensive details. In this way they hope to make these tools

comprehensible to the nonspecialist, and to invite their further application.

As you read these overviews you will become increasingly familiar with a series of new terms--from gene chip, knockouts, and viral vectors, to

genome scans and pharmacogenetics. These terms are becoming commonplace, and are already scattered throughout this book. By the time the next

volume in this series appears, it is likely that the methods that they refer to will be so widely used in our field that a separate section about them will

no longer be needed.
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18
Using Human Genomics to Advance Neuropsychopharmacology

L. Alison Mcinnes

Nelson B. Freimer

L. A. McInnes and Nelson B. Freimer: Department of Psychiatry and Neurogenetics Laboratory, University of California–San
Francisco, San Francisco, California 94143.

Genomics, the study of genomes, includes gene mapping, sequencing, and investigation of gene functions. This field will advance
neuropsychopharmacology in two complementary ways. First, it is hoped that application of genomics technologies to pedigree and
population samples of patients with psychiatric disorders will allow the identification of genes contributing to the etiology and
pathogenesis of these diseases and provide a rational basis for new drug development. Second, variations in the sequence of known
genes whose products are the targets of current psychotropic drugs may influence the likelihood that an individual patient will have a
therapeutic response to these drugs or experience a side effect. Identification and functional characterization of these sequence
variants in large populations of patients of various ethnicities constitutes the discipline of pharmacogenomics. The scope of
psychopharmacogenomics, however, is currently restricted by our limited knowledge of the genes that contribute to psychiatric
disorders and the neural pathways altered by psychotropic agents. Fortunately, data and technologies provided by the U.S. Human
Genome Project (HGP), including provision of the complete sequence of the human genome, will greatly facilitate identification of such
genes (1 ,2 ). This chapter describes how technologic advances in genomics will shape the future of psychiatric genetics and
psychopharmacogenomics, fields that may establish an objective basis for the restructuring of the nosology, diagnosis, and treatment of
psychiatric disorders. The results of genetic studies of particular psychiatric disorders and of responses to specific drugs are considered
in other parts of this book.

• IDENTIFYING GENES FOR PSYCHIATRIC DISORDERS

• PHARMACOGENOMICS

• SUMMARY

• ACKNOWLEDGMENTS

IDENTIFYING GENES FOR PSYCHIATRIC DISORDERS

Part of "18 - Using Human Genomics to Advance Neuropsychopharmacology "

Rational strategies for the advancement of psychopharmacology are dependent on furthering our currently sparse knowledge of the
pathophysiologic basis of psychiatric disorders. To this end, human genetic approaches offer a promising alternative to traditional
biochemical and neurophysiologic investigations as twin, family, and adoption studies all support the heritability of many psychiatric
syndromes. Unfortunately, attempts to first map (i.e., localize a unique region of DNA shared by patients with a particular disorder)
and then identify genes predisposing to psychiatric disorders have been frustrated by the complexity of the genetic mechanisms
underlying behavioral phenotypes.

A phenotype is the observable physical manifestation of genetic variation at a particular site or locus in the DNA, whereas a genotype
refers to the actual DNA sequence, at the responsible genetic locus. With single gene disorders (also referred to as mendelian disorders)
there is a simple, direct relationship between variation in a single gene and the phenotype that results. Thus, all patients with a given
mendelian disorder, such as cystic fibrosis, will carry abnormal genotypes at a single disease locus. In contrast, the relationship
between phenotype and genotype is not straightforward for complex genetic traits. In this setting, multiple different susceptibility
genes and environmental factors interact in varying combinations within individuals who appear to have clinically indistinguishable
phenotypes. This means that in any given sample of patients diagnosed with a particular psychiatric disorder, the number of individuals
actually sharing a disease gene or genes in common might be very small such that the “effective” sample size does not provide enough
power to detect the responsible genes.

Fortunately, there are strategies for finding genes contributing to complex traits that have been successfully applied to the genetic
dissection of other nonpsychiatric, genetically
 P.232

heterogeneous disorders. One approach is to try to reduce genetic heterogeneity in the patient sample by studying genetically
isolated populations or by narrowing the affected phenotype under study based on criteria of severity or the presence of a biological
marker for the disease. Another approach is to greatly expand the sample size and number of DNA markers used in genetic
association studies to increase the power to detect multiple possible genes contributing to disease in subsets of the sample. In
either case, both pedigree and population-based genetic mapping studies are expected to yield more promising results in the future
due in part to the extensive characterization of the human genome provided by the HGP. The HGP, begun in 1990, is a joint effort
coordinated by the U.S. Department of Energy and the National Institutes of Health, with the cooperation in recent years of
international entities such as the Wellcome Trust in Great Britain (3 ). One of the HGP's main goals is to finish the complete human
genome sequence by the end of 2003 while concomitantly identifying all the estimated 100,000 genes in human DNA and creating
the most dense and accurate genetic maps for genome screening studies. The next subsection describes how innovations in genetic
maps and the structure of genetic mapping studies may eventually lead us to identify the as yet elusive genes for psychiatric
disorders.

Genetic Maps
At the time of this writing, commonly used genetic maps consist mostly of microsatellite DNA markers (usually repeats of two, three,
or four nucleotides that vary in length among individuals) that occur with fairly even spacing across the entire genome. These maps
contain several thousand markers spaced at roughly 1 to 5 centimorgans (cM); 1 cM is a unit of genetic distance equivalent to a
recombination frequency between two loci of 1%, i.e., one recombination would occur per hundred meioses. Much denser maps are
under construction now, however, as part of the HGP. In fact, a major goal of the HGP is to characterize the extent of genetic
variation that exists among humans in order to create a map of several hundred thousand markers to enable high-density genome
screening studies of complex traits (4 ). Differences in single bases of DNA known as single nucleotide polymorphisms (SNPs) are
thought to constitute roughly 90% of sequence variation in humans. Occurring at an average spacing of 1 SNP per 1,000 base pairs (1
kilobase, kb), they will thus constitute the majority of the markers in the planned high-density map. To facilitate identification of
these SNPs, the National Institutes of Health (NIH) recently assembled cell lines and DNA from a collection of 450 anonymous,
unrelated individuals representing the major ethnic groups of the United States; this collection is known as the DNA Polymorphism
Discovery Resource (DPDR) (5 ). The DPDR is available to investigators to facilitate detection of population genetic variation in their
loci of interest, with the expectation that they will share this information with the scientific community. For instance, SNP variants
in the coding or regulatory regions of genes (cSNPs) may cause functional differences in gene expression. With such variants
catalogued in advance, it will be relatively straightforward to test multiple candidate genes for association with a disease phenotype
or a pharmacogenetic effect. The enormous task of identifying and scoring SNPs in large samples, or performing the projected high-
density genome screening studies, has necessitated the development of high-throughput technologies such as DNA chips (6 ), which
are discussed elsewhere in this volume.

Principles of Genetic Mapping: The Search for Identity by Descent

Genetic mapping methods are based on the expectation that a proportion of patients in a study population, whether members of an
extended pedigree or more remotely related descendants of a common ancestor, will share segments of DNA identically by descent
(IBD) in the vicinity of the disease gene under study. The principle behind this expectation is best illustrated by considering
genetically isolated or founder populations (7 ,8 ). A founder population descends from a small number of ancestral individuals and
grows in relative isolation with little admixture, so it is genetically homogeneous. The premise is that a disease mutation is
introduced into a population on a particular “founder” chromosome, which is then transmitted to patients descended from a
common ancestor carrying this mutation. Over subsequent generations, recombination will reduce the size of the segment of DNA
that patients share around the disease gene. The shared segment includes the markers that flank the disease gene. These markers
are said to be linked to the gene since, because of their close physical proximity, they seldom recombine with each other and hence
are transmitted as a unit or segment. Detection of linkage is the goal of pedigree-based genetic studies. Alternatively, if alleles at
markers co-occur more frequently than expected given the known allele frequencies and recombination fraction between the
markers, they are said to be in linkage disequilibrium (LD). Evidence of LD between markers also indicates that they are probably
close to each other. If LD is observed between the same markers over a region greater than occurs at random in a sample of patients,
it may indicate that they share this segment of DNA IBD and that it harbors a disease gene. Detection of LD is a goal of population-
based genetic studies.

The length of the IBD segment around a disease gene is inversely proportional to the number of generations by which patients are
separated from a common ancestor (Fig. 18.1 ), and thus genomic screening strategies to detect such a segment will depend on the
“age” of the study population (9 ). For example, a sample of affected individuals separated by roughly 15 generations from their
common ancestors
 P.233

might be expected to share segments around a disease gene detectable in genome screens using current microsatellite maps with
markers spaced at 3 to 5 cM. In contrast, the Finnish population is a founder population in which present-day individuals are
separated from their common ancestors by up to 100 generations. In this case, shared DNA segments harboring a particular disease
gene may be so small that one would have to screen the genome with a much denser marker map (e.g., markers every 0.1 cM) to
find them. Such screening studies in a nonhomogeneous population such as that of the United States, wherein common ancestors
must be located in the very remote past, will require use of the planned SNP map of around several hundred thousand markers in
order to detect regions of LD, which, it has been hypothesized, may be as small as 3 kb (10 ).
FIGURE 18.1. Genetic mapping studies in isolated populations take
advantage of the fact that many recombination events separate affected
individuals in the present day from a common disease ancestor. As a
result, the majority of patients should share a segment of DNA around
the disease gene that is longer than any other DNA segment they might
share by chance, but still small enough for positional cloning purposes.

Alternatively, identification of much larger chromosomal regions that are IBD among a sample of patients may also be carried out in
extended pedigrees wherein the small number of meioses separating affected individuals leads to a greater length of IBD sharing
around the disease gene. Such regions may be easier to detect in pedigrees with genome screens using markers spaced at broader
genetic intervals (on the order of 5 to 10 cM). It can be difficult, however, to find recombinant individuals that will allow
refinement of the candidate interval to a sufficiently small region to facilitate positional cloning. We review the relative strengths
and weaknesses of pedigree- and population-based genetic studies below.

Pedigree-Based Mapping Studies: Problems and Solutions

Linkage analysis of pedigree data has been a very successful method of mapping genes for rare single gene disorders with distinct
phenotypes such as cystic fibrosis (11 ). There are many limitations on the use of linkage analysis for complex traits, however.
Linkage analysis is a statistical means of quantifying the likelihood that the observed segregation of marker alleles within a family
supports the hypothesis of linkage versus nonlinkage to a disease gene. Traditional linkage analysis is model-based or parametric,
meaning that it requires specification of disease inheritance parameters that are not easily estimated for psychiatric disorders, such
as the frequency of the disease allele, the genotype specific penetrance, or even the number of genes likely involved. The lack of
knowledge of these parameters means that data must be analyzed under a number of different models. This process of multiple
testing diminishes the strength of statistical conclusions such that it may be nearly impossible to distinguish between background
“noise” and true but weak signals coming from multiple genes of small effect (12 ). However, several strategies have been
successfully implemented to increase the power of linkage analysis for complex nonpsychiatric illnesses by attempting to reduce
genetic heterogeneity in the patient sample or by using methods of statistical analysis that do not require specification of genetic
model parameters.

Solution 1: Reduce Genetic Heterogeneity in the Sample

Strategies for reducing genetic heterogeneity include studying a small number of large, multiply affected pedigrees ascertained, if
possible, from a founder population and narrowly defining the phenotype understudy. The premise in the former case is that the
number of genes contributing to a particular disease phenotype within one or a few large families may be less than in many small
families or the population at large. This premise is also more likely to hold if the number of disease loci in the population as a whole
is limited, as may be the case for founder populations (7 ,8 ). Furthermore, a multiply affected family may indicate that the gene or
genes involved are highly penetrant (penetrance refers to the likelihood that a person who has a disease gene will manifest the
disease phenotype) and may be easier to find.

For nonpsychiatric complex traits, refinement of the affected phenotype can be accomplished in several ways. For instance, limiting
the affected phenotype to include only the most extreme or distinct form of the illness under study has also been critical to the
success of mapping studies for complex traits, as such phenotypes are expected to reflect a more homogeneous genetic etiology
than more broadly defined phenotypes (13 ,14 ). Illustrating these points, a gene
 P.234

for a severe form of Alzheimer's disease (AD) characterized by early age of onset was detected in a few large multiply affected
pedigrees ascertained from a genetically isolated population of German descent (15 ,16 ). Although such a gene may not contribute
significantly to AD in the general population, it may still provide clues as to relevant biological pathways that might suggest
candidate genes for other mapping studies.

Another way to refine an affected phenotype is to require the presence of an objective measure associated with the disorder such as
elevated immunoglobulin E (IgE) levels in patients with asthma (17 ). However, we still have no comparable biological markers for
psychiatric disorders at least when these disorders are defined by current nosology. Hence, investigators are attempting to find
endophenotypes or subcomponents within psychiatric syndromes that may be objectively measured and inherited in a more
straightforward fashion than the constellation of symptoms that constitute the full psychiatric syndrome (18 ,19 and 20 ). What
differentiates this strategy from other attempts to refine traditional psychiatric phenotypes is that family members who have not
received a psychiatric diagnosis may still be segregating the trait of interest and may display an endophenotype that allows them to
be considered “affected” for genetic study.

Many of the efforts to investigate familial transmission of endophenotypes have focused on schizophrenia. For example,
investigators have hypothesized that abnormalities of sensory gating are biological markers for attentional dysfunction, which seems
to be a core phenotypic characteristic of schizophrenia or psychosis. Abnormal ocular movements and failure to suppress evoked
responses to auditory stimuli after a cue (the P50 response) are both thought to be transmitted within families of schizophrenic
probands whether or not family members have a psychiatric diagnosis (21 ,22 ,23 ,24 and 25 ). Two studies have examined the same
set of families for linkage to either an abnormal P50 response alone or in combination with abnormal oculomotor movements
(26 ,27 ). Evidence for linkage to each phenotype implicated different loci (the α7-nicotinic acetylcholine receptor subunit gene and
a region in chromosome 22q11-12, respectively) and was stronger than evidence for linkage of the schizophrenia phenotype alone.

Few endophenotypes have been characterized so far for mood disorders; however, a possible endophenotype is that of suicide (28 ).
Roy et al. (29 ) studied the monozygotic and dizygotic co-twins of twin suicide victims and found that significantly more monozygotic
co-twins than dizygotic co-twins also attempted suicide, possibly arguing for a genetic component to this behavior. Investigators are
continuing to develop brain banks of suicide victims for postmortem studies including genetic screens and searching for relevant
biochemical markers (30 ).

Solution 2: Alternative Analytic Models

As an alternative to model-based methods, nonparametric or “model-free” methods can be utilized to detect linkage and may be
more robust when the true mode of inheritance is unknown. These methods were originally developed for samples of affected sibling
pairs but have now been modified for analysis of other types of relative pairs or whole pedigrees. Simply stated, nonparametric
methods are designed to calculate the amount of IBD sharing of marker alleles among affected relatives where the null hypothesis is
that transmission of alleles is independent of transmission of disease. For any pair of affected relatives, the probability that the pair
will share zero, one, or two alleles IBD can be calculated based on their degree of relationship. Linkage is detected if the sharing of
marker alleles among affected relatives is increased over the sharing expected given their relationship.

Demonstration that patients share a series of alleles over multiple markers on the same chromosome, also known as haplotypes, can
definitively establish that a segment of DNA has been inherited IBD and thus likely harbors a disease gene. Methods that can
quantitate the significance of haplotype sharing among affected individuals are thus particularly useful tools for determining
candidate gene intervals in both pedigree and population samples, although the inheritance patterns within extended pedigrees
need to be fully characterized to avoid misinterpretation of the allele sharing data.

In addition to the problems with statistical detection of linkage, another major shortcoming of pedigree and affected relative pair
studies is that investigators may detect a disease gene but be able to localize it only to a very broad genetic interval. The extent to
which a genetic interval containing the disease locus can be narrowed to a small-enough interval for positional cloning purposes
depends on the number of opportunities for recombination of the disease haplotype in affected persons; in pedigrees where
individuals are separated by only two or three generations, opportunities for recombination of the disease haplotype are limited.
Although affected relative pairs are usually much easier to collect than multiply affected pedigrees, very large numbers are required
to detect linkage, and the accuracy of gene localization is usually much less than that provided by pedigrees.

Finally, when studying complex traits it is very likely that some individuals will be phenocopies, which means that they exhibit the
phenotype under study but due to different genetic or environmental factors. In this case, one may be misled by apparent
recombination events even in a single individual, and may therefore incorrectly delineate the candidate interval for a disease gene
in a region.

All of these factors can seriously impede localization and positional cloning efforts for disease genes in pedigree samples.
 P.235

Population-Based Mapping Studies

Given the limitations on pedigree studies described above, analysis of a population-based sample is frequently a preferred strategy
for high-resolution mapping of disease loci (31 ). One reason for this is that many meioses (and therefore opportunities for
recombination) have occurred since the patients in such a sample were separated from their common ancestor, so there is a better
chance to narrowly define a candidate gene interval (Fig. 18.1 ). Risch and Merikangas (32 ) also proposed that population mapping
strategies might be a more efficient means of initially localizing disease genes (given a sufficient sample size and an appropriately
dense marker map), particularly for loci of relatively small effect, as the sample sizes needed for affected relative pair strategies
may be huge and thus not feasible.

Association studies are designed to be case-control or family based (see ref. 33 for review). In case-control association studies,
allele frequencies at a particular marker are compared between a sample of patients and a sample of controls matched as closely as
possible to cases in terms of ethnicity, age, gender, and other relevant socioeconomic variables. Unfortunately, perfect matching
can never be guaranteed, and unknown population stratification can occur if many of the cases or controls share an uninvestigated
variable. In this setting, the alleles of cases might appear to differ markedly from controls at a particular genetic locus because of
such an unknown variable and not because of the presence of the disease phenotype; this could lead to a false-positive result. Such
stratification can occur even within distinct ethnic groups. For example, an association study of type 2 diabetes mellitus in a Native-
American tribe seemed to indicate that a particular allele of the immunoglobulin complex was protective against diabetes (34 ).
However, after extensive genealogic examination of Native Americans with this allele, it appeared that they all had distant
Caucasian relatives. As the allegedly associated allele was common in Caucasians, and diabetes was less common in Caucasians than
in the tribe studied, overrepresentation of this allele in nondiabetic Native Americans reflected only the presence of Caucasian
admixture and not a true protective effect from diabetes.

Alternatively, affected individuals and their parents are ascertained for family-based studies that utilize the alleles on the
nontransmitted chromosomes of parents as controls for the patients’ alleles to prevent ethnic mismatching. One commonly used
approach to analyzing such family data is the transmission disequilibrium test (TDT) (35 ). In this test each allele of a heterozygous
parent is measured to see if it is transmitted to an affected offspring significantly more often than the expected 50% by chance. In
this case, the implicated allele would be both associated and linked to the disorder, obviating the possibility that the allele is falsely
associated through population stratification. Other approaches for analyzing family-based association data use nontransmitted
parental alleles as controls, but do not evaluate actual transmission of these alleles to the patient and do not exclude data from
homozygous parents. One such approach is the likelihood-based method of Terwilleger (36 ). A disadvantage of family-based LD
methods is that it can often be difficult to sample parents of affected individuals, especially for adult-onset disease.

The LD tests described above are often used to examine the association at single markers individually, which can also be
problematic because a very large number of markers must be used for LD genome screening studies, even in isolated populations,
and statistical correction for multiple testing is necessary. Interpreting the significance of single-point association tests in this
setting becomes extremely difficult (37 ). Fortunately, the development of multipoint statistical methods for quantifying the
significance of haplotypes shared over multiple markers could help to increase the power to detect even weak LD signals coming
from a subset of the sample. Such approaches are inherently more powerful than single-point tests of association and will be
essential for the evaluation of data generated from SNP maps.

One promising LD method, termed ancestral haplotype reconstruction (AHR), assesses the likelihood that a sample of patient
haplotypes have descended from a common mutation-bearing founder haplotype (38 ). This method is currently being modified so
that it will be useful both for genome screening and subsequent fine-mapping studies. At the genome screening stage, markers are
generally spaced at sufficient distances such that they can be considered to be in linkage equilibrium with each other in distantly
related affected persons. Detecting LD between two or more markers in this setting should thus point to the candidate gene interval
as long as the underlying assumption is met that the markers tested are not in LD with each other independent of the disease
phenotype (so-called background or random LD); it is still not certain how such background LD is distributed within the genome and
between different populations. Once a candidate region has been identified by LD analysis, the next step is to type as many markers
as closely spaced as possible within the area to determine the minimal interval of maximal IBD sharing that should contain the
disease gene. Although this step can be accomplished in some cases just by observation (39 ), a statistical method that can assign
some level of significance to the observed data would be very useful, especially if the disease haplotype is relatively common and a
large sample is required to detect its contribution to the phenotype. However, multipoint analysis of markers typed at high density
for fine mapping (or for genome screens with dense SNP maps) is complicated because, since the markers are so closely spaced, one
cannot assume that these markers are not in LD with each other independent of the disease phenotype. Multipoint LD methods such
as AHR will need to take into account the possibility that significant background LD could occur between
 P.236

closely spaced markers in order to distinguish this background LD from what may be a very subtle increment of LD surrounding the
true disease locus.

In summary, the ability to localize disease genes using LD methods in a given population sample depends on the amount and extent
of LD present, the number of disease predisposing alleles at a given locus (allelic complexity), the degree to which the disease locus
increases the likelihood of manifesting the affected phenotype, and the power of current statistical methods to measure existing LD.
For an excellent review of the strengths and weaknesses of current statistical approaches for analyzing LD, see ref. 40 .

Identification of a Disease Gene

Should psychiatric geneticists overcome the many obstacles facing them and succeed in mapping a disease locus to a specific
interval, the next step would be to identify the disease gene within it, a process termed positional cloning or, given the completion
of genome sequencing, the positional candidate approach (41 ). Positional cloning in its purest sense is the process of identifying a
disease gene based only on knowledge of its chromosomal location as determined by linkage analysis or LD mapping, without any
knowledge of the gene's function. This process involves laborious efforts to build a physical map and sequence the region. Physical
maps are made by isolating and linking together yeast and/or bacterial artificial chromosomes (YACs, BACs) containing fragments of
human DNA from the region. These fragments are then sequenced and ordered so that the genomic DNA sequence across the
candidate gene region is known. Then comes the arduous process of identifying all the genes in the interval and performing mutation
detection. Advances in physical mapping and gene-finding technologies arising from the HGP have greatly speeded up this process,
however. For instance, near-complete genomic sequence data for the region of interest may already have been deposited in the
public databases, obviating the need for extensive genomic sequencing. Next, an investigator may explore large databases of partial
complementary DNA (cDNA) sequences also known as “expressed sequence tags” (ESTs) to find most or all of the genes mapping
within the area. In addition, there are already a multitude of Web-based sequence analysis programs that provide a host of
information such as exon and promotor prediction, open reading frames, and protein homologies for translated sequence. This
computer exercise is part of the positional candidate approach. Once all the ESTs have been identified, the complete sequence of
the corresponding genes (including definition of intron/exon boundaries and the promotor, if possible) are elucidated and mutation
detection begins. If the causative mutation is not detected in coding sequence, however, it can be very difficult to detect disease-
causing mutations in the surrounding noncoding DNA or introns, as these regions are large and likely to display more natural
variation than highly conserved coding sequence. One possible strategy in this situation is to search for an orthologous (similar) gene
in a model organism such as the mouse. Comparison of these sequences may highlight strongly conserved regions of DNA outside of
coding sequence that may be functionally relevant and important to examine closely for disease predisposing variants (42 ).
Provision of the complete sequences of the genomes of model organisms amenable to genetic manipulations such as flies, mice,
roundworms, and yeast by the HGP could speed understanding of comparable gene structure and function in humans and serve as a
molecular confirmation and/or supplement to current gene prediction programs (43 ,44 ).

PHARMACOGENOMICS
Part of "18 - Using Human Genomics to Advance Neuropsychopharmacology "

As genes contributing to the development of psychiatric disorders are discovered, they will be added to the known array of
neurotransmitters, receptors, and transporters that are already considered candidate genes for pharmacogenetic analysis.
Pharmacogenetics is the study of the genetic mechanisms determining an individual's responsiveness to drugs (45 ). In this approach,
genes involved in drug metabolic pathways and sites of action, or disease processes if known, are examined for naturally occurring
variants or polymorphisms, which may then be shown to affect the expression of that gene. This effect on gene function may then
be linked to the efficacy of the drug and/or a predisposition to particular side effects in individuals with that genotype. For example,
the apolipoprotein (apo) E4 allele at the apo E locus has been shown to be associated with late-onset AD, as well as to a poor
response to cholinesterase inhibitor treatment of AD (46 ). The downside of this strategy is that it is limited by the paucity of
candidate genes with proven association to psychiatric phenotypes. Fortunately, investigators in the emergent field of
pharmacogenomics are seeking to identify previously unsuspected genetic markers of drug responsiveness by using a variety of high-
throughput genomics technologies to examine drug-induced changes of gene expression in different tissues throughout the body. We
provide here a brief overview of the principles of pharmacogenetics and pharmacogenomics (see ref 47 . for review), focusing on
how these approaches are being applied in an effort to provide a more rational basis for pharmacotherapy of psychiatric disorders
than the trial-and-error approach we currently employ.

The impact of genetic variation on drug response is characterized broadly by changes in pharmacokinetic and pharmacodynamic
parameters. Pharmacokinetic studies assess the processes of absorption, distribution, first-pass and general metabolism, and
elimination of drugs. Transport processes in renal, intestinal, and hepatic epithelia and drug metabolizing enzymes exhibit genetic
variability, which will in many cases be likely to influence the pharmokinetics of
 P.237

relevant drugs. In the latter situation, the cytochrome P-450 system has been best studied (48 ) beginning with the classic example
of the poor metabolism of the antihypertensive drug debrisoquine due to several mutant alleles of the polymorphic CYP2D6 gene
also known as debrisoquine/sparteine hydroxylase. This enzyme is responsible for the metabolism of roughly a quarter of all drugs
including most antipsychotics and antidepressants (49 ). This enzyme is also inhibited potently by fluoxetine. About 7% of Caucasians
and an even greater percentage of Asians are poor metabolizers of such drugs due to polymorphisms in this enzyme. On the other
hand, some persons carry different alleles and/or multiple copies of this gene, which predispose to more rapid metabolism; up to 13
copies have been documented in a single individual. A study of nortriptyline metabolism in these individuals clearly demonstrated
that clearance of nortriptyline was proportional to the number of copies of the CYP2D6 gene, especially the CYP2D6*2 allelic form
(50 ,51 ). Knowledge that a person has a genotype predisposing to unusually slow or rapid metabolism could guide appropriate drug
choice and dosing regimen. Unfortunately, despite intensive study, no definitive relationship between polymorphisms in cytochrome
P-450 enzymes and drug efficacy or predisposition to side effects of antidepressant drugs has yet been discovered (52 ).

Pharmacodynamics concerns the relationship between the concentration of a drug and response at its site of action, for example at
receptors and transporters for neurotransmitters. Pharmacodynamic effects may also vary temporally, and so both the acute and
chronic nature of response to the drug must be considered. In the acute phase of responsiveness, receptor polymorphisms could
alter any of the myriad steps in a pathway from receptor-drug binding through the cascade of signals that result; such variants may
determine who is more prone to immediate drug reactions, for example the malignant hyperthermia that may occur in response to
antipsychotics. Genetic variation could also play a role in the drug-induced neural plasticity that occurs as a result of the chronic
treatment required for alleviation of psychiatric symptomatology as well as chronic use of addictive substances. Adaptive responses
to drugs will vary among individuals, and genetic factors may predict such phenomena as waning of drug response over time, and
proneness to side effects such as tardive dyskinesia induced by antipsychotics (53 ).

From a pharmacogenetic perspective, one of the most obvious candidates for studying psychiatric drug responsiveness identified to
date is the serotonin (5-hydroxytryptamine, 5-HT) transporter (5-HTT) (52 ). This transporter plays a critical role in the termination
of serotoninergic transmission and is the target of the most widely prescribed family of antidepressants, known collectively as the
selective serotonin reuptake inhibitors (SSRIs). Heils et al. (54 ) identified in 1996 a variant of 5-HTT with a 44–base pair (bp)
insertion within the promoter region, which is commonly referred to now as the long (versus the short) allele. Lesch et al. (55 ) then
demonstrated that baseline levels of transcription of the long variant were more than double that of the short variant in transfected
cells and that this difference was reflected in altered 5-HTT expression and 5-HT reuptake). Since then, investigators have studied
depressed patients to see if they can correlate SSRI response with alleles at the 5-HTT promoter. Smeraldi et al. (56 ) presented
data suggesting that patients with delusional depression responded better to fluvoxamine if they were homozygous for the long
allele of the 5-HTT promoter. Other investigators have obtained similar findings in a samples of depressed patients treated with
paroxetine (57 ), although in this study rapidity of response was improved in persons homozygous for the long allele, while overall
outcome at 12 weeks was the same for all genotypes.

Besides the serotonin transporter, there are several thousand other genes for neuromodulatory molecules that have been cloned and
expressed in some type of cellular system and that are viable candidates for drug targets (58 ). Pharmacogenomic technologies may
aid in prioritizing these candidates for examination, or identifying yet more candidates, by determining those genes that are
activated or deactivated in tissues during an acute psychiatric episode and in response to treatment. One approach to evaluating
gene expression involves hybridization of fluorescent or radioactively labeled messenger RNA (mRNA) samples taken from the
relevant cell population to cDNA arrays. Changes in gene expression (up, down, or none) can then be compared between different
samples at a single time point or within a sample overtime. This technique is known as serial analysis of gene expression (SAGE) (59 ).
However, if the changes in gene activation induced by disease or by drugs are localized to a specific population of cells in
inaccessible tissues such as that of the brain, rather than, say, in fibroblasts from skin biopsies, the SAGE technique will not be
helpful. Alternatively, large-scale analysis of proteins within clinical samples is also predicted by some to become a useful means of
identifying biological markers indicative of a response to drugs (60 ). Again, any changes in protein expression would need to be
detectable in easily obtainable fluids such as blood or urine to be of use in the evaluation of psychiatric disorders, and the process
of informed consent for such experimentation would need to be reviewed thoroughly.

SUMMARY
Part of "18 - Using Human Genomics to Advance Neuropsychopharmacology "

Genomics has great potential to advance the field of psychiatry in general and neuropsychopharmacology in particular. This will
occur through the identification of genes involved in the etiology of psychiatric disorders and the identification and characterization
of genes involved in the response to psychotropic agents. Although we have not made much headway as of yet in either of these
fields, impressive advances
 P.238
in our knowledge of the genomes of human and model organisms as well as access to the technologies developed to provide this information are likely to stimulate swift progress.
Success in these endeavors will lead to more efficient identification and development of drug targets, the provision of an objective basis for choice of drug, “custom drugs” based on
an individuals genotype, improved diagnosis of disease, and earlier detection of genetic predispositions to disease.

ACKNOWLEDGMENTS
Part of "18 - Using Human Genomics to Advance Neuropsychopharmacology "

L. Alison McInnes is supported by National Institute of Mental Health (NIMH) grant K01 MH01748-01. Nelson B. Freimer is supported by NIMH grants R01-MH49499 and K02-MH01375. We
would also like to thank Susan Service for her original drawing.

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19
Gene Targeting and Transgenic Technologies

Laurence H. Tecott

David S. Johnson

Laurence H. Tecott and David S. Johnson: Department of Psychiatry, Langley Porter Psychiatric Institute, University of California–
San Francisco, San Francisco, California 94143-0984.

Recent progress in the development of molecular genetic methods enables the manipulation of genes in intact mammalian organisms.
The power of such techniques to elucidate complex biological systems was initially recognized and exploited by developmental
biologists and immunologists. More recently, the utility of these approaches for examining neural gene function in the context of the
intact organism has led to their use in neuropsychopharmacology. Since the publication of the previous edition of this book, there
has been an explosion in the application of molecular genetic technologies to study the regulation of complex behavior and its
modulation by psychoactive drugs.

For several decades, the ability to manipulate genes in organisms such as yeast, fruit flies, and nematodes has produced important
insights into the regulation of a wide variety of complex biological processes. Limitations in the use of such organisms for research in
neuropsychopharmacology arise from the marked organizational differences between the mammalian brain and the systems that
govern behavior in these organisms. By contrast, a substantial degree of homology exists in the organization of the central nervous
system (CNS) and in the complement of genes expressed across mammalian species. Currently, the mouse genome is by far the most
accessible mammalian genome to manipulation. Procedures exist in the mouse for introducing new genes, expressing elevated levels
of endogenous genes, and eliminating or altering the function of identified target genes.

Mutant mouse models may be used for a number of purposes relevant to neuropsychopharmacologic research. For example, the
impact of genetic mutations on the behavior of mutant mice may be examined, providing insights into the functional significance of
particular gene products. In some cases, the manifestations (phenotypes) of these mutations may resemble features of human
neuropsychiatric diseases, providing animal models for studying neural processes relevant to such disorders. Furthermore, as genes
that confer susceptibility to human diseases are identified, it will be possible to introduce corresponding mutations into the mouse
genome, generating useful models for studying disease pathophysiology and treatment. Finally, genetic models will be useful for
investigating mechanisms through which nonselective drugs influence neural function and behavior. For example, the contribution of
a particular receptor subtype to the actions of a nonselective drug may be examined by studying its actions in animals with targeted
loss-of-function mutations of that receptor gene.

This chapter provides an overview of the transgenic and gene targeting approaches used to manipulate the mammalian genome. We
have divided these techniques into three categories: (a) transgenic technologies, in which exogenous gene sequences are inserted
into the mouse genome; (b) gene targeting technologies, in which mutations are targeted to inactivate or otherwise modify an
endogenous gene of interest; and (c) conditional genetic manipulations, in which mutations are restricted to particular stages of
development or to particular regions of the CNS. In addition to a brief description of these technologies, examples of their
application to neuropsychopharmacology are provided, as well as discussions of the benefits and limitations of each approach.

 TRANSGENIC PROCEDURES
 GENE TARGETING PROCEDURES
 PROCEDURES FOR ENGINEERING CONDITIONAL MUTATIONS
 SUMMARY

TRANSGENIC PROCEDURES
Part of "19 - Gene Targeting and Transgenic Technologies "

The ability to insert an exogenous (or foreign) gene into the mouse genome by direct injection into the pronuclei of zygotes was
achieved just two decades ago (1) . The term transgenic was applied to mice expressing exogenous DNA that had been produced
using this technique (2) . With this method, the gene of interest is inserted into a random locus in the mouse genome, and is
expressed “in trans,” i.e., not in its usual genetic locus. The techniques required for introducing
 P.242

transgenes into the mouse genome have been highly refined, permitting their widespread use. Since the development of this
technique, many thousands of lines of transgenic mice have been generated, and it has been the most widely utilized technique of
genetic manipulation in mice.

Methods of Production of Transgenic Mice

Techniques for producing transgenic mice involve the microinjection of DNA constructs into fertilized mouse eggs (Fig. 19.1) . DNA
constructs used for the generation of transgenic mice typically consist of a gene of interest located 3′ to promoter sequences
selected to produce a desired distribution of gene expression. The maximum length of the DNA sequence that may be successfully
incorporated into the mouse genome is not known, and up to 70 kilobase (kb) DNA fragments have been successfully integrated. The
transgene is linearized and purified from prokaryotic vector sequences. For optimal integration efficiency, about 1 to 2 picoliter (pL)
of DNA at a concentration of 1 to 2 ng/μL (corresponding to a few hundred molecules of a 5-kb DNA fragment) is microinjected into
the male pronucleus of a fertilized mouse egg. Although labor intensive, direct injection of DNA into the pronucleus results in much
higher rates of integration of transgenes than other known methods of transformation. After microinjection, the embryos are
surgically transferred into the oviduct of pseudopregnant mice. Pseudopregnant females are generated by matings with
vasectomized males. The act of copulation initiates the endocrine changes of pregnancy, providing a suitable uterine environment
for the survival and implantation of the transferred embyros. The foster mothers give birth 19 to 21 days after oviduct transfer. For
genotyping, DNA is typically isolated from mouse tail biopsies and screened for the presence of the transgene by Southern blotting
or polymerase chain reaction (PCR). Typically, about 20% to 40% of the mice that develop to term possess the transgene. In the
majority of cases, integration of the transgene occurs during the one-cell stage, so that the transgene is present in every cell of the
transgenic mouse. Integration usually occurs at a single random chromosomal location, and, for reasons that are not fully understood,
there are usually multiple copies of the transgene inserted as head-to-tail concatamers. Mice identified to possess the integrated
transgene are referred to as founders. The founders are typically used in a breeding strategy to produce animals that are
homozygous for the transgene insertion.

FIGURE 19.1. Procedure for production of transgenic mice. A: One-celled fertilized zygotes located in the oviduct ampullae of
pregnant donor mice are surgically harvested. B: DNA encoding the gene of interest is microinjected directly into the
pronucleus of the zygotes. C: Injected zygotes are surgically transferred into the oviducts of pseudopregnant female mice. D:
DNA from the progeny can be analyzed by Southern blot or polymerase chain reaction (PCR) for the presence of the transgene.

Uses of Transgenic Mice

Because transgenic mice often possess multiple copies of the transgene, this method can be used to produce animals with increased
levels of expression of particular genes, i.e., mice that “overexpress” genes of interest. In addition, it can be used to express
altered forms of a gene product in the distribution of the endogenous gene. One example is a transgenic line bearing a transgene
composed of the Ca2+/calmodulin-dependent protein kinase α subunit (CaMKIIα) promoter driving expression of a mutant form of
CaMKIIα that conferred Ca2+-independent activation. These mice exhibited an increased stimulation threshold for the induction of
synaptic plasticity in the hippocampus, as well as deficits in spatial memory (3, 4) . Studies of these animals led to an enhanced
understanding of the role of CaMKIIα in synaptic plasticity and spatial memory acquisition.

In many cases, it is desirable to express a gene with an anatomic distribution that does not mirror its native expression pattern in
the mouse. Such ectopic expression of a gene may be achieved using a transgenic construct in which the gene of interest is preceded
by promoter elements that direct expression in an anatomic distribution characteristic of another gene. An example of this approach
is a transgenic line in which the D1 dopamine receptor promoter was used to drive expression of a cholera toxin subunit (which
constitutively activates Gs) in cells that express D1 dopamine receptors (5) . Studies of these animals revealed that chronic
overstimulation (by constitutively activated Gs) of forebrain neurons expressing D1 receptors results in an abnormal behavioral
phenotype that was likened to human compulsive
 P.243

behaviors. For most genes, the promoter elements necessary to reproduce the native patterns of expression are not well defined. A
useful approach for identifying important promoter elements for genes of interest involves the generation of transgenic mice in
which putative promoter sequences are used to direct expression of reporter genes, whose expression is readily determined in brain
tissue. Comparisons may then be made between the pattern of reporter gene expression and that of the gene of interest (6, 7 and
8) .
It is also possible use transgenic approaches to reduce the expression of a particular gene product. This may be achieved using
“dominant-negative” mutations, mutations that induce loss of function of a gene product when expressed in the heterozygous state.
For example, transgenic constructs may be designed to express antisense RNAs that hybridize to native messenger RNA (mRNA)
sequences, thus decreasing production of the gene product of interest (9, 10 and 11) . Alternatively, the function of gene products
that aggregate into multimeric complexes may be disrupted by dominant-negative mutations that produce dysfunctional subunits of
the complex. The most prevalent approach used to generate loss of function mutations, gene targeting, is described in the next
section.

Considerations in the Interpretation of Transgenic Mouse Phenotypes

An important factor that frequently complicates the interpretation of studies with transgenic mice is the difficulty that may be
encountered in achieving a desired anatomic distribution of transgene expression. Promoter elements are often quite large, and
additional regulatory elements may at times be located great distances from the gene of interest. In addition, the site of integration
often affects the pattern and level of transgene expression, so that founder mice generated with a common targeting construct may
display different expression patterns. It may therefore be difficult to accurately duplicate a promoter's endogenous pattern of gene
expression in the setting of a transgenic mouse. Commonly, expression patterns are assessed in multiple founders, and those with
the most appropriate transgene expression would then be selected for a particular experiment.
Several additional considerations in the interpretation of phenotypes in transgenic mice warrant mention. For example, the number
of copies of the transgene incorporated into the genome varies between founder mice. In some cases, concatamers can be unstable
and susceptible to deletion of one or more copies of the transgene. In addition, the integration of the transgene may occasionally
disrupt an endogenous gene. This could lead to the development of a phenotypic abnormality unrelated to the function of the
transgene—this is estimated to occur in 5% to 10% of transgenic mice (12) . This possibility may be assessed by determining whether
similar phenotypes are present in animals derived independently from different founders, because the likelihood of two founders
possessing the same transgene integration site is minimal.

GENE TARGETING PROCEDURES

Part of "19 - Gene Targeting and Transgenic Technologies "
A mutational approach has proved to be invaluable to investigators examining the roles of gene products in complex biological
processes within prokaryotic and cultured eukaryotic cells. Recently it has become possible to apply this approach to a mammalian
system. Gene targeting procedures enable the precise (site-specific) introduction of a mutation to one of the estimated 100,000
murine genes. Typically, mutations have been designed to eliminate gene function, resulting in the generation of “knockout” or
“null mutant” mice. The introduction of mutations that produce more subtle alterations in gene function has also been achieved.
Two major developments have made gene targeting experiments feasible: (a) the generation of totipotent embryonic stem (ES) cells,
and (b) the elucidation of techniques to achieve homologous recombination in mammalian cells.

Gene Targeting Methods

ES cells are derived from 3.5-day-old mouse embryos, at the blastocyst stage of development (Fig. 19.2) . Blastocysts are cultured
individually under conditions that permit the proliferation of the inner-cell mass cells, which are those cells that would normally
become the fetus. These cells are then disaggregated, and individual ES cells clones are grown. Under optimal conditions, ES cells
retain the ability to contribute to all of the tissues of the developing fetus. The derivation of ES cells was pioneered using embryos
derived from the 129/Sv strain of mice, a strain that has been most commonly used in studies of early embryonic development.
Although this mouse strain is not ideal for the study of behavior (see below), most ES cell lines in current use are 129/Sv-derived.

FIGURE 19.2. Gene targeting in embryonic stem (ES) cells. A:

ES cells are derived from the blastocyst inner cell mass. A
targeting construct is introduced into ES cells by
electroporation. Cells are subjected to drug selection to enrich
for homologous recombinant clones (striped cells). Homologous
recombinant clones are isolated for blastocyst injection. B: A
targeting construct is produced in which the second exon of the
gene of interest is replaced by a neomycin resistance cassette
(Neo). A thymidine kinase (TK) cassette is included for negative
selection. Homologous recombination results in the
incorporation of engineered mutation into endogenous gene
locus. Arrows indicate the junction of construct sequences and
native locus.

Homologous recombination is the process by which a mutation is targeted to a precise location in the genome. A targeting construct
is generated that typically consists of a target gene sequence into which a loss-of-function mutation has been engineered (Fig. 19.2) .
Most targeting constructs are designed to achieve homologous recombination events in which recombination at the target locus
results in replacement of native target sequences with construct sequences. In mammalian cells, fragments of DNA preferentially
integrate into the genome at random locations, at rates that greatly exceed homologous recombination. Therefore, targeting
constructs are designed for use in selection strategies that enrich for ES clones in which homologous recombination has occurred. In
the commonly used positive-negative selection strategy (13), a portion of a protein-coding exon is replaced by sequences that confer
resistance to the drug neomycin. This mutation serves two purposes: (a) to inactivate
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the gene product, and (b) to provide a marker that enables the selection of cells that have integrated the construct. This exogenous
DNA fragment is flanked by regions of DNA that are homologous to the native gene. Adjacent to one of these homologous regions is a
gene encoding thymidine kinase. Treatment with the drug ganciclovir will kill cells that express this gene.

The targeting construct is typically introduced into ES cells by electroporation. In this step, cells are subjected to an electric current
that facilitates the internalization of the DNA construct. Those cells that failed to incorporate the targeting construct are killed by
the addition of neomycin to the culture medium (positive selection). The majority of the remaining cells have incorporated the
entire DNA construct (including the thymidine kinase gene) at random sites throughout the genome. By contrast, during homologous
recombination, nonhomologous regions of the construct that are not flanked by homologous sequences are excluded from the
integration event. Therefore, homologous recombinant clones will not contain the thymidine kinase gene. Thus, the addition of a
second drug, ganciclovir, will selectively kill cells that have randomly incorporated the construct (negative selection), thereby
enriching for targeted clones. ES cell clones that survive this double drug selection are then screened for homologous recombination
by PCR or Southern blot analysis. The homologous recombinant clones, which are heterozygous for the introduced mutation, are
then used to generate chimeric mice.

Following the isolation of homologous recombinant ES cell clones, these cells are microinjected into the fluid-filled blastocoele
cavity of 3.5-day-old embryos at the blastocyst stage (Fig. 19.3) . The injected embryos are then surgically transferred into the
uterus of pseudopregnant females. These animals will then give birth to chimeric mice, which are derived partly from the injected
ES cells and partly from the host embryo. For example, ES cells derived from a brown strain of mice are often injected into embryos
derived from black C57BL/6 mice, resulting in chimeras with coats
 P.245

containing black and brown patches. The extent to which the ES cells have colonized the animal may be roughly approximated by
the extent of the brown contribution to the coat. It is most important that ES cell derivatives colonize the germ cells of the chimera,
so that the targeted mutation can be propagated to subsequent generations. If chimeras are mated with C57BL/6 mice, then the
germ line transmission of ES cell–derived genetic material is indicated by the generation of brown offspring. Half of these brown
mice will be heterozygous for the targeted mutation. These heterozygous mice are then bred to produce homozygous mutant mice
that completely lack the normal gene product.

FIGURE 19.3. Generation of gene targeted mice. A: Homologous recombinant ES cells are injected into the blastocoele cavity.
B: Injected blastocysts are surgically transferred into the uterus of pseudopregnant female mice for the production of chimeric
mice. C: In this example, chimeric mice are bred with C57BL/6 animals. Germ-line transmission is indicated by coat color.
One-half of animals with the coat color indicative of the ES cell line will be heterozygous for the targeted mutation and the
other half wild type. Heterozygous animals may be bred for the production of homozygous mutant mice.

Uses of Gene Targeted Mice

Studies of null mutant mice provide novel insights into the functional roles of neural genes and, in some cases, animal models
relevant to human neuropsychiatric disorders. An illustrative example is a recent study of mice lacking the hypothalamic
neuropeptide orexin (14) . Observations of homozygous mutant mice revealed an unanticipated phenotypic abnormality. The
mutants displayed frequent episodes of inactivity characterized by the sudden collapse of the head and buckling of the extremities.
Subsequent electroencephalogram (EEG) analysis revealed these episodes to be similar to narcoleptic attacks observed in humans
and in a strain of narcoleptic dogs. Moreover, a mutation of an orexin receptor gene was found to underlie the canine syndrome
(15) . Thus, the null mutant phenotype revealed a novel role for orexin in sleep regulation. In addition, this line of mice represents
an important animal model for examining the pathophysiology and treatment of narcolepsy.

Another example illustrates the potential utility of null mutant mice to uncover mechanisms underlying the behavioral effects of
psychoactive drugs. The nonselective serotonin (5-hydroxytryptamine, 5-HT) receptor agonist m-chlorophenylpiperazine (mCPP)
interacts with several subtypes of 5-HT receptors. Although this compound typically reduces locomotor activity in rodents, it
produced a paradoxical hyperlocomotor response in a line of 5-HT2C receptor null mutant mice (16) . This response to mCPP was
blocked by pretreatment with a 5-HT1B receptor antagonist, indicating that the absence of 5-HT2C receptors unmasked a
hyperlocomotor effect of mCPP on 5-HT1B receptors in mutant mice. These results provide a model whereby genetic endowment may
contribute to the development of a paradoxical drug response. When a compound alters the function of multiple gene products with
opposing influences on behavior, then mutations or allelic variation of these genes may lead to paradoxical effects.

Although gene targeting techniques are most commonly used to generate animals with null mutations, subtle mutations may also be
introduced that alter, but do not eliminate, function. The benefits of such an approach are highlighted by a mutation of a gene
encoding the α1 subunit of the γ-aminobutyric acid A (GABAA) receptor (17) . The mutation produced a single amino acid change,
rendering the α1 subunit–containing subpopulation of GABAA receptors insensitive to benzodiazepines, without affecting their
responsiveness to GABA. The resulting animals exhibited reduced sensitivity to the sedative and amnestic effects of diazepam, but
no change in sensitivity to the anxiolytic-like effects of this drug. These results indicate that benzodiazepine site ligands devoid of
activity at α1 subunit–containing GABAA receptors may act as anxiolytics devoid of some of the side effects typically associated with
benzodiazepines, a prediction borne out by a recent report of the behavioral effects
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of such a compound (18) . These insights would not have been obtained using a conventional gene targeting approach, because a
null mutation of the α1 subunit gene would profoundly perturb brain GABA signaling.

Considerations in the Interpretation of Targeted Mutant Phenotypes

In interpreting behavioral phenotypes, attention must be paid to the effects of genetic background. The phenotypic consequences of
many targeted mutations may be influenced by modifying genes that differ among various inbred strains (19) . In some cases,
phenotypic abnormalities have been lost when mutations were bred to a new genetic background (20) . It may therefore be useful to
examine the persistence of mutant phenotypes in the context of several genetic backgrounds. In one example, three groups
independently generated lines of mice with null mutations of the 5-HT1A receptor subtype (21, 22 and 23) . Interestingly, although
each group placed this mutation on a different genetic background, all observed enhanced anxiogenic-like behaviors in the mutant
lines. Thus, particularly strong evidence is provided for a contribution of the 5-HT1A receptor to the regulation of anxiety.

Another potential problem arises from the common use of ES cells derived from 129/Sv mice. Mice of this strain are susceptible to
structural abnormalities of the CNS, such as agenesis of the corpus callosum, and are impaired in several behavioral assays (19, 24) .
This potential problem may be addressed through breeding programs to place targeted mutations on different inbred backgrounds,
and by the generation of ES cell lines derived from other inbred strains. It has been recommended that the C57BL/6 and DBA strains
be used as standards, due to the extensive body of data relating to the behavioral characterization of these strains (19) .

In addition to the above strain considerations, the standard application of gene targeting technology has several inherent limitations.
The null mutations engineered into knockout mice are typically constitutive, i.e., they are present throughout embryonic and
postnatal development. Therefore, the potential for developmental perturbations is a major caveat to the interpretation of mutant
phenotypes in adult animals. It may be difficult to determine whether a mutant phenotype reflects a normal adult role for the
targeted gene or an indirect effect of the mutation attributable to perturbed development. Such an effect may lead to an
overestimation of the functional significance of the gene product in the adult animal. Conversely, if significant compensation for the
loss of a gene product occurs during development, then the severity of the mutant phenotype may underestimate the functional
significance of the gene product. The nature of such compensatory mechanisms and the extent to which they exist may be difficult
to determine. The above considerations also pertain to the analysis of transgenic mice carrying constitutive mutations.

Another limitation of the standard gene targeting technology is that the mutations are ubiquitous, present in all of the cells of the
animal. Thus, if a neural gene of interest is also expressed in peripheral tissues, then the absence of the gene product peripherally
could lead to a lethal or altered phenotype, independent of its neural role. Moreover, for genes that are widely expressed in the CNS,
it may also be difficult to anatomically localize the brain region(s) that underlie the mutant phenotypes. New techniques to
overcome these problems by achieving region-specificity and inducibility of targeted mutations are under development, and are
described in the next section.

PROCEDURES FOR ENGINEERING CONDITIONAL MUTATIONS

Part of "19 - Gene Targeting and Transgenic Technologies "

New technologies are under development for circumventing the limitations of standard gene targeting approaches by creating
mutations that may be induced in adult animals and/or restricted to particular brain regions. Although these strategies are not yet
in widespread use, it is likely that rapid advances in this area will lead to an exponential increase in the generation of such
“conditional mutations” over the next several years.

Cell Type–Specific Mutation Strategies

When a null mutation of a gene results in a mutant phenotype, limitations in the interpretation of that phenotype can arise because
the gene is inactivated in every cell in which it was expressed in the mouse in the wild-type (WT) state. Therefore, the observed
abnormal phenotype may arise from the absence of the functioning gene product in a peripheral organ system, the peripheral
nervous system, or the CNS—i.e., in any of those regions in which the gene is normally expressed. It is possible that the absence of a
gene product in the periphery may lead to embryonic lethality, precluding use of the mutant for the studies of neural function. For
genes that are widely expressed within the CNS, it may be difficult to identify neural circuits through which mutations produce
behavioral perturbations. The ability to inactivate genes in restricted subpopulations of the cells that normally express them will be
a valuable asset in studies to uncover the neural mechanisms underlying neural phenotypes.

Recent efforts have focused on a mutational strategy developed to exert spatial control over the pattern of expression of genetic
changes introduced into mice. This approach utilizes somatic cell recombination rather than germ cell (or embryonic stem cell)
recombination to inactivate a gene in restricted populations of cells or tissues. In this approach, a tissue-specific promoter is used to
direct expression of one
 P.247

of the site-specific recombinases (25) to limit gene inactivation to only those cells expressing the recombinase. The two
recombinase systems that have been utilized for genetic manipulation in mice have been the Flp-frt system from yeast (26), and the
Cre-lox system from bacteriophage P1 (27, 28 and 29), with the large majority of reports using this technique utilizing the Cre-lox
system.
Cre (cyclization of recombination) recombinase is a 38-kd protein from bacteriophage P1, which recognizes and catalyzes reciprocal
DNA recombination between two loxP (locus of crossing over of P1) sites. The loxP site is the 34–base pair (bp) recognition sequence
for Cre composed of a palindromic 13-bp sequence separated by a unique 8-bp core sequence (Fig. 19.4A) . A gene or gene segment
with flanking loxP (“floxed”) sites will be excised by homologous recombination in the presence of Cre, leaving a single loxP site
marking the point of excision and re-ligation of upstream and downstream DNA (Fig. 19.4B) . This approach, then, involves
generating two independent lines of mice—a line bearing loxP sites, and a transgenic line in which Cre expression is driven by a
tissue-specific promoter. Animals with a gene or gene region of interest flanked by loxP sites (floxed) are generated by gene
targeting. Because the loxP sites are relatively small and placed in intronic regions, they do not typically interfere with normal gene
transcription. Of course, WT patterns and levels of expression need to be documented in these floxed mouse lines, because
inadvertent placement of lox sites into promoter elements or RNA splice sites could disrupt gene expression. The Cre mice are most
commonly generated by creating a transgenic line of mice in which Cre expression is driven by a tissue-specific promoter. As
discussed above in the section on transgenic mice, variability in transgenic expression patterns requires several lines of Cre mice
that need to be generated and assayed for patterns of Cre expression. An alternative strategy is to use gene targeting procedures to
place Cre under the control of an endogenous promoter (30) . The advantage of this approach is that Cre expression should closely
approximate the WT expression pattern of the gene it is replacing because the original gene's promoter remains in its endogenous
location. A potential disadvantage is that Cre may disrupt expression of the gene into which it has integrated. Once a line exhibiting
the desired pattern of Cre expression is identified, it is crossed with an appropriate floxed line to commence a breeding strategy
resulting in the generation of animals with a restricted pattern of gene inactivation (Fig. 19.4C) .

FIGURE 19.4. Strategy for cell-type–selective mutations using Cre-

mediated recombination. A: The loxP DNA sequence indicating the core
region and the inverted repeats that constitute Cre binding sites. B: In
the presence of Cre, a gene flanked by tandomly oriented loxP sites
(floxed gene) will be excised by homologous recombination. The
recombination occurs in the core region of the loxP site, leaving a single
recombinant loxP site in the genome after Cre excision. C: Use of the
Cre-lox system to generate cell-type–specific gene inactivation in mice.
A “floxed mouse” is generated by gene targeting to introduce loxP sites
flanking a gene of interest. The wild-type expression pattern of this gene
is shown (black) in the coronal section beneath the floxed mouse. In this
example, there is expression of the gene in the cortex (ctx), striatum
(str), and hypothalamus (hypothal). This floxed mouse can be crossed to
a transgenic mouse expressing Cre in a distribution dictated by the
promoter used in the transgene construct. In this example, Cre
expression is shown (gray) and is limited to the striatum. A breeding
program is pursued to produce animals in which expression of the floxed
gene is normal except in the striatum, where the expression of Cre
results in the excision of the floxed gene.

Several lines of Cre mice have been reported in which expression is restricted to subpopulations of cells within the CNS (31, 32, 33,
34 and 35) . The first example of this approach was the inactivation of the glutamate receptor subunit NMDAR1 in CA1 pyramidal
neurons of the hippocampus, with expression in other brain areas mostly intact (31) . NMDAR1 is the predominant N-methyl-D-
aspartate (NMDA) receptor subunit and is widely expressed in most CNS neurons. It had been previously demonstrated that
widespread gene inactivation in NMDAR1 null mutants resulted in perinatal lethality (36, 37) . When the mutation was restricted to
hippocampal CA1 neurons, animals were viable and exhibited impaired spatial learning and impaired plasticity at CA1 synapses (31) .
Thus, spatial restriction of neural mutations can be used to uncover particular brain regions or cell type through which gene
inactivation alters behavior.
The utility of this approach for producing cell-type–specific inactivation of genes is enhanced by the fact that the components of the
system produced in one laboratory can be “mixed and matched” with components from another laboratory. That is, Cre lines
generated for use with a particular floxed gene may also be used with other floxed genes when a similar pattern of gene inactivation
is desired. Collaborative efforts to generate databases of Cre and floxed lines will speed and simplify the production of animals with
restricted patterns of gene inactivation.

Inducible Mutation Strategies

As described above, the absence of a gene product throughout development complicates the interpretation of mutant phenotypes.
Efforts are currently under way to overcome this limitation through the use of gene expression systems that may be induced in the
adult animal. Strategies are under exploration for achieving this goal using a variety of compounds, such as tetracycline, steroid
receptor antagonists, and ecdysone to induce gene expression. Although these approaches have yet to be optimized for general use,
this development is likely to be close at hand. The tetracycline system has been the most utilized and best developed approach to
inducible gene regulation.
Since the introduction of the Tet system by the Bujard laboratory in 1992, many laboratories have validated the utility of this
approach to inducible gene regulation, and many refinements/improvements in the system have been introduced (38) . This system
is based on the regulatory elements of a tetracycline resistance operon of Escherichia coli, in which the transcription of tetracycline
resistance genes is negatively regulated by the tetracycline repressor (tetR) (Fig. 19.5) . When tetracycline is present, tetR binds
the tetracycline and loses its capacity to bind to the operator sequences (tetO) located within the promoter of the tetracycline
resistance operon, and transcription is permitted. By creating a fusion protein composed of the tetR and a potent transcriptional
activator, VP16, a tetracycline-dependent transactivating factor (tTA) was produced that retained the DNA binding and activation
specificity of the tetR. The desired regulatable gene of interest is placed under tetO plus a minimal promoter (Pmin), that contains
the basic promoter elements required for transcription in all cell types. Activation of this system requires the binding of the tTA to
the tetO operator sequence (39) . The presence of tetracycline,
P.248
 P.249

or other suitable ligand such as doxycycline, prevents tTA from binding to tetO and activating transcription of the gene of interest.
This is referred to as the tet-off system—that is, when tetracycline is present, transcription is off. A tet-on system has also been
developed, in which tetracycline induces transcription of the gene of interest. It utilizes a reverse tetracycline transcriptional
activator (rtTA), designed so that it would bind to tetO and activate transcription only in the presence of tetracycline-related
compounds (38) . Doxycycline is most frequently used because it is a potent regulator in both the tet-off and tet-on systems (38),
and can be easily supplied to mice through their water or food supply (40, 41) .

FIGURE 19.5. Tetracycline-regulated expression systems. A: Tet-off system. The tetracycline-controlled transactivator, tTA, is
a fusion protein consisting of the tetracycline repressor (tetR) domain and a transcriptional activation domain (VP16). tTA
homodimerizes, and in the absence of tetracycline (or the tetracycline analogue doxycycline) activates transcription of the
gene of interest that has been placed downstream from tetO and a minimal promoter. Binding of doxycycline (Dox) to the tTA
dimer prevents the binding of tTA to tetO, and transcription of the gene is prevented. Therefore, the tTA system has been
called the Tet-off system, because in the presence of doxycycline, transcription is prevented. B: Tet-on system. In the Tet-on
system, the tetracycline-controlled activator has been mutated to reverse the action of doxycycline on the transactivator. By
contrast with tTA, doxycycline binding to rtTA enables the complex to bind to tetO and activate gene transcription. In the
absence of doxycycline, rtTA is unable to bind to tetO and cannot activate transcription. Therefore, the rtTA system is also
called the Tet-on system, because doxycycline activates transcription of the regulated gene.

The tet-off and tet-on systems are binary systems—i.e., they require two genetic elements to be introduced into mice. First, a
tissue-specific promoter can be used to express tTA or rtTA in a region or cell-type specific manner; then the gene of interest is
inserted behind tetO and a minimal promoter. This can be achieved by creating two separate transgenic lines of mice and then
cross-breeding to produce bigenic lines. In these lines, expression of the gene of interest may be induced by doxycycline (tet-on) or
by the discontinuation of doxycycline treatment (tet-off). For example, the tet-off system has been used to investigate the effects
of the transcription factor ΔFosB on psychostimulant responses. A line of mice was generated in which expression of a ΔFosB
transgene was suppressed by continuous doxycycline treatment throughout development. Discontinuation of treatment in adult
animals led to overexpression of the transgene in the nucleus accumbens and to augmentation of the rewarding and locomotor
stimulant properties of cocaine (42) . The utility of the tet-on system has also been demonstrated. For example, a line of mice was
developed to examine the role of the Ca2+-activated protein phosphatase calcineurin in synaptic plasticity. Treatment of these
animals with doxycycline induced calcineurin overexpression in restricted forebrain regions, associated with deficits of neuronal
plasticity and spatial learning (41) .

Rather than generating regulatable gain of function mutants with the Tet system, regulatable loss of function mutants can also be
generated by combining the Tet system with the Cre-lox system (43, 44) . In this arrangement, a cell-type–specific promoter drives
rtTA expression and Cre is linked to tetO and a minimal promoter. In the presence of doxycycline, Cre is expressed in the cell type
specified by the promoter used to drive rtTA expression, and somatic
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cell recombination excises floxed DNA fragments in those cells—achieving an inducible cell-type–specific knockout. This inducible
knockout approach may be utilized to circumvent concerns discussed above in the interpretation of gene knockout phenotypes.
In these inducible knockout mice it must be remembered that although the excision of the floxed gene can be induced relatively
quickly, the appearance of any phenotype resulting from the absence of the gene product will occur gradually, depending on the
degradation rate of the relevant mRNA and the half-life of the protein. Another important limitation of strategies utilizing the Tet
system relates to the inherent “leakiness” of the tetO operator; i.e., low levels of unwanted gene expression may occur during
periods in which gene expression is expected to be turned off. This may be problematic when the inducible transgene is toxic or has
significant effects even when expressed at very low levels. Recent findings with tetracycline controlled transcriptional silencers
indicate that it may be possible to modify the tet system to substantially reduce unwanted gene expression (45) . In addition, work
has begun on alternative inducible gene expression systems with low levels of basal expression. One such system utilizes the insect
hormone ecdysone as an induction signal.

SUMMARY
Part of "19 - Gene Targeting and Transgenic Technologies "
The development of transgenic and gene targeting technologies is significantly enhancing understanding of cellular and molecular
functions of genes and their contributions to neural processes relevant to clinical disorders. In some instances, novel roles for
receptor subtypes have been revealed by the observation of unexpected phenotypic abnormalities in mutant animals. Mutant strains
are also providing models for studying the pathophysiology and treatment of particular neuropsychiatric diseases. In addition, some
mutant mouse models are useful for investigating the mechanisms of action of psychoactive drugs. Although mutant mouse models
represent powerful tools in neuropsychopharmacology, they do not replace older methods of investigation. These models may be
best used as components of integrated multidisciplinary research efforts spanning multiple levels of analysis.
This chapter briefly surveyed the most common strategies used for manipulating the mouse genome, and cited the advantages and
limitations of each approach. Progress in the development of conditional mutagenesis strategies will address many of the current
limitations, and facilitate uncovering the neural mechanisms through which mutations alter neural systems to impact behavior.
Although this field is still in its infancy, an exponential increase in the application of molecular genetic technologies is anticipated
to contribute substantially to our understanding of brain function in health and disease.

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20
Gene Delivery into the Brain Using Viral Vectors

Rachael L. Neve

William A. Carlezon Jr.

Rachel L. Neve and William A. Carlezon, Jr.: Department of Psychiatry, Harvard Medical School and McLean Hospital, Belmont,
Massachusetts 02478.

The delivery of recombinant genes into the brain is becoming an increasingly important strategy for answering questions about the
molecular mechanisms of brain function. Answers to these questions may be applied to many of the disorders that affect the brain.
For example, an understanding of the mechanisms by which repeated exposure to drugs of abuse increases their stimulant and
rewarding properties in animal models will almost certainly lead to new ways of treating addiction in humans. If we are able to
decipher the molecular events underlying long-term changes in neurotransmitter release, we will find new approaches to diseases
such as the epilepsies, in which neurotransmitter release is altered. Knowledge of the molecular means by which neurotransmitters
shape neuronal development and plasticity, or how trophic factors regulate neuronal health, will lead to insights into how defects in
these pathways cause specific psychiatric and neurodegenerative diseases.

Unfortunately, the brain does not yield easily to genetic intervention. The terminally differentiated state of most neurons in the
brain precludes the use of vectors, such as conventional retroviruses, that are dependent on cell replication for stable maintenance
in the cell. In addition, the molecular mechanisms of specific brain disorders may be restricted to subsets of neurons at specific
times during development and maturity. Therefore, strategies for manipulating gene expression in the brain must utilize vectors
that persist stably in postmitotic cells and that can be targeted both spatially and temporally in the nervous system. A number of
such gene delivery systems have been developed over the last decade. Rather than giving a superficial overview of the field, this
chapter highlights the use of herpes simplex virus (HSV) as a vector for gene transfer into neurons, with emphasis not only on its
potential but also on its flaws; compares and contrasts HSV-mediated genetic intervention in neurons with that mediated by other
viral vectors; and gives detailed examples of the practical uses of this technology by describing the use of HSV vectors to study the
molecular basis for drug addiction.

 HERPES SIMPLEX VIRUS: THE PROTOTYPIC VECTOR

 COMPARISON OF HSV-1 WITH ALTERNATIVE VECTORS FOR GENE TRANSFER INTO NEURONS
 GENE DELIVERY INTO THE BRAIN AS A TOOL FOR NEUROPSYCHIATRIC RESEARCH: DRUG ADDICTION
 GENE DELIVERY INTO THE BRAIN AS A MEANS FOR GENE THERAPY
 ACKNOWLEDGMENTS

HERPES SIMPLEX VIRUS: THE PROTOTYPIC VECTOR

Part of "20 - Gene Delivery into the Brain Using Viral Vectors "

HSV possesses multiple features that make it an ideal vector for delivery of genes into the nervous system. In particular, it accepts
large molecules of exogenous DNA; it infects nondividing cells from a wide range of hosts with high efficiency; it enables strong
expression of foreign genes; it is episomal, and thereby does not cause integration effects; its infection of postmitotic cells is
persistent; and HSV-1 particles can be concentrated to relatively high titers. Because of these characteristics of HSV-1, and because
it is neurotropic, it is currently one of the best viral vectors available for functional analysis of genes in the nervous system.

Amplicon vs. Genomic HSV-1 Vectors

There are two types of replication-deficient HSV vectors: those in which the foreign DNA of interest is cloned into the viral genome
itself (genomic vectors), and those that are composed of a plasmid carrying minimal HSV sequences that allow it to be packaged into
virus particles with the aid of a helper virus (amplicon vectors). A number of genes within the wild-type HSV genome are dispensable
for its growth in cells in vitro. This knowledge was used to create “crippled” recombinant HSV-1 viruses that could be used as
vectors for gene transfer into cells (1) . This type of genetically engineered genomic vector has been used by a number of
investigators and is described in detail by Fink et al. (2) .

The idea of the amplicon vector originated with the discovery of defective HSV-1 particles (3, 4) that appeared in and interfered
with HSV-1 stocks that were passaged at high multiplicities of infection (MOIs). Examination of the
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genomes of these defective HSV-1 particles revealed that they carried only a minimal subset of DNA sequences from the wild type
genome (3, 4 and 5) . These sequences included an origin of DNA replication and a cleavage/packaging site (the “a” site). It was
found that incorporation of these two sequences into a plasmid (the “amplicon”) gave the plasmid the ability to be replicated and
packaged into virus particles when it was introduced into a cell that was superinfected with wild-type virus (which supplied HSV
replication and virion assembly functions in trans). The plasmid sequences that were packaged into virus particles consisted
primarily of 150-kilobase (kb) concatamers of the original plasmid (3, 4, 5 and 6) .

The chief advantage of this amplicon type of vector, which is now packaged with replication-defective helper viruses, is that cloning
manipulations are relatively easy due to the small size (5 to 10 kb) of the plasmid. One disadvantage is that production of amplicon
vectors requires a co-propagated HSV helper virus, resulting in viral stocks that are a mixture of helper and amplicon viruses. In the
past, cytotoxic effects of these stocks limited the amount of vector that could be used to infect cells. Even though the replication-
incompetent helper viruses could not cause lytic infections in normal cells, cytopathic effects resulted both from proteins present in
the HSV-1 particles and from expression of HSV-1 immediate-early (IE) genes (7) . Occasionally, wild-type HSV-1 revertants appeared
during the amplicon packaging process, exacerbating the cytotoxicity of the virus preparations (7, 8) . However, recent
improvements in the amplicon packaging procedure, which are discussed in the following section, have largely overcome these
problems. The development of a helper virus–free packaging system for the HSV vector (9) has virtually eliminated any lingering
cytotoxicity in the preparations, although the helper-free system yields relatively low titers of virus and still needs improvement in
that area.

Present-Day Amplicon Vectors: Advantages and Disadvantages

Genomic and plasmid defective HSV-1 vectors have been used to manipulate neuronal physiology both in vitro and in vivo. These
studies have been promising, but they have also revealed limitations of the current HSV vector systems. Because HSV is too fragile to
purify on cesium chloride gradients, it could not be concentrated in the same way that encapsulated viruses such as adenovirus were
concentrated. Moreover, as noted above, nonspecific cytopathic effects of the defective vectors restricted the number of viral
particles that could be used to infect neurons. Finally, lack of persistence of high expression levels from the viral recombinants has
hampered long-term in vivo studies and has limited the usefulness of the vectors for both experimentation and gene therapy.

Recent improvements in the amplicon packaging procedure (Table 20.1) have corrected some of the limitations listed above. The
most widely used second-generation helper virus was HSV-1 tsK, with a temperature-sensitive single-base mutation in the ICP4 (IE3)
gene. Because revertants of this mutant arose at a finite frequency during the packaging procedure, lytic virus was present in some
preparations (7, 8) . The frequency of revertants was decreased with the development of an efficient packaging system using a
deletion mutant of IE3 (8) as helper virus. However, occasional lytic virus particles continued to appear, albeit at a greatly reduced
frequency, presumably as a result of recombination between the helper virus and the sequences flanking both sides of the IE3-
containing fragment present in the permissive host.

A key breakthrough was made by Lim et al. (10) when they compared three replication-defective HSV-1 mutants [KOS strain 5dl1.2,
deleted in the ICP27 (IE2) gene; and strain 17 D30EBA and KOS strain dl120, both deleted in the ICP4 (IE3) gene] for their usefulness
as helper virus for packaging an amplicon vector. Historically, IE3 mutants have been preferred because they express fewer HSV-1
genes under nonpermissive conditions than do ICP27 mutants. However, Lim et al. found that use of the IE2 mutant 5dl1.2 yielded
higher vector titers than did use of the IE3 mutants, with no increase in cytotoxicity. In addition, wild-type lytic virus was virtually
absent in stocks made using 5dl1.2 in conjunction with the permissive host 2-2 cells, likely due to the fact that 5dl1.2 is a more
complete deletion than D30EBA, sharing little sequence with the IE2-containing fragment present in the permissive host.

To achieve a favorable ratio of recombinant vector to helper virus, the stocks derived from transfection of the packaging cells
followed by superinfection with helper virus are passaged three times on the permissive host. The recombinant vector is packaged
as long concatamers, which contain multiple origins of replication, conferring a selective replicative advantage on the vector-
containing virus relative
 P.255

to the helper virus. Therefore, the efficiency of the initial transfection of vector DNA into the packaging line is critical to the
success of the packaging. Lim et al. (10) showed that the transfection of the vector DNA at the start of the packaging procedure was
significantly more efficient using Lipofectamine (Life Technologies) than using calcium phosphate, and thereby achieved a favorable
ratio (≥1) of vector to helper. Since then, we have achieved vector/helper ratios greater than 100.

For a viral vector to have utility for gene therapy, cytopathic effects of the virus must not outweigh the beneficial effects of the
transgene. Unfortunately, in the past, investigators have had difficulty generating nontoxic HSV-based replication-defective vectors.
They often were toxic to neurons in vitro (7) . Significant necrosis, often accompanied by inflammation and gliosis, was identified at
injection sites with some genetically engineered HSV-1 vectors used for in vivo studies (see, e.g., ref. 11) . However, the
achievement of a more favorable ratio of vector to helper, and the virtual elimination of wild-type virus in the vector preparations
(10) has greatly reduced the cytotoxicity of present-day defective HSV-1 amplicon vectors (see, e.g., refs. 12, 13 and 14) . An
additional improvement to the packaging procedure, the banding of the virus on a sucrose step gradient, followed by a high-speed
centrifugation to pellet the virus, has reduced further the cytotoxicity of the virus preparations. It simultaneously removes toxic
factors present in the crude cell lysates, and enables concentration of the vector to titers exceeding 108/mL.

A troubling problem that has not yet been resolved for any viral vector used in the brain, except perhaps for the lentiviral vector
(see below), is that of persistence of expression. Numerous investigators have had the experiences reported by During et al. (15) and
Lim et al. (10), in which an initial peak in expression of an HSV transgene in vivo or in vitro, respectively, has been followed by loss
of the bulk of the expression by 1 to 2 weeks postinfection. Interestingly, superinfection with helper virus 5dl1.2 1 week
postinfection rescued expression of a transgene expressed under the control of the IE 4/5 promoter (10) . Apparently,
transactivating factors provided by the helper virus reactivated transcription of the transgene.

Two recent developments suggest that the problem of persistence of expression is not insoluble. Use of a 9-kb fragment of the
tyrosine hydroxylase promoter to drive reporter gene expression in an HSV-1 amplicon vector resulted in prolonged gene expression
in vivo (16), suggesting that neuronal, unlike viral, promoters in HSV-1 vectors have the potential to produce stable gene expression.
Additionally, the development of hybrid amplicons that incorporate elements that allow autonomous replication of the episome (17)
or that incorporate adeno-associated virus (AAV) elements for genomic integration of the amplicon (18, 19 and 20) have resulted in
vectors that support long-term gene expression both in vitro and in vivo.

An improvement in gene transfer methods in general has been the incorporation of the gene for the green fluorescent protein (GFP)
into many vectors. Coexpression of GFP allows the investigator to detect cells infected by the vector with a fluorescence microscope,
whether they are fixed or alive. There are at least three ways to “mark” vector-infected cells with GFP (Fig. 20.1) . In the example
shown, the objective is to coexpress GFP with the AMPA receptor GluR1. They can be expressed on a single transcript (HSV-IRES/GFP)
by putting an internal ribosome entry site (IRES) between the two genes, which are then transcribed from the same promoter. The
IRES enables independent translation of the two coding sequences even though they are present on the same messenger RNA (mRNA).
In theory, the two coding sequences should be expressed at similar levels, but in practice the translation of one of the two coding
sequences on the mRNA often occurs at the expense of the other. Alternatively, they can be expressed from two independent
transcriptional units, in a bicistronic vector. The addition of an extra transcriptional cassette to the vector makes it larger and more
unwieldy to use; and the level of transcription from one promoter is independent of the level of transcription from the other, so
that the transgenes may be expressed at very different levels. Third, the GFP can be fused to the transgene product, so that they
are expressed
 P.256
as a single protein. This is a trickier construction, since the two coding sequences must be placed in frame with each other. However, the added benefit of being able to track the subcellular
location of the transgene product makes this the option of choice in many instances.
FIGURE 20.1. Different vector constructions for coexpressing two genes from a viral vector. Glutamate
receptor GluR1 and green fluorescent protein (GFP) are used as examples.

COMPARISON OF HSV-1 WITH ALTERNATIVE VECTORS FOR GENE

TRANSFER INTO NEURONS
Part of "20 - Gene Delivery into the Brain Using Viral Vectors "

Despite the problems that remain with the HSV-1 vector, it is a gene delivery system that has come of age. In addition, numerous alternative and increasingly user-friendly means of gene transfer into the
brain are now available (Table 20.2) . Adenovirus vectors, like HSV-1 vectors, infect postmitotic cells and can enter a broad range of mammalian cell types. They have the additional advantages that they
can be concentrated to very high titers (≥1010/mL). However, the use of adenovirus vectors continues to be restricted by the robust host immune response that they elicit (21, 22) .

TABLE 20.1. CHRONOLOGY OF IMPROVEMENTS IN AMPLICON VECTOR PACKAGING SYSTEM

TABLE 20.2. COMPARISON OF VIRUSES USED TO MANIPULATE GENE EXPRESSION IN THE BRAIN

At present, HSV amplicon vectors can accommodate larger pieces of foreign DNA, on the order of 15 kb, than can the adenovirus vectors, which can only contain a maximum of 6 to 8 kb (although
the new “gutless” adenovirus vectors can take up to 37 kb of foreign DNA). Foreign genes are cloned into easy-to-manipulate amplicon vectors that can be packaged directly into viral particles as
head-to-tail repeats in the presence of the helper virus, with no intermediate recombination step required. This enables rapid construction of a large number of recombinant vectors simultaneously,
and is particularly useful for those who are doing mutation analysis, and who wish to work with multiple genes. Such ease of cloning is not possible with the genomic HSV and adenovirus vectors.

Direct in vivo transfer of genes into the brain has been achieved using not only herpes virus and adenovirus vectors, but also adeno-associated virus vectors (see refs. 23, 24 for review) and
lentivirus vectors (see ref. 25 for review). In contrast to other viral vectors, adeno-associated virus vectors do not cause an immune response or toxicity. Interestingly, the ability of adeno-
associated virus vectors to transduce and express transgenes is not equivalent in all regions of the brain (26, 27) . Only in some regions will neurons bind and internalize the virus, with resultant
long-term expression. Stability of expression of lentivirus vectors in the brain is their greatest advantage. Long-term expression of β-galactosidase and GFP was observed in rat neurons for at least 9
months following intracerebral injection of the vectors, with no sign of tissue pathology or immune response (28, 29) . Progress has been made in achieving biosafety with these vectors, by
eliminating viral sequences nonessential for transduction.

GENE DELIVERY INTO THE BRAIN AS A TOOL FOR NEUROPSYCHIATRIC

RESEARCH: DRUG ADDICTION
Part of "20 - Gene Delivery into the Brain Using Viral Vectors "

Overview
The bulk of the published work on gene transfer technologies has focused on their use for gene therapy (see ref. 30 for an excellent review). To date, the delivery of recombinant genes into the
brain as a strategy for answering questions about the molecular mechanisms of brain function has utilized primarily HSV vectors. One of the most successful uses of this strategy has been in the field
of addiction research. Exposure to drugs of abuse causes many changes in gene expression within the brain. A major challenge in addiction research is to determine which of these changes have a
direct influence on behavior. Viral vectors offer the ability to study individual changes in gene expression in
 P.257

discrete brain regions. In the case of addiction, it is thus possible to mimic certain aspects of the drug-exposed state without ever
administering the drugs themselves. The ultimate goal of such studies is to understand the “biobehavioral” mechanisms of addiction,
that is, to establish direct, causal relationships between drug-induced changes in biology and drug-induced changes in behavior.
Examples of behavioral changes in addiction that may result from drug-induced alterations in biology (gene expression) are
compulsive drug use (drug-taking) and craving (drug-seeking).

Biobehavioral Studies of Addiction

Addiction Circuitry
Much research on the neuronal circuitry involved in drug addiction has focused on the mesolimbic dopamine (DA) system. The
dopaminergic projection from the ventral tegmental area (VTA) of the midbrain to the nucleus accumbens (NAc) of the forebrain has
been implicated in the habit-forming (rewarding) effects of many types of abused drugs, including stimulants (cocaine,
amphetamine) and opiates (heroin, morphine) (31, 32) . However, the neural events that mediate the acute rewarding effects of
abused drugs are not understood, nor are the neuroadaptations that presumably underlie the transition from occasional drug use to
compulsive drug use. In rats, repeated exposure to drugs of abuse appears to cause increases in sensitivity (“sensitization”) to the
rewarding effects of drugs (33, 34 and 35), a phenomenon that may contribute to the addiction process (36) . This altered
sensitivity is presumably a consequence of altered gene expression, and the VTA-NAc circuitry is a logical starting point for
biobehavioral studies. Because several robust and reliable drug-induced neuroadaptations have been discovered within this circuitry
(37), it has been the focus of gene transfer studies in which the behavioral significance of altered gene expression has been assessed.
To date, the biobehavioral significance of three specific, drug-induced changes in gene expression have been studied using viral-
mediated gene transfer—the ability of drugs (cocaine, morphine) (a) to increase expression of the AMPA (glutamate) receptor
subunit GluR1 in VTA, (b) to alter expression of GluRs in the NAc, and (c) to increase the activity of the transcription factor CREB
(cAMP response element binding protein) in the NAc.

GluRs in the VTA

AMPA receptors are made up of various combinations of the subunits GluR1, GluR2, GluR3, and GluR4 (collectively called GluRs) (38,
39) . Repeated intermittent exposure to morphine selectively elevates expression of GluR1 in the VTA (40) . Relative levels of GluR1
and GluR2 expression in the dopamine neuron-rich VTA are important because the subunit composition of AMPA receptors controls
their function. High expression of GluR1 favors the formation of Ca2+-permeable (GluR1-homomeric) AMPA receptors (13, 38, 39),
which presumably increases sensitivity to the excitatory (depolarizing) effects of glutamate (12) . Conversely, high expression of
GluR2 favors the formation of Ca2+-impermeable (heteromeric) AMPA receptors, since this subunit contains a motif that blocks Ca2+
conductance (38) . Because repeated drug exposure is known to selectively increase the electrophysiologic responsiveness of VTA
dopamine neurons to AMPA receptor agonists (41), it is possible that drug-induced alterations in GluR1 expression in this region
contribute to drug-induced behavioral changes, such as sensitization. However, this question is difficult to address using traditional
methods. For example, AMPA agonists and antagonists cannot be used to study the relationship between GluR1 expression and
sensitized drug responses because they affect AMPA receptor function generally and thus do not mimic morphine's selective effects.
This biochemistry-behavior relationship could be studied by performing intra-VTA microinjections of HSV-GluR1, which caused ˜75%
increases in GluR1 expression within this region (13) .

It was first necessary to determine if viral-mediated elevations in GluR1 expression within the VTA would increase sensitivity to the
locomotor-stimulating effects of morphine, a hallmark of behavioral sensitization (36, 42) . Low doses of morphine induced
significantly more activity in rats with viral-mediated elevations in GluR1 expression within the VTA. Furthermore, increased activity
in rats given HSV-GluR1 was seen only in response to morphine, and was not evident after saline. The transient nature of the HSV-
mediated elevations in transgene expression allowed the behavioral adaptations to be correlated with the time course of GluR1
expression: when morphine was given only on days 7 and 8 after microinjection—when elevations in GluR1 expression had
dissipated—the rats that were given HSV-GluR1 were no longer more sensitive to the stimulant effects of morphine. When some rats
given HSV-GluR1 were tested with morphine on days 3 to 4 and on days 7 to 8, significant increases in sensitivity to the locomotor-
stimulating effects of the persisted in rats given HSV-GluR1, despite the fact that GluR1 labeling in the VTA had dissipated. Thus
when morphine is given while GluR1 expression in the VTA is elevated, increased sensitivity to morphine outlasts viral-induced
increases in GluR1 expression. These data suggest that altered expression of GluR1 in the VTA underlies, at least in part, the
development and expression of sensitized behavioral responses to morphine.

The effect of elevated GluR1 expression in the VTA on the rewarding effects of morphine was examined using place conditioning, a
classic conditioning procedure in which rats learn to associate the rewarding effects of a drug with a distinctive environment. Rats
given HSV-GluR1 into the rostral (anterior) portion VTA spent more time in morphine-associated environments than did control rats,
indicating
 P.258

an increase in morphine reward (13, 43) . Since prior treatment with morphine intensifies its rewarding actions in the place-
conditioning paradigm (33), these data suggest that the behavioral consequences of morphine preexposure are mimicked by HSV-
mediated expression of GluR1 in the VTA.

Together, these data demonstrate that specific changes in motivational states can result from altered expression of a single,
localized gene product. Drug-related increases in GluR1 expression in the VTA, a region known to be involved in the induction of
sensitization (42, 44), may themselves be sufficient to explain sensitization (13, 41), or they may lead to Ca2+-dependent adaptations
(45) that also contribute to changes in drug sensitivity (Fig. 20.2) . Thus these studies have added strength to the hypothesized
association between the VTA and sensitization, and identified biobehavioral relevance for the drug-induced regulation of the GluR1
protein in the VTA.

FIGURE 20.2. Simplified schematic of putative reward circuitry; see ref. 39 for detailed discussion. Treatments that increase
excitation in the ventral tegmental area (VTA) (A) are rewarding, presumably because they promote the inhibitory actions of
dopamine (a D2-like receptors) in the nucleus accumbens (NAc) (B). Inhibition of NAc GABAergic output neurons, in turn,
decrease inhibitory influences on reward processes in other areas of brain reward circuitry (C), including the ventral pallidum
(VP) and peduncular pontine nucleus (PPN) (63). Elevations in GluR1 expression in the VTA (A) increase drug reward,
presumably because the accompanying changes in Ca2+ flux increase the excitability and/or neuronal function of VTA
dopaminergic neurons (as in ref. 49). Conversely, elevations in GluR1 in the NAc decrease drug reward (B), presumably because
the accompanying changes in Ca2+ flux increase the excitability of NAc GABAergic neurons that normally inhibit reward
processes in distal regions (C). (Based on refs. 13, 43, and 55.)

GluRs in the NAc

There are two important reasons for interest in how altered expression of GluRs in the NAc affects the actions of drugs of abuse.
First, GluR2 is a target gene of ΔFosB, a stable and long-lasting variant of the fos family of transcription factors that is regulated in
the Nac by drugs of abuse (46) . Second, GluR1 expression is elevated the NAc in cocaine-sensitized rats during long-term drug
withdrawal (47) .

Exposure to cocaine and other drugs of abuse causes the rapid and transient expression of the immediate-early gene c-fos in the
striatum (including the NAc) (48, 49) . Repeated drug exposure decreases expression of the transient forms of c-fos, but increases
expression of a more stable and long-lasting form of the transcription factor, ΔFosB (50, 51) . The accumulation and sustained
transcriptional activity of ΔFosB in the NAc could mediate long-lasting neural and behavioral adaptations that accompany repeated
drug exposure (37) . Consistent with this notion, inducible transgenic mice that express ΔFosB spontaneously (i.e., without prior
drug treatment) in the NAc during adulthood have increased sensitivity to the locomotor-stimulating and rewarding effects of
cocaine (46) . The proximal cause of the increase in drug sensitivity is presumably not increased expression of ΔFosB per se, but
rather increased expression of a target gene (or genes) whose transcription is regulated by this factor. The ΔFosB-overexpressing
mice also had large increases in GluR2 expression in the NAc, implicating this AMPA receptor subunit in the increased drug sensitivity.
To examine whether elevated GluR2 expression in the NAc was sufficient to cause increases in sensitivity to the rewarding effects of
cocaine, rats were tested in the place-conditioning paradigm after microinjections of HSV-GluR2 into this region (46) . This
treatment dramatically increased sensitivity to the rewarding effects of cocaine, mimicking the effects of increased expression of
ΔFosB. Together, these findings provide strong evidence that the increase in cocaine sensitivity seen in ΔFosB transgenic mice is
attributable, at least in part, to elevated expression of GluR2 in the NAc.

For comparison, rats were given microinjections of vectors expressing other GluRs into the NAc, and tested with cocaine in the
place-conditioning paradigm. Rats given microinjections of HSV-GluR1 into the NAc spent dramatically less time than control rats in
the cocaine-associated environments, suggesting that elevated expression of this AMPA receptor subunit in this region increases
sensitivity to the aversive effects of the drug. Additionally, some rats were tested after intra-NAc microinjections of HSV-GluR2Q,
which expresses unedited GluR2. This form of GluR2 lacks the final transcriptional edit (Q → R) that produces the motif that blocks
Ca2+ flux (38, 39) . Use of this construct showed that the ability of GluR2 to increase cocaine reward appears to be directly related to
diminished Ca2+ permeability, because overexpression of GluR2Q does not increase cocaine reward, but rather causes effects that
more closely resemble those of increased GluR1 expression (as would be expected).

There are many possible explanations for how altered Ca2+ flux in the NAc might influence drug reward, considering the role of Ca2+
in cellular functions including membrane depolarization, neurotransmitter release, signal transduction, and plasticity (52, 53) .
Certainly, cocaine-induced changes in the excitability of NAc neurons have been reported:
 P.259

repeated exposure to cocaine makes neurons in this region significantly less excitable than normal at short (3-day) withdrawal
periods (54) . Studies with ΔFosB (46) suggest that these electrophysiologic adaptations are associated with increases in the
rewarding efficacy of cocaine, because elevations in GluR2 expression (which would be expected to minimize Ca2+ flux and/or
neuronal excitability) increase cocaine reward, whereas elevations in GluR1 (which would be expected to increase Ca2+ flux and/or
neuronal excitability) decrease (or oppose) cocaine reward.

Together, these data support the working hypothesis (55) that altered Ca2+ flux and/or neuronal excitability in the NAc has important
consequences on motivated behaviors (Fig. 20.2) . Moreover, they suggest that altered GluR1 expression in this region seen during
long-term (3-week) cocaine withdrawal (47) might also be associated with important changes in the rewarding efficacy of the drug.
Regardless, the use of HSV vectors has identified biobehavioral relevance for the drug-induced regulation of GluRs in the Nac.

CREB in the NAc

Chronic cocaine exposure increases 3′,5′-cyclic adenosine monophosphate (cAMP) formation and protein kinase A (PKA) activity in the
NAc (37) . Direct stimulation of PKA in the NAc counteracts the rewarding properties of cocaine (56), suggesting that drug-induced
up-regulation of the cAMP system is a neural mechanism of drug tolerance. Increased PKA activity leads to increased CREB
phosphorylation, which activates CREB-mediated gene transcription and could be an important step in producing long-lasting
neuroadaptations. To determine the functional role of CREB and its transcriptional consequences in the NAc, its expression in this
region was increased directly by microinjecting HSV-CREB (57) . In other rats, a dominant negative mutant CREB (mCREB) was
overexpressed, which is transcriptionally inactive and competes with endogenous CREB for cAMP response element binding sites
(CREs) (58) .

The effects of elevated CREB expression in the NAc on cocaine reward was studied with place conditioning. Although the effects of a
low (threshold) dose of cocaine were not altered by control treatments, this dose established dramatic conditioned place
preferences in rats given bilateral microinjections of HSV-mCREB (which acts as a CREB antagonist) into this region. This rewarding
effect was “inversed” to aversion in rats with elevated expression of CREB in the NAc; rats given HSV-CREB avoided drug-associated
environments, suggesting that this dose of cocaine was made aversive by gene transfer. When cocaine was administered a week
(rather than 3 days) after microinjections of the HSV vectors into the NAc, cocaine was devoid of rewarding or aversive effects. This
finding confirms that the behavioral consequences of HSV viral vectors are transient and reversible, and have a time course of
efficacy that parallels that of transgene expression.

Dose-response analyses revealed that microinjections of HSV-mCREB and HSV-CREB in the NAc were producing, respectively,
approximately parallel leftward (more rewarding) and rightward (less rewarding) shifts in the effects of cocaine. At a high dose of
cocaine, there were no differences in the preferences for the drug-associated environment between rats given HSV-mCREB and
those given vehicle, consistent with observations that there is an upper limit to the magnitude of place preferences that can be
established (59) . Treatment with high doses of cocaine established place preferences in some rats given HSV-CREB, suggesting that
the aversive consequences of increased levels of CREB in the NAc can be counteracted by more drug.

One explanation for these findings is that elevated CREB expression in the NAc increases local dynorphin function. Dynorphin is the
endogenous ligand for κ opioid receptors (60), and κ opioid agonists have aversive actions in the nucleus accumbens shell (NASh)
(61) . To determine if dynorphin is involved in the cocaine aversion caused by HSV-CREB, brain receptors for dynorphin were blocked
with the long-lasting κ receptor antagonist norBNI. Treatment with norBNI [intracerebroventricular (ICV)] before cocaine place
conditioning blocked the aversive effects associated with cocaine in animals given HSV-CREB into the NAc, but not in rats given
microinjections of vehicle or HSV-mCREB. The fact that only the aversive properties of cocaine are altered significantly by nor-
Binaltorphimine (norBNI) suggests that microinjections of HSV-CREB into the NAc enhance the aversive aspects of cocaine via
increased stimulation of κ opioid receptors by dynorphin.

These results suggest that drug-induced increases in CREB activity (62) is a homeostatic change that opposes drug reward. Mimicking
increases in CREB activity by increasing levels with HSV-CREB or by stimulating PKA-induced phosphorylation (56) decreases the
rewarding effects of cocaine. Moreover, these data implicate κ opioid receptors in the valence (reward versus aversion) of cocaine
action, and suggest that CREB-mediated transcription in the NAc is a “drug reward rheostat” (Fig. 20.3), in part via effects on
dynorphin expression. These data also suggest a sequence of D1 receptor–mediated intracellular events, culminating with altered
gene transcription, through which exposure to cocaine influences subsequent responsiveness to the drug. Augmented release of
dynorphin could inhibit local DA release through actions at κ opioid receptors on terminals of mesolimbic DA neurons that innervate
the NAc (61) . Diminished release of dopamine in the NAc may itself be aversive, or it may unmask other actions of cocaine that are
aversive or that oppose drug reward. Regardless, these viral vector studies have identified biobehavioral relevance for alterations in
CREB function in the NAc.

FIGURE 20.3. Schematic depiction of CREB activity in the NAc functioning as a cocaine “reward rheostat.” Elevated expression
of CREB increases CREB-mediated transcription of dynorphin (a measure of CREB activity). Elevated dynorphin, in turn,
decreases cocaine reward at high doses of drug, and makes cocaine aversive at low doses of drug. Conversely, disruption of
CREB activity by overexpression of dominant-negative CREB (mCREB) decreases dynorphin transcription, which increases
cocaine reward. (Based on ref. 57.)
 P.260

Conclusions
The use of viral-mediated gene transfer in addiction research is leading to an understanding of where certain changes in gene
expression occur within the cascade of molecular events that lead to the addicted phenotype. This approach complements and
extends the predominantly pharmacologic approaches that previously have been used in addiction research, and in fact can be
thought of as “genetic pharmacology.” Understanding the molecular basis of addiction will facilitate the development of effective
pharmacologic treatments that specifically target proteins, enzymes, or transcription factors within the addiction cascade. These
therapeutics may be the prototypes for a new generation of “smart” pharmacotherapies that are designed to negate or even reverse
changes in the molecular structure of the brain that characterize specific brain disorders.

GENE DELIVERY INTO THE BRAIN AS A MEANS FOR GENE THERAPY

Part of "20 - Gene Delivery into the Brain Using Viral Vectors "

The recent rapid advancements in gene transfer technologies have raised hopes that central nervous system (CNS) gene therapy, the
introduction of genes into the brain to ameliorate neuropsychiatric diseases, is closer to reality. However, a number of major
methodologic advances must be made before it can become a reality. First, and most important, stability of transgene expression
must be achieved. Second, not only stability but also inducibility and regulatibility of transgene expression are a priority, since the
level of transgene product is often critical. Third, the transgene capacity of most vectors must be increased, so that not only the
gene(s) of interest but also appropriate regulators or inducible promoters can be delivered to the brain. Fourth, because of the
small volume of material that can be delivered stereotactically, it will be necessary to increase both the viral titers and the
transduction efficiencies for all the known vectors. Fifth, a high degree of cell specificity of gene transfer must be achieved, by the
use of targeted vectors that selectively infect particular cell types, cell-specific promoters, and routing via normal neuronal
projections in the brain. Finally, nontoxic vectors that do not induce an immune response must be developed.

The development of gene therapy for neuropsychiatric diseases suffers, in addition, from many of the same problems that drug
therapeutics research on these disease does. Neurodegenerative disorders such as Alzheimer's disease pose particular problems for
gene therapy because neurons in the CNS cannot undergo regeneration. Therefore, gene therapeutic approaches must target the
remaining brain cells, provide suitable replacements for the dying cells, or enable regeneration of CNS neurons. Another set of
hurdles arises from the complex etiology of most neuropsychiatric disease. It is not clear that a single gene product will cure any of
these diseases. In addition, the molecular mechanisms of different neuropsychiatric diseases may be restricted to subsets of neurons
at specific times during development and maturity. Consequently, as noted above, optimal strategies for gene therapy must utilize
vectors that persist stably in postmitotic cells and that can be targeted both spatially and temporally in the nervous system.
Present-day viral vectors have come of age for use in basic research, but vectors useful for gene therapy in the brain are still a work
in progress.

ACKNOWLEDGMENTS
Part of "20 - Gene Delivery into the Brain Using Viral Vectors "

We thank Dr. Frederick Boyce for helpful discussions. This work is supported by National Institutes of Health (NIH) grants AG12954
and HD34563 to R.L.N.

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21
Neuropsychopharmacology of Worms and Flies

William R. Schafer

William R. Schafer: Division of Biology, University of California–San Diego, La Jolla, California 92093-0349.

Pharmacologic agents often biochemically interact with multiple receptor or channel proteins, and induce multiple changes in
cellular physiology and signal transduction. Thus, identifying the biologically relevant targets and effectors of a given neuroactive
substance can be a challenging problem. This chapter describes how genetic analysis in simple model organisms, primarily worms or
flies, has been used to identify molecules that mediate drug responses in the nervous system.

Essentially all the studies described here rely on the same general strategy. The drug of interest is tested for its ability to affect
worm or fly behavior. Once a behavioral response is defined for wild-type animals, it is then used as a behavioral assay to identify
mutant worms/flies that exhibit abnormal drug responses and thus define genes whose products are involved in the drug's
mechanism of action. Once these genes are identified and cloned, human homologues can be identified based on sequence similarity,
and tested for involvement in human drug responses. This sort of approach has a number of potential advantages. For one,
phenotype-driven genetic screens essentially make no prior assumptions about the types of molecules involved in the process being
studied; any gene that is not essential for life and affects the behavioral response to a drug is in principle equally likely to be
identified in a mutant hunt. Thus, this approach is well suited for identifying previously unknown receptors or signal transduction
molecules that participate in drug responses. Furthermore, modern molecular genetics provides the ability to manipulate specific
gene products in an intact animal, often in a cell-type–specific manner. By making it possible to assess a particular protein function
within the context of an intact nervous system, this approach can provide a most compelling demonstration of in vivo function.

Among organisms with nervous systems, two are particularly amenable to genetic analysis: the nematode Caenorhabditis elegans,
and the fruit fly Drosophila melanogaster. These organisms share a number of advantages that make them especially well suited for
classic and molecular genetics. For example, both have short generation times (2 weeks for Drosophila, 3 days for C. elegans), can
be maintained easily and in large numbers in the laboratory, and are amenable to germline transformation. In addition, detailed
genetic maps of both organisms are available, and the genome sequences of both organisms are now virtually complete. Although
both organisms contain relatively simple nervous systems, they differ significantly in scale and level of characterization. The C.
elegans nervous system consists of exactly 302 neurons, whose precise position, cell lineage, and anatomic connectivity are known
(1, 2 and 3) . Consequently, it is possible to identify the roles of specific neurons and muscle cells in behavior using techniques such
as single-cell laser ablation, and to thereby understand in a precise manner how the action of a particular gene product in a defined
set of neurons influences the whole animal's behavior (4) . C. elegans is particularly suitable for genetic analysis of basic
intracellular processes in neurons because the worm's nervous system is nearly dispensable for growth in the laboratory. Thus, even
mutants with defects in basic neuronal functions such as neurotransmitter release are often viable and fertile (5) . The Drosophila
nervous system is somewhat more complex, and contains approximately 105 neurons. Consequently, it is somewhat less well
characterized at the cellular level than the C. elegans nervous system; however, the increased behavioral complexity afforded by
this bigger nervous system also makes it perhaps better suited for investigating more complex forms of behavior and learning (6) .

 STUDIES OF DRUG MECHANISMS IN MODEL ORGANISMS

 QUESTIONS AND FUTURE PROSPECTS

STUDIES OF DRUG MECHANISMS IN MODEL ORGANISMS

Part of "21 - Neuropsychopharmacology of Worms and Flies "

Genetic pharmacology has historically been a powerful approach for neurobiological studies in C. elegans and Drosophila. Many
studies of drug-resistant flies or worms have made use of pesticides or antihelminthic drugs that target
 P.264

the insect or nematode nervous system. For example, screens for C. elegans mutants resistant to the pesticide (and cholinesterase
inhibitor) aldicarb have been used with notable success to identify genes involved in synaptic function; molecules first studied in this
way include the vesicular acetylcholine transporter and the synaptic proteins UNC-13 and UNC-18 (7, 8) . Likewise, studies of C.
elegans mutants resistant to the anthelminthic ivermectin have provided insight into the functions of the invertebrate-specific
family of glutamate-gated chloride channels (9) . More recently, attention has turned to the possibility of using genetic
pharmacology to study the mechanisms of action for psychotropic drugs, including therapeutic agents and drugs of abuse. The
following sections describe some examples of drugs whose mechanism of action has been studied in worms and/or flies, and the
information that these studies have provided so far.

Therapeutic Agents

Lithium
Lithium salts are widely used for the treatment of bipolar affective disorder (manic-depressive illness). Lithium remains among the
most effective treatments for acute mania, and it is also an effective mood-stabilizing agent for the prevention of both manic and
depressive episodes. However, lithium has a number of side effects; for example, it is a known teratogen in vertebrate embryos, and
can mimic the action of insulin in inducing synthesis of glycogen (9a) . However, although lithium has been shown to affect a number
of molecular and cellular processes in neurons and other cells, the mechanisms through which it exerts its therapeutic effects on
mood are not well understood.

One of the most prevalent theories for lithium's mechanism of action, first proposed by Berridge et al. (10) , is the inositol depletion
hypothesis. According to this model, the critical functional consequence of lithium treatment is to reduce the intracellular
concentrations of inositol, a key component of the phosphoinositide signal transduction cycle that mediates the effects of many
neuromodulators, including serotonin (11) . Lithium ions are uncompetitive inhibitors of both inositol monophosphatase (IMPase),
the enzyme that catalyzes the conversion of inositol monophosphates (IMPs) to inositol, and inositol polyphosphatase (IPP), the
enzyme that converts inositol 1,4-bisphosphate to inositol 4-monophosphate (Fig. 21.1) . Inositol is required for the generation of
phosphatidyl 4,5-inositol bisphosphate (PIP2), whose cleavage by phospholipase C yields the calcium mobilizing agent inositol 1,4,5-
trisphosphate (IP3) and the protein kinase C activator diacylglycerol (DAG). Since both of these phosphoinositide-derived second
messengers are critical signal transduction molecules that mediate the effects of diverse neurotransmitters and neuromodulators, a
severe depletion of intracellular inositol would be expected to dramatically alter neuronal function. Thus, it has been proposed that
lithium exerts its psychoactive effects by depleting intracellular inositol pools and thereby attenuating phosphoinositide signaling in
neurons and other cells. However, experiments in rats suggest that while clinically effective concentrations of lithium are sufficient
to inhibit IMP activity in the brain, they result in only a modest decrease in inositol levels (12) . Thus, it is not clear that inositol
depletion can account for the psychotropic effects of lithium.

FIGURE 21.1. The phosphoinositide signaling pathway.

Recent genetic studies using simple eukaryotes has provided two plausible alternative hypotheses for lithium's mechanism of action.
Interestingly, many of the key genetic findings on lithium response mechanisms have come from studies of a unicellular eukaryote
that lacks a nervous system altogether, the slime mold Dictyostelium discoideum. Despite its considerable evolutionary divergence
from the metazoa, many of the signal transduction mechanisms in Dictyostelium show remarkable conservation with those in human
neurons. Dictyostelium usually exists as a free-living amoeba; however, during times of nutrient deprivation, these amoebae
aggregate into a multicellular mass, or slug, which then develops into a fruiting body consisting of differentiated stalk and spore
cells. Lithium has two effects on Dictyostelium development (13) . At high concentrations, lithium blocks the aggregation of
amoebae. In contrast, low concentrations of lithium permit aggregation, but block spore cell differentiation, causing cells that
normally would form the spore head to instead form stalk cells. This latter effect of
 P.265

lithium on spore differentiation is mimicked by a mutation in the gene gskA (14) , which encodes a homologue of the signaling
molecule glucogen synthase kinase 3 (GSK-3). GSK-3 molecules are conserved signaling molecules originally identified as negative
regulators of glycogen synthesis, and subsequently implicated in the regulation of gene expression and cell movement. Since
lithium's effects on both Dictyostelium development and glycogen synthesis were identical to those caused by inhibition of GSK-3,
Klein and Melton (15) investigated whether lithium might affect GSK-3 signaling. They subsequently demonstrated that vertebrate
GSK-3 is directly inhibited by lithium in Xenopus oocytes, and that GSK-3, but not IMPase, is responsible for the teratogenic effects
of lithium on the embryo. Thus, at least some of the side effects of lithium, such as its teratogenic and insulin-mimetic effects, are
almost certainly phosphoinositide-independent and instead mediated through the GSK-3 pathway. Because GSK-3 molecules are
abundant in the brain, it is also possible that this pathway might also mediate some of lithium's therapeutic effects on mood.

However, other studies in both Dictyostelium and Drosophila support a link between the phosphoinositide pathway and the mood-
altering effects of lithium. One such study concerned the mechanism of lithium's aggregation-inhibiting action in Dictyostelium, an
effect that is independent of the gskA gene. A number of genes required for this high-concentration response to lithium were
identified in genetic screens. One of these genes, dpoA, was shown to encode a proline oligopeptidase (PO), an enzyme involved in
the degradation of bioactive peptides (16) . Interestingly, dpoA appeared to act via the phosphoinositide signaling pathway, since
both mutations in dpoA and treatment with PO inhibitors elevated the levels of intracellular IP3, but had no effect on GSK-3 activity.
The elevation of IP3 in dpoA mutants was a consequence of increased dephosphorylation of IP5 (inositol 1, 3, 4, 5, 6-pentaphosphate),
an alternate source of IP3 utilized by both Dictyostelium and animal cells. Thus, inhibition of PO compensated for the decrease in
PIP2 levels induced by lithium by activating an alternative pathway for the production of IP3 (and by extension inositol). Interestingly,
abnormalities in PO activity have been observed in patients with both bipolar and unipolar depression (17, 18) . Thus, these results
in Dictyostelium raise the possibility that PO may be linked to depression and mania through its effect on inositol signaling, and that
lithium's efficacy in the treatment of depression may result from its ability to exert compensatory effects elsewhere in the inositol
pathway.

However, the mechanism by which lithium-induced changes in the inositol pathway affect neuronal function may not involve
inositiol depletion per se. This conclusion rests in part on a study of mutant flies defective in the enzyme IPP, a lithium-sensitive
enzyme in the inositol pathway involved in the conversion of IP3 to inositol (19) . ipp mutants were shown to be completely defective
in the IPP activity, since they were unable to degrade I(1, 4) P2, the IPP substrate. However, contrary to the prediction of the
inositol depletion model, the phosphoinositol signaling pathway (which is necessary for Drosophila phototransduction) remained fully
functional in photoreceptor neurons of the ipp mutant. Similar effects were seen when photoreceptor neurons were treated with
lithium; IPP activity was inhibited, yet the inositol-dependent phototransduction cascade was still functional. Thus, neither genetic
nor pharmacologic inhibition of IPP resulted in a depletion of inositol pools sufficient to interfere with the phosphoinositide signaling
cascade. The ability to maintain high levels of inositol in the absence of IPP was apparently due to an alternate pathway involving
synthesis and dephosphorylation of inositol 1,3,4,5-tetrakisphosphate (Fig. 21.1) . However, although ipp mutations and lithium
treatment did not affect phosphoinositide signal transduction, they had unexpected and dramatic effects on synaptic function.
Specifically, in ipp mutant and lithium-treated wild-type photoreceptor neurons, the probability of vesicular release was greatly
increased, and affected neurons were unable to maintain a synaptic response to a prolonged tetanic stimulus. A variety of molecules
involved in synaptic fusion and vesicular traffic, including synaptotagmin and adaptor protein 2 (AP2), are regulated through specific
physical interactions with inositol polyphosphates (e.g., IP4, IP5, and IP6) (20) . Thus, the effects of lithium on neuronal function in
Drosophila as well as in humans may stem not from defects in inositol signaling per se, but from defects in synaptic function and
plasticity due to alterations in inositol polyphosphate pools.

In summary, lithium provides a good example of the power of genetic neuropsychopharmacology in simple model systems. Studies in
Dictyostelium were instrumental in identifying the GSK-3 pathway as a possible mediator of lithium's deleterious side effects, and
have also provided insight into a possible link between neuroactive peptides and depression. Work in Drosophila has provided an
important lead into discovering how lithium's effects on phosphoinositide signaling affect neuronal function. Future studies in both
organisms have the potential to provide further insight into lithium's mechanism of action, in particular to address more precisely
how lithium-induced changes in inositol lipid content alter synaptic transmission and plasticity in neurons.

Fluoxetine and Other Antidepressants

Another group of drugs that have been the subject of research in simple eukaryotes are those used in the treatment of unipolar
depression. Such drugs include the monoamine oxidase (MAO) inhibitors, the tricyclic antidepressants (e.g., imipramine and
clomipramine), and the selective serotonin reuptake inhibitors (SSRIs; e.g., fluoxetine). A common property of many of these
molecules is their ability
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to potentiate serotoninergic neurotransmission, either by interfering with reuptake of serotonin from the synapse (tricyclics and
SSRIs) or by blocking enzymatic degradation of serotonin (MAO inhibitors). Thus, the therapeutic actions of all of these molecules
are usually explained in terms of a model for depression known as the serotonin hypothesis. According to this model in its simplest
form, levels of serotoninergic neurotransmission in the forebrain are a key determinant of mood, with high activity leading to
euphoria and low activity to dysphoria. Thus the chronic dysphoria experienced by depressed patients could be a consequence of
chronically low serotoninergic transmission, which could be compensated for by interfering with serotonin degradation. This
serotonin hypothesis, or variations thereof, represents the most widely accepted explanation for antidepressant action (21, 22) .

However, the serotonin hypothesis, at least in its simplest form, fails to account for a number of observations about antidepressants.
For one, a direct correlation between the level of serotoninergic transmission and mood has not been demonstrated; normal
individuals treated with serotonin reuptake blockers do not typically experience euphoria, nor does dietary serotonin depletion
induce depression in individuals not already prone to depression (23) . Moreover, the mood-altering effects of serotonin reuptake
blockers in depressed patients occur on a different time scale from their effects on serotoninergic transmission; whereas SSRIs and
most tricyclics elevate synaptic serotonin levels within hours, their effects on mood are not apparent for 2 to 6 weeks. Finally, a
number of effective antidepressants appear to function independently from serotonin, including selective norepinephrine reuptake
inhibitors (SNRIs) such as desipramine, MK869, which antagonizes substance P receptors, and bupropion, whose target is unknown
(24, 25) . Because of these observations, many current models hypothesize that SSRIs are effective against depression not because
of their acute effects on serotoninergic transmission, but because of long-term adaptive changes in monoamine neurotransmission
that arise from chronic inhibition of serotonin reuptake (21) . An appealing feature of this type of model is that long-term activation
of different direct targets by different classes of antidepressants (the serotonin transporter by SSRIs, other targets by atypical
antidepressants) could in principle lead to a common set of adaptive responses in the brain. Alternatively, it is possible that
antidepressants might act, at least in part, at serotonin-independent direct targets.

Studies in C. elegans have provided insight into potential serotonin-dependent and -independent activities of antidepressants.
Nearly all antidepressants have at least two clear effects on C. elegans behavior: stimulation of egg laying and hypercontraction of
muscles in the nose. Whereas the stimulation of egg laying by antidepressants is primarily due to potentiation of serotoninergic
transmission (see below), the effect of antidepressants on the nose muscles appears to be independent of serotonin, since serotonin
itself does not cause nose contraction, whereas antidepressants still contract the noses of serotonin-deficient mutants. Mutations
conferring resistance to the induction of nose contraction by fluoxetine have been identified in seven genes, designated Nrf genes,
for nose resistant to fluoxetine (26) . All the Nrf mutations are recessive and confer resistance to several chemically disparate
antidepressants in addition to fluoxetine; thus, the products of the Nrf genes might potentially represent common, serotonin-
independent antidepressant targets. So far, two Nrf genes have been cloned, nrf-6 and ndg-4. These two genes define the first
members of a novel gene family, and encode predicted multipass integral membrane proteins that are expressed in the nasal
epidermis and the intestine. nrf-6 and ndg-4 have been shown to be defective in the transport of yolk proteins across the intestinal
membrane, suggesting that NRF-6 and NDG-4 may be components of a complex that transports molecules across epithelial
membranes. Based on this result, it is reasonable to suppose that the fluoxetine resistance of nrf-6 and ndg-4 mutants might reflect
a defect in drug uptake rather than the absence of a functional drug target in the neuromuscular system. However, while NRF-6 and
NDG-4 (and by extension their yet unidentified vertebrate homologues) may not represent antidepressant targets per se, they might
represent molecules that function in transport of antidepressants across the blood–brain barrier.

Another C. elegans molecule that clearly represents a serotonin-independent antidepressant target is encoded by the gene egl-2.
egl-2 was originally defined by the dominant gain of function mutations that impaired the activity of the vulval muscles (which
mediate egg laying) and enteric muscles (which mediate defecation) (27, 28) . Both of these defects in muscle activation could be
relieved by treatment with the tricyclic antidepressant imipramine, though not by serotonin or fluoxetine. Thus, imipramine
appeared to act through a serotonin-independent target to suppress the egl-2 muscle activation phenotype (29) . The nature of this
target was revealed when egl-2 was cloned and shown to encode a potassium channel homologous to the Drosophila ether-a-go-go
(eag) channel (30) . Studies on EGL-2 channels expressed in Xenopus oocytes demonstrated that the imipramine-suppressible
dominant alleles of egl-2 encoded mutant channels that opened inappropriately at low voltages. Remarkably, imipramine was shown
to function as a specific antagonist of both the EGL-2 channel and its mammalian homologue MEAG. Thus, this class of calcium
channels appears to represent a conserved target of tricyclic antidepressants in both worms and humans. Interestingly, an important
side effect of tricyclic antidepressants is a type of cardiac arrhythmia called long QT syndrome, a disorder which has also been
linked to mutations in potassium channel genes (29a, 29b) . Thus, the blockade of eag-related potassium channels by tricyclics
provides a likely explanation for this clinically important side effect of tricyclics.

Studies in C. elegans may also provide insight into the serotonin-dependent mechanisms of antidepressant action.
 P.267

The ability of antidepressants (other than tricyclics) to stimulate egg laying in C. elegans depends on their ability to potentiate
serotoninergic neurotransmission (29) , and can be mimicked by exogenous serotonin itself (31) . Serotonin is released from egg-
laying motor neurons called HSNs (27) , and appears to function as a neuromodulator that modifies the functional state of the egg-
laying muscles to potentiate contraction (32) . Serotonin also inhibits locomotion, apparently by inhibiting neurotransmitter release
from excitatory motor neurons (32, 33) . The signal transduction mechanisms that mediate both of these actions of serotonin have
been analyzed genetically, and in both cases the phospholipase C (PLC) homologue egl-8 is required for serotonin response. In the
egg-laying muscles, the effects of PLC appear to be mediated through the protein kinase C homologue tpa-1, whereas in the motor
neurons the most important mediator appears to be the diacylglycerol-binding synaptic protein UNC-13. The involvement of the
phosphoinositide signaling pathway in serotonin signal transduction in both the egg-laying muscles and the motor neurons of C.
elegans has an interesting parallel in mammals, since a number of mammalian serotonin receptor subtypes also signal through
activation of PLC.

The apparent conservation between the signaling pathways mediating serotonin response in C. elegans and humans raises the
possibility that the long-term effects of elevated serotoninergic transmission might also be accessible to genetic analysis in C.
elegans. As noted previously, the alleviation of depression by serotonin-potentiating antidepressants is thought to involve adaptive
signaling pathways that are activated by prolonged elevation of serotoninergic neurotransmission. In C. elegans, prolonged exposure
to serotonin has been shown to lead to adaptive down-regulation of egg-laying behavior and recovery from serotonin-induced
paralysis (34) . Genes encoding possible components of serotonin adaptation pathways have been identified on the basis of serotonin
hypersensitive or adaptation-defective phenotypes (35) ; however, at present little is known about how these or other genes affect
long-term responses to serotonin. Future analysis of serotonin adaptation genes may provide insight into the molecular mechanisms
underlying long-term responses to elevated serotonin transmission that may be important for the therapeutic action of
antidepressants.

Volatile Anesthetics
A variety of volatile molecules, including diethyl ether, halothane, and isoflurane, are capable of inducing general anesthesia, a
behavioral state involving loss of consciousness, analgesia, amnesia, and loss of motor activity. Although these agents have been
widely used in surgery for over a century, their mechanism of action remains poorly understood. General anesthesia appears to
result from defects in synaptic transmission rather than axonal firing; however, it is not clear whether anesthesia results from
potentiation of inhibitory synapses, inhibition of excitatory synapses, or both. The potency of a given volatile anesthetic shows a
very strong correlation to its lipid solubility; this observation, known as the Meyer–Overton rule, has led to the hypothesis that
volatile anesthetics act by disrupting hydrophobic interactions between proteins and/or lipids in neurons. However, the biologically
relevant targets for volatile anesthetics have not been conclusively identified. In principle, this problem appears ideally suited to
attack by a phenotype-driven genetic approach; by identifying mutants that are resistant or hypersensitive to anesthetics and
cloning and sequencing the mutant genes, it should be possible to identify anesthetic targets that are essential for anesthesia in vivo.
In fact, such screens have been conducted in both Drosophila and C. elegans, and a variety of genes affecting sensitivity have been
identified (36) . At present, none of the Drosophila anesthetic response genes have been cloned; thus, molecular information about
their gene products is not available. However, the recent cloning of several C. elegans genes with quantitatively large effects on
anesthetic sensitivity raises the possibility that they might define conserved molecular targets important for anesthetic action.

C. elegans has two distinct responses to volatile anesthetics. At lower concentrations (similar to the alveolar concentrations used in
human anesthesia), volatile anesthetics rapidly induce abnormalities in the pattern of locomotion (37) . Although this effect is
behaviorally quite dissimilar from anesthesia, it is similar to the effect of many mutations that affect synaptic transmission in C.
elegans. In fact, treatment with volatile anesthetics confers resistance to the behavioral effects of cholinesterase inhibitors (38) , a
hallmark of defective neurotransmitter release (7) . Thus, at these concentrations, volatile anesthetics appear to act presynaptically
to interfere with synaptic transmission in C. elegans. A number of mutants with altered sensitivity to these low-concentration
effects of volatile anesthetics have been identified. Potentially the most informative with respect to anesthetic mechanisms contain
mutations in genes encoding components of the SNARE complex, the presynaptic machinery that mediates synaptic vesicle fusion.
Recessive mutations in at least three SNARE genes, unc-64 [encoding C. elegans syntaxin (39) ], snb-1 [encoding VAMP/synaptobrevin
(40) ], and ric-4 (encoding SNAP-25), confer significant hypersensitivity on the effects of both halothane and isoflurane on
coordinated movement. Furthermore, a novel mutation in unc-64, which affects a spice receptor site and consequently leads to the
production of truncated syntaxin peptides, confers strong resistance to the effects of volatile anesthetics on both coordinated
movement and cholinesterase sensitivity (38) . These results suggest that volatile anesthetics interfere with synaptic transmission
through direct interaction with one or more members of the SNARE complex.

At approximately 10-fold higher concentrations, volatile anesthetics induce reversible paralysis in C. elegans, a behavioral
 P.268

effect qualitatively reminiscent of anesthesia. Interestingly, none of the synaptic mutations affecting the low-concentration effects
on coordinated movements affect this high-concentration paralytic response. However, a different, nonoverlapping group of genes
has been identified that confers resistance or hypersensitivity to paralysis by anesthetics in C. elegans. Several of these genes have
been cloned, including unc-1, which encodes a homologue of stomatin (41) , and unc-8, which encodes a subunit of the
degenerin/ENaC family of passive sodium channels (42, 43) . Both unc-1 and unc-8 are expressed in neurons, and both genes can be
mutated to confer either resistance or hypersensitivity to halothane (44) . Allele-specific genetic interactions between unc-1, unc-8,
and the yet uncloned unc-79 and unc-80 genes suggest that their products may physically interact in a multimeric channel complex
specifically involved in anesthetic responses. Since stomatin has been shown to function as a negative regulator of cation channels in
erythrocytes, a reasonable hypothesis is that UNC-1/stomatin may modulate influx through UNC-8 degenerin channels in neurons
that respond to anesthetics. Homologues of both stomatins and ENaC channels have been identified in mammals, and are known to
be expressed in the central nervous system; thus, in principle stomatin-regulated ENaC channels could also affect anesthetic
responses in humans.

In summary, there are two distinct sets of genes that affect responses to volatile anesthetics in C. elegans, which affect different
behavioral responses to different concentrations of anesthetics. At present, it is not clear which of the two (or whether both) might
encode homologues of biologically relevant human anesthetic targets. Although the genes involved in synaptic function alter
anesthetic responses at clinically relevant concentrations, the behavioral responses they affect are qualitatively quite different from
general anesthesia. Conversely, although the stomatin/degenerin genes affect a paralytic response that closely resembles anesthesia,
the response also has a relatively long time delay and occurs at concentrations well above those clinically relevant in humans. Given
the effective drug concentrations for these two behavioral responses, it is possible that the synaptic genes might encode targets
relevant to anesthesia, while the stomatin/degenerin genes might encode targets relevant for side effects of anesthetics.
Alternatively, it is possible that genes affecting high-concentration anesthetic responses do define molecules involved in anesthesia,
especially since the nematode cuticle is relatively impermeant and presents a significant barrier for the entry of many drugs. Since
well-defined mammalian homologues exist for both classes of anesthetic response genes, it should be possible in the future to
examine these issues directly in mammalian systems.

Drugs of Abuse

Ethanol
Unlike many neuroactive substances, ethanol is not believed to have a single molecular target in neurons; rather, a number of
receptors and channels, including the N-methyl-D-aspartate (NMDA), serotonin, and γ-aminobutyric acid (GABA) receptors and
various voltage-gated ion channels, appear to be modulated by the presence of ethanol (45) . Very little information exists
concerning the relative importance of each of these putative direct targets for the psychoactive effects of ethanol; however, a
variety of experiments in cultured cells suggest that a critical short-term effect of ethanol is to enhance receptor-mediated
synthesis of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP). Conversely, long-term ethanol exposure appears to
decrease intracellular cAMP levels. Both the acute and chronic effects of ethanol have also been linked to changes in dopaminergic
neurotransmission (46) . In particular, ethanol has been shown to promote release of dopamine in the mesolimbic pathways of the
brain, in particular the so-called reward pathway synapses between the ventral tegmental area (VTA) and the nucleus accumbens
(NAc). At present, the in vivo significance of these findings with respect to the psychoactive effects of ethanol in mammals remains
to be determined. Moreover, although sensitivity to both the acute and chronic effects of ethanol are clearly affected by genetic
factors, the nature of the genes affecting human ethanol sensitivity are not known.

Recent work in Drosophila has provided support for both the dopamine and cAMP hypotheses of ethanol action. Ethanol vapor has a
number of effects on Drosophila behavior, including hyperactivity, disorientation, uncoordination, and ultimately immobilization.
Using an instrument called an inebriometer (47) , lines of mutant flies have been identified that exhibit abnormal sensitivity to
volatilized ethanol. Among the mutants showing significant hypersensitivity to ethanol were those containing a mutation in the
learning gene amnesiac, which encodes a homologue of the mammalian pituitary adenylyl cyclase activating peptide (PACAP) (48,
49) . Consistent with the implications of this homology, the effects of amnesiac on ethanol response appeared to involve the
adenylyl cyclase pathway, since the adenylyl cyclase activator foskolin blocks the ethanol sensitivity associated with amnesiac loss-
of-function mutations. Moreover, several other loss-of-function mutations affecting cAMP pathway components, including the
adenylyl cyclase gene rutabaga and the cAMP-dependent protein kinase gene DCO, also conferred ethanol sensitivity. Although one
might suppose based on these results that the response to ethanol is simply a function of the level of cAMP signaling in the relevant
neuronal targets (with increased ethanol response corresponding to low cAMP signaling), a variety of data are inconsistent with this
simple model. For example, genetic or pharmacologic activation of the cAMP pathway does not lead to ethanol resistance.
Nonetheless, these genetic data provide the first conclusive link between the activity of the cAMP pathway and the behavioral
effects of ethanol in an intact organism; the precise nature of that link remains to be determined, but should be accessible to
further genetic analysis.
 P.269

Some of the behavioral effects of ethanol on Drosophila have also been shown to be dependent on dopamine (50) . Ethanol has
varying effects on fly locomotion depending on the duration of exposure. During the first 7 to 10 minutes of ethanol treatment,
animals become hyperactive and move at a greatly increased rate; subsequently, they become increasingly uncoordinated and
eventually become completely immobile. When flies are depleted of dopamine through ingestion of a tyrosine hydroxylase inhibitor,
they become significantly less susceptible to this stimulation of motor activity by ethanol. However, these dopamine-depleted flies
exhibited no abnormalities in their sensitivities to ethanol-induced uncoordination or immobilization. Thus, the stimulation of motor
activity by ethanol may involve ethanol-induced enhancement of dopaminergic transmission in brain areas controlling locomotion,
whereas the other behavioral effects of ethanol are likely to involve other neurotransmitter systems.

The genetic analysis of ethanol response mechanisms in Drosophila is still in its early stages. However, it is already clear that
mutants with altered responses to ethanol can be identified in straightforward genetic screens, and at least in some cases analyzed
in the context of well-defined neuronal signaling cascades. Perhaps the greatest promise for future studies is the possibility that
novel ethanol response genes, possibly including the direct molecular targets of ethanol, can be identified in ethanol-resistant or
ethanol-hypersensitive screens.

Nicotine
Tobacco has been implicated in more deaths than any other addictive substance (51) , yet the biochemical basis for compulsive
tobacco use remains poorly understood. The substance most responsible for the addictive properties of tobacco is nicotine, a potent
stimulant and cholinergic agonist. Long-term exposure to nicotine is known to cause adaptive changes in the activity and number of
nicotinic receptors in the brain, which are thought to be important for nicotine addiction (52) . For example, nicotinic receptors
exist in multiple functional states, some of which are relatively refractory to channel opening though they retain affinity for agonists.
Chronic exposure to nicotine or other agonists results in an increased fraction of receptors adopting the lower activity states,
leading to an attenuation of the overall nicotine response (53) . Long-term nicotine treatment also causes a long-lasting functional
inactivation of some nicotinic receptors (54) , which has a slower time course and is much longer lasting than the rapid, receptor-
intrinsic desensitization induced by acute agonist exposure. Depending on the receptor and cell type, long-term nicotine treatment
can also either increase or decrease the number of nicotinic receptors on the cell surface, effects that appear to be mediated at the
level of protein turnover (55, 56) . The cellular pathways that promote these changes are not well understood; for example, little is
known about the cellular pathways that regulate receptor turnover, or the molecular mechanisms that regulate the switching
between different nicotinic acetylcholine receptor (nAChR) states.

Genetic analysis in C. elegans may provide insight into the mechanisms underlying long-term responses to nicotine. Both acute and
chronic nicotine treatment have striking effects on the behavior of C. elegans, including hypercontraction of body wall muscles,
stimulation of egg laying, and increased pharyngeal pumping. The effects of nicotine on the body and egg-laying muscles are
mediated through a nicotinic receptor known as the levamisole receptor (57, 58) . The antihelminthic drug [and ganglionic nAChR
agonist (59) ] levamisole is a potent agonist of this receptor; like nicotine, levamisole causes body muscle hypercontraction and (at
high doses) spastic paralysis. Although the levamisole receptor is found on nematode muscle, its pharmacologic profile generally
resembles that of ganglionic nicotinic receptors of vertebrates. By screening for levamisole-resistant mutants, it has been possible
to identify genes affecting the function of the levamisole receptor (60) . Mutations conferring strong resistance to levamisole have
been identified in six genes. Three of these genes, unc-38, unc-29, and lev-1, encode nicotinic receptor subunits (61, 62) . The UNC-
38 protein is most similar to the insect α-like subunits ALS and SAD (49% amino acid identity); among vertebrate receptor subunits,
the closest similarity is to neuronal α subunits (61) . UNC-29 and LEV-1 are closely related proteins whose closest homologues in
vertebrates are neuronal non-α subunits (approximately 55% sequence similarity). Three additional genes conferring strong
levamisole resistance, unc-50, unc-74, and unc-63, have not been cloned, but have been shown to be required for assembly of a
functional levamisole receptor as assayed in vitro (63) . In addition to conferring resistance to levamisole (and other nicotinic
agonists), mutations in these genes cause defects in the coordination of body movement (60) . Mutations in three additional genes
(lev-8, lev-9, and lev-10) confer weaker resistance to levamisole, do not cause defects in locomotion, and have no detectable effect
on the biochemical properties of the receptor as assayed in vitro (60, 63) . Thus, the proteins encoded by these genes have been
hypothesized to regulate the activity of the receptor indirectly.

Long treatments with nicotine and other nicotinic receptor agonists lead to adaptation (57) . Animals treated with exogenous
nicotine initially hypercontract to the point of spastic paralysis; however, after several hours in the presence of nicotine, they
recover their ability to move and regain much of their body length. In some C. elegans strains (for example, strains with weakly
crippled nAChRs), long-term nicotine treatment eventually leads to almost complete inactivation of the response to nicotine.
Moreover, when nicotine-adapted animals are removed from nicotine, their locomotive behavior becomes uncoordinated and
resembles that of mutants with strong defects in the levamisole receptor (i.e. an unc-29 or unc-38 null mutant). Thus, long
treatments with nicotine cause nicotine dependence in addition
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to nicotine tolerance in the C. elegans body muscle. Long-term nicotine treatment also down-regulates levamisole receptors in the
egg-laying muscles. Overnight treatment with nicotine leads to an almost complete attenuation of levamisole sensitivity with
respect to egg laying, and this attenuation of levamisole response persists for up to 24 hours after removal from nicotine. This loss
of levamisole responsiveness is accompanied by a corresponding decrease in the abundance of UNC-29–containing receptors in the
vulval muscles, an effect that may be mediated at the level of protein turnover (64) . Interestingly, the nicotine-dependent
decrease in UNC-29 receptor abundance requires the activity of TPA-1, a vulval muscle-expressed PKC isoform. Since UNC-29 and
other nicotinic receptor subunits contain consensus sequences for PKC phosphorylation, this raises the possibility that direct
phosphorylation of nicotinic receptors might represent a signal for increased turnover. In the future, it should be possible to test
this hypothesis, as well as identify other genes required for long-term responses to nicotine in C. elegans.
Another set of genes, the weak levamisole-resistance genes lev-8 and lev-9, appear to represent positive regulators of nicotinic
receptor activity. Mutations in these genes confer partial resistance to levamisole and nicotine with respect to body muscle
contraction and strong resistance with respect to egg laying (65) . However, lev-8 and lev-9 mutations do not affect the assembly of
levamisole-binding nicotinic receptors as assayed in vitro (58) , and the abundance of UNC-29 receptors in the vulval muscles is not
significantly reduced by mutations in these genes (65) . lev-8 and lev-9 may therefore encode regulatory proteins that stimulate the
activity of nicotinic receptors in vivo, but are not subunits or essential accessory proteins. In principle, the inhibition of the lev-8 or
lev-9 gene products might represent a plausible mechanism for functional inactivation of nicotinic receptors. Once lev-8 and lev-9
are cloned, it will be interesting to determine whether mammalian homologues exist for these molecules, and if so, whether they
are involved in regulating the functional activity of nicotinic receptors in human neurons.

Cocaine
Cocaine is a potent psychostimulant, and among the most widespread addictive drugs of abuse. The psychoactive effects of cocaine
are thought to result largely from its ability to potentiate aminergic neurotransmission in the limbic pathways of the brain. Cocaine
inhibits the reuptake transporters for dopamine, serotonin, and norepinephrine, which leads to accumulation of monoamine
transmitters at the synapse. The dopaminergic synapses of the nucleus accumbens are thought to be particularly important for
cocaine addiction, since pharmacologic inhibition or surgical lesioning of these areas confers significant resistance to both the short-
term and long-term effects of cocaine in rodents (46) . However, dopamine is probably not the only neurotransmitter involved in
cocaine addiction, since mice lacking the vesicular dopamine transporter will still self-administer cocaine after repeated
administration of the drug (66, 67) . Although dopaminergic transmission in the limbic reward pathways has been implicated in the
reinforcing properties of a wide range of addictive substances in addition to cocaine, the molecular and cellular mechanisms that
lead to addiction in these neurons are not well understood.
Recent work in Drosophila suggests that the mechanisms of cocaine action may be accessible to genetic analysis. When flies are
exposed to volatized free-base cocaine, they exhibit dose-dependent stereotypical behaviors that are surprisingly reminiscent of
cocaine's psychostimulant effects in mammals (68) . For example, at low doses treated flies become hyperactive and exhibit
compulsive, continuous grooming behavior. At intermediate doses animals move more slowly and display stereotyped locomotive
behaviors such as circling. Finally, at high doses animals undergo tremors, spastic paralysis, and finally death. Repeated treatment
of flies with low doses of cocaine results in an increased behavioral response, a phenomenon known as sensitization; cocaine
sensitization also occurs in mammals and is thought to underlie some aspects of addiction in humans. Interestingly, male flies are
more sensitive to cocaine than females, a sexual dimorphism that also holds true in mammals (69) . Thus, cocaine has both short-
term and long-term effects on fly behavior that are remarkably analogous to its effects on mammals.
These behavioral similarities between cocaine's action on flies and mammals raise the possibility that they might share a common
functional basis as well. In fact, recent evidence indicates that cocaine's actions on fly behavior also involve effects on aminergic
neurotransmission. Insects contain cocaine sensitive reuptake transporters for dopamine, serotonin, and octopamine (an
invertebrate neurotransmitter chemically similar to norepinephrine); thus, cocaine at least in principle could increase synaptic
levels of multiple monoamine neurotransmitters in the fly brain (70, 71 and 72) . The monoamine most convincingly implicated in
cocaine's acute effects on flies is dopamine. Dopamine receptor antagonists have effects on grooming and locomotive behaviors that
are the converse of the effects of cocaine, and these antagonists can also block the effects of cocaine and cocaethylene on these
behaviors in decapitated Drosophila preparations (Fig. 21.2) (69, 73) . Moreover, when flies are depleted of endogenous dopamine
using tyrosine hydroxylase inhibitors, they acquire resistance to the acute effects of cocaine treatment (50) . Paradoxically,
however, transgenic animals in which dopamine and serotonin release is blocked by ectopic tetanus toxin expression are actually
hypersensitive to cocaine (74) . Thus, although dopaminergic neurotransmission is clearly involved in behavioral responses to cocaine
in Drosophila, the specific role that it plays in these responses is not completely clear.

FIGURE 21.2. Biogenic amines and their biosynthesis.

 P.271

Surprisingly, cocaine sensitization in Drosophila has been linked to a different biogenic amine—tyramine. Tyramine is present only in
trace quantities in mammalian nervous systems; however, in insects it is a somewhat more abundant molecule and also serves as a
precursor for the important neuromodulator octopamine (Fig. 21.3) . Mutants with defects in this biosynthetic pathway have been
identified in Drosophila behavioral screens. For example, inactive mutants have low levels of the enzyme tyrosine decarboxylase,
and consequently fail to efficiently synthesize both tyramine and octopamine; in contrast, TβH mutants are defective in the
tyramine β-hydroxylase enzyme, and thus synthesize tyramine but not octopamine. Interestingly, while inactive mutants display an
essentially normal acute response to cocaine, they are strongly defective in sensitization (75) . This sensitization defect can be
rescued by feeding the mutant flies tyramine but not octopamine; moreover, TβH mutants (which lack octopamine but not tyramine)
and Ddc mutants (which fail to synthesize dopamine) show normal cocaine sensitization. Furthermore, cocaine actually increases the
levels of tyrosine decarboxylase activity in treated flies, suggesting that cocaine sensitization may actually occur at least in part
through induction of tyramine synthesis. Remarkably, both the induction of tyrosine hydroxylase activity by cocaine and cocaine
sensitization itself require the activities of the period, clock, and double-time genes, three members of the conserved signal
transduction pathway that controls circadian rhythms in animals and fungi (76) .

FIGURE 21.3. The adenylyl cyclase signaling pathway.

How might tyramine mediate cocaine sensitization in flies, and does it play a similar role in mammals? At present, these questions
are difficult to answer. Although the function of tyramine in insect nervous systems has not been clearly established, putative
tyramine receptors have recently been identified in both Drosophila and the honeybee (77) . Possibly cocaine might act in a period-
dependent manner to facilitate tyramine release from nerve terminals, which could then induce plasticity in other monoamine
pathways in the brain. Future studies will be needed to identify the specific tyramine receptors that might mediate such responses
and to understand the neural basis for their effects on behavior. In vertebrates, tyramine receptors have not been identified; thus,
it remains an open question whether tyramine plays a role in human sensitization to cocaine that parallels its role in Drosophila.
However, the involvement in Drosophila of the circadian clock pathway, which is highly conserved between insects and humans,
suggests that at least some components of the molecular mechanisms underlying this process may be shared between these widely
divergent organisms.

QUESTIONS AND FUTURE PROSPECTS

Part of "21 - Neuropsychopharmacology of Worms and Flies "

Perhaps the major potential pitfall of using worm or fly genetics to investigate drug mechanisms is that there is no guarantee that
those mechanisms will be conserved across the evolutionary gulf separating these disparate animals. Certainly at the anatomic level,
the brains of humans, flies, and worms are vastly different organs. Nonetheless, for most pharmacologic studies, the critical issue is
conservation at the molecular level, and with the worm and fly genomes essentially complete, it is clear that at the molecular level
the C. elegans and Drosophila nervous systems are quite similar to their human counterpart. For example, the C. elegans and
Drosophila genomes contain homologues of each of the basic types of potassium channels, calcium channels, and G proteins, as well
as putative receptors for most human neurotransmitters (78, 79) . To be sure, there are a small number of nervous system molecules
found in vertebrates and flies but not nematodes (e.g., voltage-gated Na channels), as well as molecules found in nematodes and
flies but not vertebrates (e.g., the ivermectin-sensitive glutamate-gated Cl channel). However, on the whole the nematode,
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fly, and vertebrate nervous systems appear to be remarkably similar at the molecular level given their vast differences in scale and functionality.

What are the prospects for model organism neuropsychopharmacology in the postgenomic future? The availability of substantial portions of the worm and fly genomes has already made
the rate-limiting step of classic forward genetics—cloning a mutant gene—significantly easier and more straightforward. This cloning process will become easier still as high-resolution,
single-nucleotide polymorphism maps of the worm and fly genomes become available. The imminent completion of the human genome will also provide great benefits to model
organism studies, since it will allow rapid identification of human homologues for worm or fly genes and more reliable distinction of genuine mammalian orthologues from other
members of a gene family. The great advantage of worm and fly studies for the elucidation of drug mechanisms is the ability to conduct unbiased, phenotype-driven mutant screens to
identify unknown gene products involved in drug response. Since ethical considerations will always preclude such approaches in humans, and since time, space, and cost considerations
make them inefficient even in simpler vertebrates, C. elegans and Drosophila are likely to serve as workhorses for basic neuroscience research for many years to come.

REFERENCES
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22
Beyond Binding: Molecular and Cell Biological Approaches to Studying
G-Protein–Coupled Receptors

Gabriel A. Vargas

Mark Von Zastrow

Gabriel A. Vargas: Department of Psychiatry, University of California–San Francisco, San Francisco, California 94143.

Mark Von Zastrow: Departments of Psychiatry, Cellular and Molecular Pharmacology, and Program in Cell Biology, University of
California–San Francisco, San Francisco, California 94143.

The origins of the modern concept of receptors can be traced to the beginnings of the 20th century (1 ). Almost two decades passed
until the first neurotransmitter, acetylcholine, was identified from classic physiologic studies on the vagus nerve performed by Otto
Loewi in 1921. Since these seminal discoveries the pace of advance has increased enormously. A revolution in the field began in the
1950s, with the discovery that neurotransmitter receptors are targets of clinically relevant psychotropic drugs and the development
of radioligand binding techniques (2 ,3 ). Radioligand binding methodologies remain a mainstay of modern neuropsychopharmacology,
and have facilitated the identification of receptor subtypes as well as the discovery of novel receptors that mediate the actions of
important drugs.

The application of recombinant DNA methodologies sparked a second revolution in neuropsychopharmacology. These methods
facilitated the cloning of complementary DNAs (cDNAs) encoding distinct receptors, the identification of large families of
homologous receptors, and unprecedented insight into subtype diversity within individual receptor families (4 ,5 ).

Important families of receptors include steroid hormone receptors, receptor tyrosine kinases, ligand-gated ion channels, and G-
protein–coupled receptors (GPCRs). GPCRs comprise the largest class of signal-transducing receptors, with well over 1,000 members
identified in humans. In some organisms, genes encoding GPCRs comprise 1% of the genome (6 ). GPCRs mediate the actions of the
majority of neurotransmitters and neuromodulators, as well as other important biological ligands. These receptors are also critically
important drug targets. Indeed, the majority of psychopharmaceuticals presently in use either bind directly to specific GPCRs (e.g.,
antipsychotics) or indirectly influence GPCR function by modulating the availability of endogenous ligands (e.g., selective serotonin
reuptake inhibitors, SSRIs). Therefore elucidating mechanisms of GPCR function and regulation is of central importance to
understanding the actions of clinically relevant drugs.

During the past several years there has been a great deal of progress in elucidating specific mechanisms of GPCR function and
regulation. Much of this progress can be attributed to the application of newer molecular and cell biological techniques, which have
complemented previously developed pharmacologic approaches for probing receptor function. This chapter discusses some of these
molecular and cell biological approaches for isolating and studying cloned receptors, focusing specifically on GPCRs expressed in a
variety of systems. Although we restrict our scope in this chapter to representative approaches applied to GPCRs, these methods
have broad potential application and have been used to study other important receptor families.

 ISOLATION AND IDENTIFICATION OF RECEPTORS

 EXPRESSION AND PURIFICATION OF CLONED GPCRS
 MECHANISMS OF LIGAND BINDING AND ACTIVATION
 REGULATION OF RECEPTOR SIGNALING
 EMERGING HORIZONS
 SUMMARY AND CONCLUSIONS

ISOLATION AND IDENTIFICATION OF RECEPTORS

Part of "22 - Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors "

The identification of GPCRs by biochemical purification is a challenging task because of the generally low abundance of these
proteins in cells and tissues, and because GPCRs are highly hydrophobic molecules that are easily denatured when solubilized in
detergent solutions. Molecular cloning techniques have greatly facilitated the identification of GPCRs. Molecular cloning takes
advantage of the ability to generate and screen “libraries” containing cDNAs corresponding to the messenger RNAs (mRNAs) that
encode cellular proteins.
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A detailed discussion of molecular cloning techniques is beyond the scope of the present review and has been described elsewhere (7 ). In general, a cDNA library is generated from a
specific tissue and animal source (such as rat brain) by purifying mRNA from the tissue, using the enzyme reverse transcriptase to generate a strand of DNA complementary to each
mRNA present in this mixture, and then using a DNA polymerase to generate double-stranded DNA from this sequence that is suitable for insertion into an appropriate plasmid or phage
vector that facilitates faithful replication of the sequences and allows selection of individual clones corresponding to a single cDNA. The main challenge in receptor cloning is to isolate
or “screen” for the appropriate receptor-encoding cDNA from a library. Several different approaches to library screening have been used successfully for cloning cDNAs encoding GPCRs.

Receptor Cloning from Protein Sequence

Early isolation and cloning of receptors relied on purifying sufficient quantities of receptor and then microsequencing peptide fragments. The hamster β2-adrenergic receptor was
cloned using partial sequence information derived from protein purified from hamster lung (8 ). The sequence of amino acids present in a GPCR fragment allows one to predict the
sequence of the corresponding region of the mRNA sequence by “back-translation” from the genetic code. This nucleic acid sequence can be searched for in the cDNA library by virtue
of the ability of complementary strands of DNA to anneal together with extremely high specificity. Because of the degeneracy of the genetic code, many amino acids can be encoded
by more than one nucleic acid codon. Therefore, nucleic acid “probes” used to screen cDNA libraries often contain mixtures of sequences representing multiple potential “spellings” of
the known peptide fragment. cDNA clones encoding the sequence of interest can be isolated by hybridization of a labeled probe or by polymerase chain reaction (PCR)-mediated
amplification from a cDNA mixture.

Receptor Cloning by Sequence Homology

Sequence homology within the GPCR superfamily of receptors enables the use of DNA sequence from a previously cloned receptor to probe libraries for other related receptors. Cloning
of the dopamine D2 receptor is an example of where sequence homology was used to clone other members of the GPCR superfamily of receptors. Olivier Civelli's group (9 ) used the
hamster β2-adrenergic receptor gene as a hybridization probe to isolate cDNA encoding the rat D2 dopamine receptor. The authors used two major criteria to determine that the cDNA
isolated encoded a functionally relevant dopamine receptor. First, they demonstrated that mRNA corresponding to their cDNA sequence was expressed in tissues that had been
previously shown by radioligand binding to express functional D2 receptors. They accomplished this by Northern blotting, a procedure by which RNAs isolated from cells or tissues is
resolved by gel electrophoresis and the specific RNAs homologous to a particular sequence is detected by hybridization of a specifically labeled probe. Second, the authors
demonstrated that the cDNA isolated from their library encoded a functional D2-class dopamine receptor. This was accomplished by applying conventional radioligand binding and
receptor signaling assays to detect functional D2 receptor activity in fibroblast cells that do not normally express dopamine receptors and that were transfected with the cDNA isolated
from library screening.

Receptor Cloning by Functional Expression

GPCRs can also be cloned based on their functional properties. The cloning of the serotonin (5-hydroxytryptamine) 5-HT1C receptor used this approach (10 ). Taking advantage of the
high expression level of the 5-HT1C receptor in the choroid plexus, the authors isolated mRNA from this source and injected this preparation into Xenopus oocytes, which allows both
injected mRNA and cDNA to be translated and expressed. The activation of the 5-HT1C receptor by its ligand leads to an increase in intracellular calcium through the inositol phosphate
signaling pathway. This rise in intracellular calcium leads to the opening of Ca2+-dependent chloride channels that was detected by means of highly sensitive electrophysiologic
techniques. Similar results could be obtained by injecting cDNAs produced from the mRNAs. Pools containing progressively smaller numbers of cDNAs were then analyzed until a single
cDNA encoding a functional serotonin receptor was isolated.

The cloning of the delta opioid receptor (11 ,12 ) was also achieved by a functional assay. A cDNA library prepared from cells that endogenously express the delta opioid receptor was
expressed in cultured fibroblast-like cells that do not normally express opioid receptors. Cells expressing opioid receptors were identified by binding of a radiolabeled opioid peptide.
cDNA isolated from the selected cell was isolated and retransfected, to allow multiple rounds of purification until a single cDNA encoding the delta opioid receptor was isolated. The
isolated cDNA was confirmed to encode a delta opioid receptor by analysis of radioligand binding properties and ligand-induced signal transduction in transfected cells.

Identification of Receptor Subtypes (Fig. 22.1 )

FIGURE 22.1. Dopamine D1 receptor two-dimensional (2D) snake diagram downloaded from the G-protein–coupled receptor database (GCRDb),
http://gcrdb.uthscsa.edu/index.html, which is a very useful site for researchers working on GPCRs (13,14). This diagram shows the seven-transmembrane structure of GPCRs
and the long carboxy terminus tail characteristic of D1-like dopamine receptors. In the GCRDb, amino acid mutations found in the receptor are listed in white in the 2D snake
diagram and hyperlinked to the GRAP mutant database. [http://tinygrap.uit.no/]. (Used by permission of the GCRDb.) See color version of figure.

A general principle in molecular pharmacology is that multiple receptors exist that can recognize the same ligand. This “one ligand, multiple receptors” principle has led to great
interest in the identification of multiple receptor subtypes.
 P.277

For example, radioligand binding techniques and assays of receptor-mediated signal transduction originally defined two classes of
dopamine receptor: D1 receptors that stimulate adenylyl cyclase, and D2 receptors that inhibit this enzyme. Molecular biological
techniques confirmed that these receptors represent distinct gene products and led to the discovery of additional, structurally
homologous subtypes of receptor protein. These subtypes of dopamine receptors would have been impossible to identify definitively
using classic pharmacologic approaches, as their pharmacologic properties are quite similar and some subtypes are expressed at very
low levels in native tissues. For example, the D4 dopamine receptor is a member of the “D2-class” of dopamine receptors that was
cloned by sequence homology to the cloned D2 receptor (15 ). D4 receptors are of great interest because they bind the atypical
antipsychotic clozapine with approximately 10-fold higher affinity than cloned D2 receptors (16 ).

Molecular biological methods also led to the discovery of additional diversity among closely related GPCRs encoded by genetic
variants or by modification of the receptor gene after transcription. For example, multiple variants of D4 receptor, which differ only
in the structure of the third cytoplasmic loop of the receptor protein, are encoded by closely related genes that are inherited in a
mendelian manner ((17 )). Two variants of the D2 dopamine receptor are
 P.278

generated from the same gene by alternative splicing, which occurs during posttranscriptional processing of the RNA. Another
interesting example of such receptor diversity is the 5-HT2C receptor, which exists in variant forms determined by a
posttranscriptional process called RNA editing (18 ). In many cases the functional significance of such variation among GPCRs is not
known. However in some cases, such as RNA editing of the 5-HT2C receptor, individual receptor variants have significantly different
functional properties.

As a further extension of the “one ligand, multiple receptors” concept, molecular biological methods led to the discovery that a
specific neurotransmitter can often bind to more than one class of receptor protein. The first example of this can be found in the
acetylcholine receptors (AChRs). The idea that distinct muscarinic and nicotinic AChRs exist was first indicated by pharmacologic
studies. Molecular cloning of the corresponding receptor cDNAs confirmed this idea and revealed very precisely the differences
between these classes of AChR: muscarinic-type AChRs are GPCRs, whereas nicotinic-type AChRs are members of the structurally
distinct family of ligand-gated ion channels (LGICs). There are now several examples of this type of diversity, including the existence
of distinct GPCRs and LGICs for serotonin, glutamate, and γ-aminobutyric acid (GABA).

Discovery of Orphan Receptors

Orphanin FQ and Its Receptor

In addition to identification of receptor subtypes, the cloning approaches discussed above have also enabled the isolation of GPCRs
before the identification of any known ligands. Putative receptors for which a ligand is not known, called orphan receptors, hinge on
sequence similarity with other GPCRs. The cloning of an opioid-like GPCR, known as ORL1 or the orphanin receptor, was
accomplished by several groups looking for additional members of the opioid receptor family by cloning structurally homologous
cDNAs (19 ,20 ). Expression of the ORL1 cDNA revealed that this putative receptor did not bind any of the typical mu, delta, or
kappa ligands with high affinity, despite having particularly high sequence homology to the kappa receptor. In addition, there are
large differences in anatomic distribution of ORL1 mRNA compared to the known distribution of opioid receptors. Later, elegant
studies led to the isolation of an endogenously expressed peptide ligand for this receptor, nociceptin or orphanin FQ, allowing the
ORL1 gene product to be clearly established as a bona fide GPCR (21 ).

Orexins and Their Receptors

Many other cDNAs encoding candidate orphan receptors have been identified by DNA sequence analysis. Current estimates suggest
that there are ˜100–200 such orphan GPCRs encoded in the human genome (not including predicted olfactory receptors). The
isolation of so many candidate orphan receptors has led some groups to attempt systematic approaches to identifying their
endogenous ligands. Identification of orexin and the orexin/hypocretin receptor was accomplished by using a cell-based detection
system using cells transfected with a large number of candidate orphan receptor cDNAs (22 ). A cDNA (HFGAN72) was identified that
was capable of causing an elevation in cytoplasmic free calcium in response to a crude peptide-containing extract prepared from
brain. A specific peptide ligand was isolated from this mixture according to its ability to activate this orphan GPCR. This peptide is
expressed highly in the lateral hypothalamus and influenced feeding behavior when introduced into rat brain, and is called orexin
(from the Greek orexis meaning appetite). The receptor encoded by HFGAN72 was named the orexin receptor. Recently, a canine
narcolepsy gene was identified by positional cloning as belonging to a subtype of orexin receptor (23 ). Thus the identification of
orphan GPCRs can lead to powerful new insights relevant to diverse areas of neuropsychopharmacology.

EXPRESSION AND PURIFICATION OF CLONED GPCRS

Part of "22 - Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors "

Expression
The ability to express cloned cDNAs in various cell types has provided powerful tools for studying the functional properties of
defined GPCRs. In many cases, receptors expressed in heterologous cell systems have remarkably similar functional properties to
those in their native tissue of origin, although this is not always the case. For example, D2 dopamine receptors differ in their
properties in pituitary GH4C1 cells and Ltk-fibroblasts. In the pituitary cells, D2 receptors fail to elicit phosphoinositide hydrolysis
and induce a decrease of intracellular calcium. In contrast, in the fibroblast cells, the D2 receptor induced a rapid stimulation of
inositol 1,4,5-trisphosphate and an increase of intracellular Ca2+ (24 ). Therefore it is important to compare results obtained from
studies of cloned receptors in heterologous systems to the properties of receptors in native tissues.

In addition to facilitating functional studies, heterologous expression can also be used to produce large amounts of receptor protein,
which is necessary for certain biophysical and structural studies. Mammalian cells are typically used for functional studies of
expressed GPCRs; however, it is sometimes preferable to use nonmammalian cells (such as insect cells or yeast cells) for large-scale
expression of GPCRs because these cells can be grown economically in very large amount. Prokaryotic expression systems (such as
Escherichia coli) have potential advantages for large-scale production but have not been used widely in GPCR research because, in
general, it has been difficult to obtain functional activity of GPCRs using these systems.
 P.279

Purification
The ability to express cloned receptors makes it possible to modify the receptor protein to facilitate purification and detection.
There are many strategies that have developed from this capability. For example, one useful approach is to insert an antigenic
epitope into the GPCR, which can facilitate detection and purification by standard immunochemical procedures using well-
characterized antibodies (see ref. 25 for review). This approach obviates the sometimes laborious path of generating antibodies
that recognize the native receptor. Epitope tags are typically short sequences (˜10 residues) that bind tightly to a highly specific
antibody. In many cases, epitopes chosen for this purpose are either synthetic (not derived from any known biological sequence) or
derived from nonmammalian sources, in an attempt to reduce the probability of cross-reaction with endogenous antigens. Popular
epitopes include a region from the hemagglutinin molecule of influenza virus (“HA” epitope) or the “Flag” epitope, a synthetic
sequence recognized by a series of well-characterized antibodies. The use of such epitopes allows the purification of tagged proteins
using commercially available reagents. Tagged receptors can be isolated from extracts prepared from cultured cells by
immunoaffinity chromatography. A column can be made by immobilizing an antibody onto a solid support that will bind to tagged
receptors as the extracts are passed over the column. Elution of the specifically bound receptor can be accomplished by changing
either the salt concentration or the pH to disrupt the antigen-antibody interaction. An example of this approach is found in the high-
level production of the human β-adrenergic receptor (26 ).

MECHANISMS OF LIGAND BINDING AND ACTIVATION

Part of "22 - Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors "

Use of Site-Directed Mutagenesis

The availability of cDNAs encoding specific GPCRs and the development of various cellular expression systems provide powerful tools
for examining the specificity of ligand binding to receptors. In particular, site-directed mutagenesis can be used to alter residues in
the GPCR structure, and then the functional consequences of these modifications on ligand binding can be examined. Many
techniques have been developed for introducing specific mutations into the receptor cDNA, as discussed in detail elsewhere (27 ).
Selected examples of the use of these techniques for understanding the structural basis of ligand binding and receptor activation are
outlined below.

Point Mutagenesis
Point mutations refer to modifications of the cDNA that result in the substitution of a single amino acid residue in the expressed
receptor protein with another amino acid. Point mutagenesis can be used to dramatically change the physicochemical properties of
a specific residue (e.g., substitution of a basic residue with an acidic one); or cause a subtle change in residue structure (such as
substitution of a serine residue with cysteine, which results in a change of a single atom in the protein structure). Point mutations
are extremely useful because they often do not cause global perturbations of receptor structure and therefore allow highly specific
analysis of the function of defined receptor residues. For example, point mutations have been used to define important
determinants of receptor-ligand interaction with considerable precision (28 ).

Point mutations can identify essential features in the receptor structure. Mutation of a single aspartic acid residue present in the
predicted third transmembrane domain of catecholamine receptors abrogates high-affinity binding of catecholamines to the β2-
adrenergic receptor. Since an aspartic acid residue is present at the corresponding position in other catecholamine receptors as well,
this residue is said to be “conserved” in the structure of multiple receptors. Point mutagenesis applied to these receptors indicates
that this aspartic acid is required for ligand binding to a number of receptors, apparently by serving a conserved function as a
counter-ion for the positively charged amine moiety of catecholamine ligands (28 ,29 ). Like the aspartic acid residue discussed
above, other amino acids that serve a specific function in the receptor generally are found to be conserved between receptors with
similar properties.

Point mutations can identify nonconserved residues that determine the specificity with which drugs bind to structurally homologous
receptors. Nonconserved residues present in the receptor sequence may play a pharmacologically important role in determining
unique properties of individual receptor subtypes. Nonconserved residues can be essential for determining the specificity with which
a drug binds to closely related subtypes of G-protein–coupled receptors. Point mutation analysis combined with appropriate
pharmacologic assays can be used to identify such divergent receptor residues that are critical for drug binding, thus providing
insight into the structural basis of ligand binding specificity that is useful for drug design.

Point mutations can provide insight into species- and population-specific differences in receptor pharmacology. For the very reason
that nonconserved residues are often not essential for basic receptor function, these residues are often not conserved across species.
Thus the pharmacology of many subtype-specific drugs can be highly dependent on the species of animal studied. For example,
three homologous genes encoding distinct subtypes of α2-adrenergic receptor are expressed in various mammalian species. However,
the pharmacology with which subtype-selective drugs bind to receptor subtypes encoded by mouse or rat receptor genes can differ
substantially from the pharmacology characteristic of the corresponding human receptor (30 ). This
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has led to considerable confusion in the correspondence between pharmacologic and molecular biological definitions of specific
receptor subtypes across species, and has important implications for the use of animal models for the development of subtype-
specific drugs for humans. Furthermore, nonconserved residues involved in subtype-specific drug binding can also differ within the
human population, as a result of random mutation and genetic drift. This concept has not yet been extensively explored but may be
an important direction for the use of pharmacogenomics in clinical medicine.

Deletion Mutagenesis
Another mutational approach useful for probing receptor structure and function is removal of certain residues from the receptor
structure entirely. Deletions of multiple residues in certain parts of the receptor protein (e.g., transmembrane helices) can be
difficult to interpret because they often lead to massive disruption of receptor structure. However, deletion of limited regions in
extracellular or cytoplasmic domains are often well tolerated and have been quite informative. For example, deletion of residues
located in the amino-terminal extracellular domain of polypeptide receptors [such as the follicle-stimulating hormone (FSH)
receptor] and the calcium receptor implicate this domain in ligand interaction. Deletion of residues located in the third cytoplasmic
loop of various receptors, such as the muscarinic acetylcholine receptors, implicated this domain in functional coupling to
heterotrimeric G proteins (27 ).

Substitution or Chimeric Mutagenesis

A very powerful approach to site-directed mutagenesis is to substitute entire series of residues from one receptor with the
corresponding residues of another. This approach is based on the idea that receptors are composed of modular structural domains,
and takes advantage of the fact that receptor domains that mediate similar functions often have conserved amino acid sequence.
Chimeric substitutions are often less disruptive than deletions to the overall structure of the receptor protein. For example,
chimeric mutagenesis has been useful for defining transmembrane residues that mediate subtype-specific and species-specific
differences in ligand binding to adrenergic receptors. Receptor chimeras between α2- and β2-adrenergic receptors defined multiple
cytoplasmic domains that contribute to the specificity of receptor interaction with their cognate heterotrimeric G proteins (4 ).

Use of Random Mutagenesis

In contrast to site-directed mutagenesis, random mutagenesis is an unbiased approach that can examine a much broader range of
modifications of the receptor protein. Random mutagenesis, therefore, has the potential to reveal unanticipated features of
receptor structure and function. Random mutagenesis typically requires functional assay of a much larger number of mutant
receptors than analyzed using site-directed mutation. The relatively low throughput inherent to traditional methods of receptor
characterization have limited the practical utility of random mutagenesis of mammalian GPCRs. This limitation has become less
significant with the recent development of higher throughput functional assays and the successful expression of mammalian GPCRs
in more genetically tractable organisms. For example, the budding yeast Saccharomyces cerevisiae has been used recently for
studying the functional properties of a large number of mutant chemokine receptors in which selected regions of the receptor
protein were mutagenized in a nonbiased manner. Analysis of these data identified residues in the receptor protein essential for
ligand binding and activation. In addition, this nonbiased screening approach yielded unanticipated information, including the
identification of mutations that constitutively activate receptors and the identification of functional mutant receptors predicted to
contain fewer than seven transmembrane domains (31 ).

Use of Biophysical Approaches

Biophysical techniques are essential for detailed examination of protein structure and conformational change. One reason these
methodologies have had limited application in the study of GPCRs is that they typically require milligram quantities of receptor, a
quantity difficult to acquire from native tissue sources. For many years rhodopsin, purified from retina, was the only GPCR that
could be generated in sufficient quantity for biophysical study. Indeed, much of what we know about GPCR structure and
conformational change has been elucidated from elegant biophysical studies of rhodopsin. Recently, the development of improved
expression and purification strategies have made it possible to obtain other GPCRs in sufficient quantity and purity for biophysical
study. Thus it is likely that biophysical approaches will play an increasingly important role in future studies of GPCR structure and
activation.

Structural Studies of Rhodopsin

High-resolution structural information can be provided by x-ray diffraction methodologies applied to ordered three-dimensional
crystals of pure protein. Rhodopsin, a GPCR mediating phototransduction in the retina, has been a favorite for such studies because
it can be purified in sufficient amount and purity to facilitate crystalization. Previous studies using electron diffraction of two-
dimensional crystals of rhodopsin obtained structural information to a resolution of approximately 7.5 Å (angstroms; 1 angstrom = 10-
10
m), revealing the relative orientation of the transmembrane helices in the lipid bilayer (32 ). Recently x-ray diffraction has
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been used to solve the structure of three dimensional crystals of rhodopsin to a resolution of 2.8 Å. This accomplishment is truly a
major milestone in the field, revealing for the first time the atomic structure of any GPCR and providing detailed information about
specific interactions between this GPCR and retinal, its physiological ligand (33 ).

Molecular Modeling of GPCR Structure

As the hydrophobic domains predicted to form transmembrane helices are extensively conserved among all GPCRs, it is believed that
the general features of rhodopsin's transmembrane structure are relevant to other GPCRs. This has motivated the use of rhodopsin's
structure as a “template” on which to predict the stucture of other GPCRs, via computational approaches that identify homologous
residues and infer thermodynamically stable conformations of extracellular and cytoplasmic loops. It remains to be determined the
degree to which specific features of diverse GPCRs are actually conserved at the level of atomic resolution. Indeed, based on well
established differences in the pharmacology of individual GPCRs, one might expect there to be significant limitations of such
homology-based predictive methods, at least with respect to structural features involved in drug binding. Nevertheless, the
available experimental data leave little doubt that this approach is an important starting point for mechanistic studies and for
rational drug development (34 ).

Biophysical Studies of Conformational Dynamics Involved in GPCR Activation

While crystallographic methods have the potential to provide detailed information regarding the relative positions of all residues of
the receptor protein, these methods are inherently limited to reporting on a static structure. Thus additional methods are required
to examine dynamic conformational transitions that mediate ligand-dependent signal transduction via GPCRs. Several biophysical
approaches have been utilized for this purpose. Classic studies of rhodopsin measured the optical absorbance properties of this
photoprotein that are highly sensitive to changes in protein conformation. Sophisticated studies using optical spectroscopy indicate
that rhodopsin cycles rapidly through a series of distinct conformational states following photon-induced activation. Many other
types of biophysical techniques have been applied to examine specific features of light-induced conformational changes of rhodopsin,
as well as to examine ligand-induced conformational changes of other GPCRs (35 ). Specific residues in the receptor protein can be
labeled with a chemical probe, typically using a combination of site-directed mutagenesis and organic chemistry techniques.
Spectroscopic methods can then be used to detect conformational changes involving the labeled residue, by measuring changes in
the local environment or mobility of the chemical probe. Approaches of this type have been applied to several GPCRs, and have
begun to yield interesting new information about the dynamic effects of clinically relevant drugs on GPCR structure (29 ).

Potential for Rational Drug Design

The availability of increasingly detailed mutational and biophysical data and the development of sophisticated molecular models
suggest that it may be possible in the future to design new classes of therapeutically useful drugs based on this information. A
precedent for such an approach is the structure-based design of the angiotensin-converting enzyme inhibitor captopril, the first drug
on the market that was designed based on its interactions with its target (36 ). Inferences about the structure of GPCR-ligand
interaction are currently used in a limited manner to guide the modification of existing drugs. However, an important goal is to
design completely new drugs de novo based on the structural basis of GPCR activation. A clue that this may be possible comes from
recent studies of mutant GPCRs, in which histidine residues have been introduced at defined positions in the receptor structure that
can be coordinated by certain metal ions. Addition of the metal ion to the receptor, by coordinating histidine residues introduced
within specific transmembrane helices, influences the receptor conformation to either activate or inactivate the receptor (37 ).
Thus the metal ion can serve either as an “engineered” agonist or antagonist for certain mutant receptors. While it is unlikely that
this strategy will directly yield clinically useful drugs, these exciting studies serve as a proof of the principle motivating further
studies of GPCR structure and conformational change.

REGULATION OF RECEPTOR SIGNALING

Part of "22 - Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors "

Methods to Examine Regulation of Receptors by Posttranslational Modification:

GPCR Phosphorylation
Many different types of posttranslational modification have been implicated in the regulation in of GPCR function, localization or
stability. A detailed discussion of this large area of research is beyond the scope of this chapter. Instead, we illustrate the use of
specific methods by discussing some aspects of protein phosphorylation, the most extensively characterized type of posttranslational
modification that regulates GPCRs.

Work by Edwin G. Krebs and his collaborators in the 1950s demonstrated that enzyme-catalyzed protein phosphorylation and
dephosphorylation reactions were involved in the regulation of glycogen phosphorylase and suggested the notion of the phosphate
group as a “covalently bound allosteric ligand” (38 ). Since these seminal studies, phosphorylation has been shown to play a critical
role in the
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regulation of a wide variety of cellular proteins, including many GPCRs. Phosphorylation of mammalian proteins typically occurs on
serine, threonine, or tyrosine residues. Serine/threonine phosphorylation is widely recognized to regulate GPCRs. Tyrosine
phosphorylation, a more recently discovered modification that is well established to mediate signaling via non-GPCR growth factor
receptors (39 ), may also play a role in regulating certain GPCRs (40 ).

A family of enzymes called G-protein–coupled receptor kinases (GRKs) are well known to attenuate GPCR signal transduction and
promote the endocytosis of certain GPCRs by clathrin-coated pits. Other kinases, such as the 3′,5′-cyclic adenosine monophosphate
(cAMP)-dependent protein kinase (PKA) and protein kinase C can also regulate GPCRs by phosphorylating distinct cytoplasmic
serine/threonine residues (41 ,42 ,43 and 44 ). Certain kinases (such as PKA) typically phosphorylate residues located within a well-
defined “consensus sequence,” making it possible to predict potential sites of regulatory phosphorylation simply by examination of
the primary structure (polypeptide sequence) of the cytoplasmic domains of the receptor. Residues phosphorylated by other kinases,
such as GRKs, are more difficult to predict because they do not conform to a rigidly defined consensus sequence. However, even in
the case of enzymes with relatively well-understood substrate specificity in vitro, there are major limitations to the use of sequence
analysis for predicting phosphorylation sites in vivo. Residues conforming to a specific consensus sequence are not always
phosphorylated under physiologic conditions, and, conversely, in some cases residues that do not conform to a well-defined
consensus sequence can be phosphorylated in the intact cell. Thus it is important to determine the phosphorylation of GPCRs when
expressed in the appropriate mammalian cells.

Analysis and Identification of Phosphorylated Proteins In Vivo

There are many ways of detecting phosphorylated proteins. A starting point for many of these methods is resolution of
phosphorylated proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). In SDS-PAGE, proteins
dissolved in SDS are loaded onto one end of a porous gel and exposed to an electric field, which causes the SDS-coated proteins to
move as “bands” in the gel according to differences in relative molecular mass. By using appropriate radiolabeled compounds (such
as inorganic phosphate added to the culture medium), it is possible to apply the technique of autoradiography to specifically detect
radioactive, phosphorylated proteins resolved by SDS-PAGE. It is also possible to use gel electrophoresis to separate proteins
according to relative charge, a property that is modified predictably by certain modifications such as phosphorylation. These types
of separation can be combined in the use of two-dimensional gel electrophoresis, which allows high resolution of proteins as “spots”
differing in relative size and charge.

Proteins resolved by gel electrophoresis can be transferred to a membrane composed of nitrocellulose or polyvinyl difluoride (PVDF).
This allows many manipulations to be performed, such as detection of a specific protein from a complex mixture by the ability of
the protein to be bound by a specific antibody. This procedure, called immunoblotting or “Western” blotting, can be used with
commercially available antibodies recognizing phosphorylated peptide sequences or phosphorylated amino acids.

GPCRs resolved by gel electrophoresis can also be analyzed by chemical sequencing, typically by a process called Edman degradation,
which sequentially cleaves residues from the amino-terminal end of the protein. Phosphorylated residues can be distinguished from
their nonphosphorylated counterparts by chromatography or by incorporation of radioactive phosphate, allowing the identification
of specific phosphorylated residues in a polypeptide sequence by the order of appearance in the eluate collected after multiple
cycles of Edman degradation. A very powerful method for determining amino acid sequence and detecting posttranslational
modifications of proteins is via mass spectrometric analysis. For example, with tandem mass spectrometry it is possible to measure
the mass of specific protein fragments with an accuracy of one part in 10,000 up to 12,000 daltons and one part in 1,000 up to 25 kd
(45 ). The impressive accuracy of this method makes it possible to detect phosphorylation as well as many other posttranslational
modifications, even those that cause subtle changes in the protein size or charge.

Chromatography, which refers to any separation based on differential behavior of a molecule between a stationary phase and a
moving phase, offers many ways of identifying protein modifications. High-performance liquid chromatography (HPLC) using reverse-
phase (e.g., C18) columns allows the precise separation of peptides derived from proteolytic or chemical fragmentation of GPCRs.
By comparing the pattern of peptide fragments derived from the native protein and the modified protein, one can identify specific
polypeptide fragments containing the modification of interest. Subsequently, these fragments can be isolated and further analyzed
by methods such as Edman degradation or mass spectrometry.

Methods to Examine Regulation of Receptors by Localization and Trafficking

It has been appreciated for many years that a critical parameter that can regulate the strength of functional signal transduction via
GPCRs is the actual number of receptors present in target tissues and, in particular, the number of receptors present in the plasma
membrane of individual cells. Indeed, disturbances in the regulation of receptor number and/or distribution may be of primary
importance in the pathophysiology
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of certain neuropsychiatric disorders. For example, long-term administration of dopamine receptor antagonists can induce
upregulation of specific receptors, which may contribute to the apparent supersensitivity of dopamine receptors associated with
tardive dyskinesia (46 ). In contrast, prolonged stimulation of certain GPCRs with agonist ligands can lead to a decrease in the
number of binding sites available on the cell surface. This phenomenon is termed receptor “down-regulation” and may contribute to
the effects of certain antidepressant drugs (47 ). Studies using radioligand binding and subcellular fractionation techniques provided
early evidence that multiple mechanisms are capable of mediating changes in the number of GPCRs present at the cell surface (48 ).
More recently developed molecular and cell biological approaches provide powerful tools for directly visualizing the subcellular
localization of GPCRs and for performing biochemical studies of specific receptor trafficking mechanisms.

Immunochemical Methods to Visualize the Subcellular Localization of Receptors

GPCRs can be detected in situ in cell or tissue preparations using immunochemical techniques and receptor-specific antibodies.
Antibodies that recognize the native receptor protein can be used to examine the localization of endogenously expressed receptors,
whereas epitope-tagging methods (see above) can be used to detect mutated versions of the receptor protein or as a means to
detect recombinant receptors for which antibodies recognizing the native receptor are not available. In either case the general
scheme is as follows: Cells or tissues expressing the receptor of interest are fixed using standard histologic methods. The fixed cells
or tissue can be “permeabilized” with a nonionic detergent, to facilitate biochemical access to receptors situated in intracellular
membranes, and then specimens are incubated with antibodies recognizing the receptor of interest. After sufficient time has
elapsed to allow antibodies to bind to their respective epitopes in the specimen (typically several hours), the specimens are washed
extensively to remove nonspecifically associated antibodies. Antibodies bound to the receptor are then detected by incubation of
specimens with a “secondary” antibody that binds specifically to the “primary” antibody (the antibody bound initially to the
receptor). The secondary antibody is typically coupled to a fluorochrome (such as fluorescein), a recognizable particle (such as
colloidal gold), or an enzyme that can produce a localized reaction product (such as horseradish peroxidase) to facilitate direct
visualization of the receptor-containing immune complex using various light or electron microscopic techniques (Fig. 22.2 ).

FIGURE 22.2. Visualization of HA epitope-tagged dopamine D1 receptors in transfected cells, using a fluorochrome-labeled
secondary antibody and fluorescence microscopy. The ability of this receptor to undergo regulated internalization is indicated
by the dopamine-induced redistribution of immunoreactive receptors from the plasma membrane (visualized as linear staining
at the cell periphery) to endocytic vesicles (visualized as punctate structures located throughout the cytoplasm). A: Untreated
cells (no ligand). B: Treated with 10 μM dopamine. (Photograph courtesy of Gabriel Vargas.)

Biochemical Methods to Assay Specific Receptor Trafficking Processes

Whereas microscopic imaging can readily provide a great deal of qualitative information about GPCR localization and trafficking, it
can be quite challenging to quantitiate
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from these data the precise amount of receptor present in a specific subcellular localization or to measure accurately the rate or
extent of specific trafficking processes. The importance of these processes has motivated the development of biochemical methods
for examining GPCR trafficking. In addition to their utility for receptor localization, antibodies specifically recognizing GPCRs
facilitate biochemical studies of GPCR trafficking using techniques adapted from other areas of cell and molecular biology. For
example, one method that has been extremely useful for quantitative studies of GPCR endocytosis is cell-surface biotinylation
coupled with immunoprecipitation of receptors. Proteins present in the plasma membrane of cells can be specifically labeled by
incubating intact cells in the presence of biotin coupled to an activated ester, which is membrane-impermeant and therefore forms
a covalent bond only with exposed amine moieties present in plasma membrane proteins. In general, biotinylation in this manner
does not adversely affect GPCR function, allowing biotinylation to be used as a chemical tag for surface receptors. The biotin moiety
is extremely useful for subsequent detection or purification of surface-tagged proteins because it binds with extremely high affinity
(Ka ˜1015 M-1) to the proteins avidin or strepatavidin. Using variations of this basic biochemistry, it is possible to measure a wide
variety of membrane trafficking processes. For example, internalization of GPCRs has been measured by the inaccessibility of
biotinylated receptors to a membrane-impermeant reducing agent that “cleaves” the biotin moiety away from tagged proteins (49 ),
and surface biotinylation has been used to measure the rate and extent of proteolytic degradation of receptors after endocytosis
(50 ,51 ).

Methods for Examining Specific Protein Interactions Involved in GPCR Function and
Regulation
A salient lesson emerging from recent cell biological studies is that GPCR signal transduction can be viewed, in essence, as a
dynamically regulated network of protein–protein interactions that occur in specific subcellular locations. Therefore, an important
goal of current and future research is to define these critical protein interactions and elucidate their temporal and spatial regulation
in intact cells and tissues. A great deal of effort is presently going into developing and applying novel methods for the study of
protein–protein interactions both in vitro and in vivo, as illustrated by the following examples.

Coimmunoprecipitation Techniques to Examine Defined Protein Interactions with

GPCRs in Intact Cells
As discussed above, it is possible to rapidly purify GPCRs from cell or tissue extracts using receptor-specific antibodies or epitope
tagging methods. In addition to being extremely useful for examining posttranslational modifications of GPCRs, in some cases it is
possible to use these techniques to isolate receptor-containing complexes that presumably reflect protein interactions occurring in
the intact cell. The basic idea is to immunopurify a specific GPCR from cell or tissue extracts (or from a partially purified subcellular
fraction prepared from a cell or tissue lysate) using an antibody recognizing the native receptor or an engineered epitope tag, and
then to analyze proteins specifically associated with this complex using a different antibody. In general, this is accomplished by
immunoprecipitation of the receptor followed by analysis of associated proteins in the complex by immunoblotting with the
appropriate additional antibody. In some cases, the protein complexes are sufficiently stable that they remain associated through
the initial immunopurification of the receptor. In other cases this is not true, and the complexes dissociate before the receptor can
be purified from the extract. In this case, various chemical agents can be added prior to cell lysis to physically “cross-link” closely
associated proteins with one another by forming covalent bonds that prevent dissociation of the complex. Coimmunoprecipitation
has been used to assay GPCR interaction with heterotrimeric G proteins (52 ) and with β-arrestins (53 ), and to examine the
regulation of these protein interactions by ligand-induced activation of the receptor.

Use of Coimmunoprecipitation to Examine Oligomerization of GPCRs

The idea that GPCRs may function in vivo as higher-order molecular complexes has been suspected for many years. Recent studies
provide strong support for this idea and, specifically, provide evidence for homo- and heterodimerization of individual GPCRs in vivo.
This principle is perhaps best established for receptor tyrosine kinases, where it is well established that oligomerization of receptors
is required for appropriate ligand-dependent signal transduction (54 ). A relatively early hint that GPCRs may also undergo
oligomerization came from studies of the β2-adrenergic receptor using epitope-tagging techniques, where it was observed that
receptors tagged with one epitope could specifically coimmunoprecipitate receptors tagged with a distinct epitope (55 ). Early
evidence suggesting a functional role of oligomerization in GPCR signaling came from mutational studies in which structural domains
present in distinct, functionally inactive mutant receptors could “complement” one another when coexpressed in cells, suggesting
the formation of a functional “hybrid” oligomeric receptor protein (56 ). More recently, evidence for oligomerization of many GPCRs
has been reported. A particularly compelling example of this is the recent observation that distinct subtypes of GABA-B receptor
hetero-oligomerize in cells, and that oligomerization is essential for the formation of recombinant receptors possessing the
functional properties characteristic
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of native GABA-B receptors observed in vivo (57 ,58 ). Recent studies using epitope-tagging and coimmunoprecipitation have
demonstrated the formation of homo- and heterodimers of opioid receptors, and suggest that receptor oligomerization may
contribute to the remarkable diversity of pharmacologic properties observed in natively expressed opioid receptors (59 ). There is
also emerging evidence that certain GPCRs may associate in vivo with completely different classes of receptor protein, such as the
dopamine D5 receptor (a GPCR) and the GABA-A receptor (a ligand-gated ion channel). In a recently published study (60 ),
glutathione S-transferase (GST)-fusion proteins encoding the C-terminal tail of the D5 receptor were shown to interact with the
GABA-A receptor present in rat hippocampal extracts. Additionally, using an antibody recognizing the dopamine D5 receptor, it was
possible to coimmunoprecipitate the GABA-A receptor from cell extracts. Interestingly, this coimmunoprecipitation was detected
only when both receptors were stimulated by their respective ligands, suggesting that this heterotypic interaction is regulated in a
ligand-dependent manner.

Identification of Novel Protein Interactions with GPCRs

In addition to known proteins that mediate and regulate GPCR signaling (heterotrimeric G proteins, GRKs, arrestins), which were
originally identified by functional assays using biochemical purification, cDNA cloning methods have facilitated the identification of
additional protein interactions with GPCRs that were completely unanticipated (61 ). These novel protein interactions, while their
functional relevance remains unclear in many cases, are of great interest and potential therapeutic importance as drug targets.

Of the many techniques for identifying novel protein–protein interactions developed over the last 10 years, interaction cloning
methods such as the yeast two-hybrid system (62 ) have been particularly useful for studies of GPCRs. In the yeast two-hybrid
system, protein interactions are detected by their ability to reconstitute the activity of a “split” transcriptional activator complex.
A transcription factor such as GAL4 can be divided into two domains: a DNA binding domain and a transcriptional activation domain.
For the transcription factor to be active, these two domains must be in close proximity to one another, so that the DNA binding
domain can bind the promoter sequence in a “reporter” gene and the activation domain can promote gene transcription. A
polypeptide sequence for which one wishes to identify putative interacting proteins (such as a sequence derived from a cytoplasmic
domain of a GPCR) is cloned into a vector coding for the isolated DNA binding domain from the GAL4 transcription factor, thereby
producing a fusion protein containing the GPCR-derived sequence as “bait” with which to search for potential protein interactions. A
cDNA library prepared from a tissue of interest is cloned into a cDNA encoding an isolated transcriptional activation domain,
producing a large number of fusion proteins containing tissue-derived polypeptide sequences as potential “prey” for protein
interaction with the GPCR-derived fusion protein. Both the bait and prey plasmids are transformed into a strain of yeast harboring a
“reporter” gene that can be transcribed only in the presence of an “intact” GAL4 transcription factor. Either “half” of the
transcription factor is not sufficient to promote efficient transcription of the reporter gene. However, if the fused bait and prey
polypeptides form a sufficiently stable protein–protein interaction, they bring their corresponding DNA binding and transcriptional
activation domains into close proximity, thus reconstituting transcriptional activation of the reporter gene. Transformed yeast cells
containing plasmids encoding the corresponding interacting protein domains can be identified by screening techniques based on
GAL4-dependent transcription of reporter genes conferring antibiotic resistance or other selectable metabolic activities, or encoding
enzymes that can be detected using colorimetric assays. Assays using the E. coli–derived lacZ gene, for example, can be used to
screen for a characteristic blue reaction product when exposed to 5-bromo-4-chloro-3-indolyl α-D-galactoside.

Protein interactions suggested to occur by the yeast two-hybrid system can be examined using various in vitro biochemical
techniques, such as affinity chromatography facilitated by GST-fusion proteins. In addition to serving as an independent assay for
previously defined candidate interacting proteins, this method can be used to identify novel protein interactions with GPCRs de novo
(63 ). In this method a DNA encoding a polypeptide sequence of interest is fused to GST using standard cDNA cloning techniques and
expressed as a recombinant protein in E. coli. The GST portion of the fusion protein allows the efficient immobilization of the
protein by binding to agarose beads covalently derivatized with glutathione. Proteins from a cell or tissue extract that bind to the
fusion protein then can be isolated as an immobilized protein complex by affinity chromatography. As an example of the use of
these methods, it was shown recently (64 ) that the third cytoplasmic loop of the dopamine D2 receptor binds specifically to
spinophilin, a large cytoskeleton-associated protein that also binds to protein phosphatase-1. This binding was initially identified
through use of the yeast two-hybrid system, and then the identification of the specific domains that mediate this protein interaction
was accomplished by affinity chromatography using GST-fusion proteins.

Methods for Examining Candidate Protein Interactions in Intact Cells

A major question regarding novel protein interactions with GPCRs, such as those identified using interaction cloning
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or protein affinity chromatography, is whether or not they actually occur in an intact cell. Immunocytochemical techniques can
provide some insight into this question by determining whether candidate interacting proteins “colocalize” in cells with the
appropriate GPCR, as expected if the proteins physically interact in vivo. However, even in the event that extensive colocalization is
observed, immunocytochemical techniques of this sort do not provide direct evidence for a physical interaction between candidate
proteins. Coimmunoprecipitation techniques, as discussed above, provide a useful method for addressing this question. However,
demonstrating that a specific protein association can occur in vivo is only the first step in the process of assessing the potential
physiologic relevance of a novel protein interaction, as this method generally does not provide any information regarding the
possible functional activity of a candidate protein interaction. Addressing this question can be a challenging task that involves
creative application of diverse techniques and functional assays. Examples of novel protein interactions with GPCRs for which
compelling functional data exist include the aforementioned interaction of the D2 dopamine receptor with ABP280 (65 ) and
interaction of the β2-adrenergic receptor with NHERF/EBP50-family proteins (51 ,63 ).

EMERGING HORIZONS
Part of "22 - Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors "

Unexpected Signaling, Cross-Talk, and Transactivation Involving GPCRs (Fig. 22.3 )

FIGURE 22.3. Schematic diagram of G-protein–coupled receptor (GPCR) signaling. A: The G-protein paradigm. Following
agonist binding, GPCRs activate heterotrimeric G proteins (G), which then regulate the activity of specific cellular effectors. B:
Beyond the G-protein paradigm. Following agonist binding, GPCRs can associate with members of diverse families of
intracellular proteins, including heterotrimeric G proteins (G), polyproline-binding proteins such as those containing SH3
domains (SH3), arrestins (Arr), G-protein–coupled receptor kinases (GRK), small guanosine triphosphate (GTP)-binding proteins
(g), SH2 domain–containing proteins (SH2), and PDZ domain–containing proteins (PDZ). These interactions allow GPCRs to
initiate multiple intracellular signaling pathways, with each subtype of receptor likely coupled to a relatively unique set of
effectors. (From Hall RA, Premont RT, Lefkowitz RJ. Heptahelical receptor signaling: beyond the G protein paradigm. J Cell
Biol 1999;145:927–932, with permission.) See color version of figure.

Another line of evidence suggesting the existence of functionally relevant, novel protein interactions involving GPCRs comes from
recent work by several labs suggesting that unanticipated functional interactions can occur between GPCRs and receptor tyrosine
kinases (RTKs), a distinct family of single-transmembrane receptors involved in growth, differentiation, and oncogenesis (66 ). The
RTK family includes the epidermal growth factor receptor (EGFR), the first receptor shown to have intrinsic tyrosine kinase activity
(67 ,68 ). Whereas the classic pathway for RTK-mediated signaling is initiated by binding of polypeptide growth factors (such as EGF)
to the extracellular domain of the RTK, it has been observed recently that certain GPCRs can initiate signaling cascades traditionally
thought to be controlled by RTKs. In this situation, the primary signal appears to be through the GPCR, which in turn
“transactivates” the RTK. For example, several GPCRs can mediate transactivation of coexpressed EGFRs, thus stimulating
mitogenesis by a similar downstream pathway as that initiated by binding of EGF directly to the EGFR (69 ). One mechanism of
GPCR-mediated transactivation involves the activation of a membrane-associated metalloproteinase, which cleaves the EGF
precursor protein to generate increased amounts of ligand for the EGFR (70 ). Another mechanism of cross-talk involves the
formation of heteromeric signaling complexes, which include components of both “classical” GPCR and RTK signaling cascades. For
example, recent studies suggest that the nonreceptor tyrosine kinase c-Src can associate with the β2-adrenergic receptor and the β-
arrestin in endocytic membranes, thus mediating mitogenic kinase activation either by a c-Src-mediated phosphorylation of
downstream effectors (71 ) or by c-Src-mediated phosphorylation of co-endocytosed EGFR (72 ).

Visualization of Protein Localization and Interaction in Living Cells

As discussed above, immunochemical methods are useful for examining the localization of proteins in intact cells.
 P.287

However, these methods are typically applied to fixed cells because they require disruption of the cell membrane and prolonged
incubation of specimens with antibodies used to detect the receptor of interest. The discovery of proteins from certain marine
animals that have high levels of intrinsic fluorescence has fostered a revolution in the ability to localize proteins in living cells.
These proteins, such as the green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria, are brightly fluorescent
molecules that can fold properly in many environments and do not require any additional chromophore for their fluorescence
(73 ,74 ). This allows them to be used to “tag” GPCRs and other important signaling proteins in intact cells. This is accomplished by
using site-directed mutagenesis to create a fusion between the GPCR polypeptide sequence and the sequence encoding the GFP tag,
analogous to the introduction of an antigenic epitope tag. The localization of the fusion protein can be examined in intact cells
using fluorescence microscopy. Examples of this methodology include the visualization of ligand-induced endocytosis of a GFP-
tagged β2-adrenergic receptor in living cells and visualizing the dynamic recruitment of GFP-tagged β-arrestin from the cytoplasm to
the plasma membrane induced by activation of various GPCRs (75 ,76 ).

While GFP has facilitated the localization of proteins in living cells, localization by itself does not necessarily indicate the
occurrence of a physical interaction of a GPCR with a specific protein. The development of mutant versions of GFP, which differ in
their excitation and emission spectra, has made it feasible to examine in vivo protein interactions using the process of fluorescence
resonance energy transfer (FRET) (77 ). FRET occurs when two suitable fluorophores are present in extremely close proximity so that
light produced from one fluorophore can be “transferred” efficiently into exciting the other. FRET can be detected in living cells
using sophisticated microscopy, making it possible in principle to detect specific protein interactions with GPCRs and study their
localization in real time. FRET imaging has not yet been used extensively for GPCR research but holds great promise for future study
of the spatial and temporal dynamics of protein interactions with GPCRs in intact cells and tissues.

SUMMARY AND CONCLUSIONS

Part of "22 - Beyond Binding: Molecular and Cell Biological Approaches to Studying G-Protein–Coupled Receptors "

We have discussed a subset of experimental approaches that have provided powerful new tools for studying GPCR function and
regulation. These approaches are responsible, in large part, for the vast explosion of new information about specific mechanisms of
GPCR biology that has emerged over the past several years. In many cases these developments have extended directly from seminal
observations made originally through classic pharmacologic approaches, which remain of central importance to understanding GPCR
function and regulation. Indeed, we view newer molecular and cell biological approaches as complementing, rather than replacing,
the sophisticated pharmacologic methods that have been developed over the years since the discovery of receptors as important
drug targets.

Molecular cloning techniques have allowed the isolation of cDNAs encoding many G-protein–coupled receptors. The isolation of
receptor cDNAs has provided insight into the remarkable structural homology among GPCRs, revealed an unanticipated level of
molecular diversity in the GPCR superfamily, allowed functional characterization of defined receptor subtypes in heterologous
systems, and made it practical to produce large amounts of receptor protein for pharmacologic, biochemical and biophysical studies.

Structural, biophysical, and molecular modeling approaches hold great promise for ultimately defining the precise atomic
determinants of receptor-ligand interaction and for understanding protein conformational changes involved in receptor activation
and regulation. Continued progress in this important area may lead to entirely new concepts and methods relevant to therapeutic
drug design. Site-directed mutagenesis techniques complement structural and biophysical approaches and have enabled, in the
absence of precise structural information, the empirical identification of residues and receptor domains important for ligand binding
and activation. Cell biological methods have elucidated mechanisms of signal transduction and regulation in impressive detail, and
have revealed a previously unanticipated level of specificity and complexity of crosstalk between signal transduction systems.
Emerging technologies for detecting protein interactions in intact cells are suggesting new insights into cell biological mechanisms of
GPCR function and regulation, and are beginning to allow real-time examination of the temporal and spatial dynamics of defined
protein interactions in living cells.

Based on the new methodologies available today and current pace of progress in using these methods for elucidating GPCR function
and regulation, we anticipate that the next several years will see even greater progress in our understanding of the fundamental
biology of GPCRs. Indeed, the field of GPCR research is rapidly moving away from a focus on any one set of experimental techniques
and has become a vanguard area of integrative structural, molecular, and cell biology. Further developments of these experimental
methods, combined with new in vivo imaging and genomics approaches that have appeared on the horizon, are likely to fuel
continued rapid progress in the field. This exciting progress is fundamentally and directly relevant to the main mission of
neuropsychopharmacology: to develop and provide effective therapies for the complex neuropsychiatric disorders that affect our
patients.

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23
Applying Functional Genomics to Neuropsychopharmacology

Michael Brownstein

M. Brownstein: National Institutes of Health, National Institute of Mental Health, Bethesda, Maryland.

“The time has come,” the Walrus said, “to talk of many things.”

--Lewis Carroll

The time has come indeed. The sequencing of the human genome and the genomes of a number of other species subject to research
(1 ,2 ,3 ,4 ,5 and 6 ) have paved the way for new sorts of studies. Soon researchers will be able to look at the response of every
human gene to specific manipulations or developmental events at multiple time points. This will require a new mindset. Researchers
will not necessarily be testing specific hypotheses as they have done in the past. Instead, they will rely on the emergence of
patterns and systematic features in their data sets (and those of others) to describe the phenomena being examined. Such patterns
may hint at functions of collections of genes, the interactions of their products, and their importance in physiologic and pathologic
processes. This chapter introduces array technology, discusses the sorts of experiments that can now be done with it, and suggests
future advances. Several reviews have already been published on this subject, and the reader should refer to them for additional
information (7 ,8 ,9 ,10 ,11 ,12 ,13 ,14 and 15 ). In addition, university, government, and commercial Web sites are valuable
sources of news, background material, reagents, arrays, software, and instrumentation
(16 ,17 ,18 ,19 ,20 ,21 ,22 ,23 ,24 ,25 ,26 ,27 ,28 ,29 ,30 ,31 ,32 and 33 ).

 EARLY STUDIES OF GENE EXPRESSION

 GENE EXPRESSION ARRAYS
 THE DEVIL IS IN THE DETAILS
 NORMALIZING RATIOS
 QUALITY CONTROL
 SMALL SAMPLES, FALSE NEGATIVES, AND FALSE POSITIVES
 BIOINFORMATICS
 INTERPRETING EXPERIMENTS
 ANNOTATING EXPERIMENTS

EARLY STUDIES OF GENE EXPRESSION

Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

The human genome is composed of approximately 3 billion DNA nucleotides encoding more than 100,000 genes (16 ). Each of these
genes must be turned on or off in the right cells at the right time for an individual to develop and prosper. The genes that are
ultimately expressed in a particular tissue define it. That is, brain is brain and liver is liver because of the particular collections of
transcripts found in their respective cells. Brain, however, is extraordinarily heterogeneous. It has been estimated that nearly half
of the genes in the genome are expressed there, distributed among the different neuronal and glial populations.

Genes are made of DNA, a nucleic acid polymer that has deoxyribose as its sugar backbone. Each sugar moiety in the chain has a
base (adenine, A; cytosine, C; guanine, G; or thymine, T) attached to it. DNA exists as a double-stranded helix. The two antiparallel
strands are bound to one another because their sequences are complementary—that is, the opposing bases are held together by
hydrogen bonds, A to T and C to G. Similarly, messenger RNA (mRNA), the transcription product of the coding region of each gene, is
complementary to the DNA strand from which it was copied and can bind to it. Northern blotting, the first method developed for
detecting single mRNA species in a cellular extract, is based on this phenomenon. In this technique, RNA samples are fractionated by
agarose gel electrophoresis, and the RNA bands are transferred (blotted) onto nitrocellulose membranes. Single RNA species can
then be detected by hybridizing a radiolabeled DNA to the blot that is complementary to the RNA of interest.

In the past, the responses of cells or organisms to environmental cues were studied on a small scale, one gene or pathway at a time.
Initially, Northern blotting was used to examine the abundance of specific mRNA species. Subsequently, other methods were chosen
because they were simpler and more sensitive, such as reverse-transcriptase polymerase chain reaction (RT-PCR) (34 ), or because
they were more comprehensive, such as SAGE (serial analysis of gene expression) (35 ,36 ). These techniques provide useful
information, but they are tedious, time-consuming, and expensive to employ.

In the last 5 years, spurred by the availability of large volumes of genomic and cDNA (EST [expressed sequence tag]) sequence data
from a variety of organisms, investigators have developed methods to study mRNA profiles in cells and tissues by means of large-
scale, high-throughput, parallel methods. In the future, it would be helpful to look at protein and small molecule profiles as well,
but the reagents required (panels of antibodies, for example) are difficult to
 P.292

assemble and utilize. Even though there is not a one-to-one correspondence between the level of a particular transcript in a cell and
that of its translation products, a great deal can be learned by performing mRNA expression profiling.

GENE EXPRESSION ARRAYS

Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

Expression profiling relies on large ordered collections of cDNAs immobilized on glass (microarrays) or synthetic oligonucleotides
immobilized on silica wafers or chips (probe arrays). Both of these methods are the conceptual descendants of target nucleic acids
immobilized on filters or membranes and detected with complementary radioactive probes. While filter-based systems are
commercially available, reasonably priced, and fairly easy to use, it is clear that they will be preempted by glass or chip arrays
developed with fluorescent probes. Glass arrays printed on microscope slides are now much cheaper to employ than chips, and many
universities and research institutes have already built facilities for printing and probing such arrays. So it is worth discussing the uses
to which such arrays have already been put, and the uses to which neuropsychopharmacologists could put them.

Few investigators have used arrays to study brain so far, preferring instead to look at mammalian cell lines and tumors
(37 ,38 ,39 ,40 ,41 ,42 ,43 and 44 ). In addition, many workers have focused on yeast (45 ,46 ) or prokaryotes (47 ,48 ) because their
genomes are small and have been completely sequenced. Consequently, every protein-encoding gene can be arrayed and examined.
This will be true of human and mouse arrays in the not-too-distant future. Meanwhile, experiments can be done with the arrays that
are available. These have between a few thousand and a few tens of thousands of elements, and with them we can begin to
catalogue genes expressed in regions of the developing and adult nervous system, and to look for alterations in expression patterns
associated with pathologic states or physiologic/pharmacologic manipulations (49 ). Consider, for example, the work that could be
done to understand the mechanism(s) of action of selective serotonin reuptake inhibitors (SSRIs) and the reason for their delayed
onset of action in depressed patients. As is known, SSRIs increase the availability of serotonin (5-hydroxytryptamine, 5-HT) to
presynaptic and postsynaptic receptors, of which there are at least 14 subtypes (50 ). Among these, 5-HT1A receptors on
serotoninergic raphe neurons are thought to play a key role in regulation 5-HT release (51 ). 5-HT1A agonists, which are used to treat
anxiety, inhibit serotonin secretion. Conversely, desensitization of 5-HT1A receptors, which could result from elevated 5-HT levels in
the synaptic space following SSRI administration, may have the opposite effect—an increase in 5-HT release by raphe neurons, and
chronic stimulation of 5-HT receptors in regions such as the hippocampus, amygdala, and septum. Despite all the research that has
been done to date, the identity of the structures and biochemical alterations that are responsible for the antidepressant actions of
SSRIs is still moot.

Array experiments will allow investigators to explore the serotoninergic system in a way that is model independent and
comprehensive, and the experiments should become easy and cheap enough to perform to permit varying many parameters and
comparing many conditions.

Initially, regional responses to a single dose of SSRI at a variety of times in one mouse strain might be examined. Subsequently,
mouse strains that differ in their behavioral reactions to SSRIs could be examined; knockout mice known to have altered responses
to SSRIs (e.g., 5-HT1A receptor knockouts) could be studied; and drugs that resemble, facilitate, or inhibit the behavioral effects of
SSRIs could be investigated. Mice would be better to use for this work than rats as of now because very big mouse arrays are
available as are genetically manipulated animals and a variety of well-characterized inbred strains. Unfortunately, mice have small
brains, and obtaining samples of minute regions (e.g., raphe nuclei) large enough to make sufficient RNA for labeling is difficult.
Help is on the way, though. Better labeling methods, dyes, and detection devices are being developed. In fact, the amount of total
RNA needed for an array experiment has already fallen well below 1 μg, and should approach 1 ng shortly.

Each array experiment will let an investigator look simultaneously at thousands of transcripts including those encoding enzymes
involved in energy metabolism, receptors, G proteins, second messengers, and ion channels, to name a few. In addition, there will
be many species represented on big arrays, the actions of which are unknown. The major task will be to assign them functions (see
below).

THE DEVIL IS IN THE DETAILS

Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

Methods for making and probing arrays and analyzing array data have developed quickly. In spite of this, the supply of arrays has not
kept up with demand, and demand should increase dramatically if the goal of using arrays is to compare many conditions and then
mine the data systematically for patterns of gene expression. Thus, as stated earlier, costly products are unlikely to gain wide
acceptance, and glass slide arrays are likely to be most commonly employed. For this reason, I now discuss their production and use.

Large collections of cDNAs and their sequences are now in the public domain. Some sets of cDNAs have been sequence verified and
are ideal to use for preparing arrays; others have not been validated and are less useful. To make arrays, plasmid DNA is prepared
from gridded sets of clones to be printed, and (typically) the 3′ end of each cDNA is amplified by PCR. The purified PCR products are
then spotted using a robotic arrayer. It is possible to fabricate one's own arrayer (18 ), but many investigators will prefer
 P.293

to buy an instrument or obtain arrays from core facilities or commercial vendors. Many thousands of 100-μM spots can be printed on a single glass microscope slide (see ref. 10 for
details).

It is important to realize that array experiments do not permit measurement of the amount of each RNA that is present in a sample. This is because the relationship between the
amount of transcript in a mixture and the intensity of the fluorescent spot it produces is a complex one—influenced by labeling efficiency, hybridization and wash conditions, and the
sequence, quality, and quantity of the printed DNA. Thus, microarrays are typically employed to measure the relative abundances of RNA species in two or more extracts. To achieve
this goal, in a two-sample experiment the RNAs are separately labeled with dyes of different colors, and then the products are mixed and hybridized to the arrayed spots (Fig. 23.1 ).
After washing, the slides are scanned with a “reader” or “scanner” and the intensities of the fluorescent signals produced by the two separate dyes are determined spot by spot.
Following background subtraction and “normalization” of the signals from the two channels (see below), a ratio of intensities of the two colors is determined for each spot, and the
relative abundance of the two input RNAs can then be estimated. Finally, “clustering” methods are used to sort and display the data.

FIGURE 23.1. To perform microarray experiments, RNA is purified from two or more samples of cultured cells or
dissected tissues. These RNAs are used to produce labeled probes. In the example given, the dye cy5, which
fluoresces red, was used to label probe from sample 1; and the dye cy3, which fluoresces green, was used to label
probe from sample 2. The labeled products are mixed and hybridized to the spots on the microarray. Following a
wash step, the array is scanned and the signals from the red and green channels are superimposed. If an RNA species
is more abundant in sample 1 than 2, the resulting spot will be red; in the reverse case, the spot will be green.
When the RNA is equally abundant in the two samples, the spot is yellow. See color version of figure.

Two sorts of experimental paradigms have been defined: type I and type II (42 ). In the former, two samples are compared to one another; in the latter, multiple samples are
compared. To look at multiple samples (e.g., time points, drug doses, developmental ages, brain regions, autopsy specimens), each sample in the set could theoretically be labeled
with a different fluorescent dye that could, in turn, be visualized with a different laser. Presently, most commercial readers have only two lasers, but four-color instruments have
already appeared on the market. (The number of dyes that it is possible to use for labeling RNA samples is dictated by a reader's ability to resolve the signal from individual dyes and
the strength of the signal each dye produces.)

To use a two-color scanner for multiple comparisons, discrete samples must be compared to a reference standard. The ideal standard would have modest amounts of each transcript
represented on the array used, because it needs to generate a nonzero denominator for the hybridization ratio. In the case of the mouse, the 17-day embryo and/or the adult brain
have been proposed as sources of standard RNA. Pools of cell-line RNAs have been used as standards for human work. It would be useful if a central source of standards existed and if
huge batches were prepared.

NORMALIZING RATIOS
Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

Since it is difficult, if not impossible, to measure the amount of RNA used to produce a labeled probe, normalizing the signals from the source RNAs is essential. To do this, a set of
“housekeeping genes” is chosen because their transcription is fairly constant across a range of conditions. The ratio of signals from these genes is set to 1. The housekeeping set needs
to be defined empirically, and in looking for candidates to include in such a set, few genes have been found that have constant expression levels. When large arrays are used, this is
not a problem; hundreds of genes (or the entire set of genes) can be used for “global normalization” (Fig. 23.2 ). When small arrays are employed, on the other hand, the size and
composition of the gene set used for normalization are very important. Just as a reference standard is urgently needed now, a normalization set supplied by a central site would be
quite valuable.

FIGURE 23.2. Normalization. Most genes, especially those with “housekeeping” functions, do not change very much from one experimental condition to another. For this
reason, housekeeping genes, or the entire collection of genes on a large array, can be used to set the ratio of signals from the two color channels to 1 as shown here. See color
version of figure.

QUALITY CONTROL
Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

While we have methods to assess the quality of DNA sequence data, for example, there is no generally accepted method for establishing the quality of an array study. In spite of this,
there are some controls that can be built into an array. As noted earlier, scientists are arraying DNAs generated by PCR from plasmid templates. It is highly desirable to use sequence-
verified cDNA sets. Amplifying these with
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specific primers would confirm the identity of each clone, but this is expensive. Assuming we can obtain a reasonably well-validated
clone set, the cheapest way to produce the 1.5- to 2.0-kilobase (kb) DNAs for printing is to use vector primers, and to analyze the
products on agarose gels. The resulting cDNAs will include T-tails of varying lengths, and repeat sequences. Hybridization of labeled
probe to these sequences is prevented by addition of a blocking solution containing oligo (dA) 20-mers, yeast transfer RNA (tRNA),
and (for human probes) Cot-1 DNA to the probe solution. To show that the blocking was successful, a number of negative controls
should be included among the samples arrayed: spotting buffer, Cot-1 and human genomic DNA, plasmid DNA, oligo (dT), and oligo
(dA).

It is useful to array targets for nonmammalian transcripts and to “spike” the samples with the corresponding polyA-tailed RNAs.
These RNAs can be added in different concentrations to crude tissue extracts, total RNA samples, or purified polyA-plus RNA to
determine extraction efficiencies and detection sensitivities. It would be a mistake to imagine that the added RNA standards can be
used to generate figures for absolute amounts of RNA in samples for the reasons given earlier. They should be used exclusively for
quality control.

An additional set of spots that have been found useful to array are “landing lights.” These DNAs, which are printed at regular
intervals, are used for orientation.

SMALL SAMPLES, FALSE NEGATIVES, AND FALSE POSITIVES

Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

Failure to detect transcripts or changes in transcripts could result from low-quality arrays or poor labeling methods. Over time, the
methods used to make and probe arrays should improve, and false negatives will grow less important. Presently, we can detect RNAs
with an abundance of about 1:300,000 in a complex sample. This translates into a few copies per cell if one is studying a
homogeneous cell line. Seeing increases in rare transcripts under these circumstances should be simple, but measuring decreases
will difficult if not impossible when one can barely detect a weak signal in the first place. Since brain samples are much more
heterogeneous than cell lines, the problem of detecting rare mRNAs is even harder. For this reason, it may be necessary to isolate
neuronal populations from brain sections by microdissection or to collect single neurons by laser capture methods to enrich and
study rare, cell-specific transcripts. To take full advantage of these dissection techniques, methods will have to be developed for
isolating and labeling picogram quantities of RNA. (One million cells yield about 5 to 10 μg of total RNA.) It is important to note that
labeling methods have to preserve the heterogeneity and relative abundances of the RNAs in the samples to be studied. Care must
be taken if PCR is used in the labeling procedure to avoid biasing the sample. Novel labeling methods can be tested using arrays and
serially diluted RNA templates.

At present investigators use Northern blotting, the TaqMan system, or in situ hybridization histochemistry to weed out false-positive
responses of selected mRNAs. When arrays are no longer limiting, this could be accomplished by studying replicate samples, but the
argument could be made that one should focus on variations in collections of genes instead of single ones, and that looking at many
conditions once may be more powerful than looking at the same condition many times.

BIOINFORMATICS
Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

Analyzing the earliest, small-scale, array experiments was simply a matter of listing the names of transcripts that appeared to
increase or decrease from control levels (Fig. 23.3 ). As the sizes of arrays increased and labeling methods improved, new algorithms
had to be developed that “clustered” the hundreds or even thousands of expression changes found in a typical experiment.
Clustering methods permit the classification of genes on the basis of similarities or differences in their patterns of expression across
multiple experiments (52 ,53 ). The output is usually in tabular form. Experimental conditions are listed across the top of a table,
and names of genes listed along the side. The response of each gene in each experimental condition is color coded—one color (red)
indicating an increase and another (green) a decrease vs. a standard signal (Fig. 23.4 ). The eye can readily detect patterns in
complex images of the sort described, and groups of genes can be identified that parallel one another. Commonly, such genes
function in concert.
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In the hypothetical SSRI study described above, one group of genes may be increased or decreased in the raphe nuclei following
chronic, but not acute, treatment with drug, and a different collection of genes is altered in areas innervated by raphe neurons such
as the hippocampus. Different sets of genes might respond to SSRI treatment when behaviorally responsive mouse strains are
compared to unresponsive ones, and the pattern of gene expression is different in knockout animals that do not respond to SSRIs as
compared with animals that do. Each additional experiment may narrow (or broaden) the list of genes of interest.

FIGURE 23.3. After normalization, a scatter plot shows that most genes fall in a ratio = 1 space. Arrows above and below this
space point at genes with altered expression. See color version of figure.

FIGURE 23.4. Clustering. The clustering algorithm has sorted the genes that were studied in a series of experiments according
to similarities in their patterns of expression. By convention, red indicates an increase from the standard used, and green a
decrease. The collections of genes that are moving up or down in parallel can be readily seen. See color version of figure.

There are ways of evaluating data that are not completely model-independent. Gene responses can be imposed on metabolic charts
or on maps of chromosomes. In the former case, increases or decreases in the utilization of certain pathways can be detected; in
the latter, deletions or changes in copy number may be recognized, or strong positional candidates identified.

INTERPRETING EXPERIMENTS
Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "

Recent studies of changes in gene expression in yeast associated with nutritional and environmental stresses, the cell cycle, or
genetic manipulations are examples of well planned and executed surveys (45 ,46 ,54 ,55 and 56 ). All of the 6,200 known and
predicted protein-coding genes in the yeast genome were arrayed on a single microscope slide, and sufficient numbers of cells were
grown to make ample amounts of RNA for labeling. Each experiment gave a richly detailed picture of molecular responses to a
physiologic process or perturbation.

Unfortunately, the brain is much more difficult to examine than yeast. It varies with age, and is composed of hundreds of different
sorts of cells that express, in aggregate, as many as half of the genes in the genome in a highly regulated manner. To determine the
properties of single populations of cells in the context of the intact structure will be difficult, but perhaps not impossible. Initially,
it would make sense to identify all the genes expressed in the developing and adult nervous system. This, in fact, is one goal of the
Brain Molecular Anatomy Project (BMAP) (57 ). After this goal is achieved, the regional and cellular localization of “brain genes” will
be determined.

In addition to cataloging the transcripts in the brain, it would be helpful to look at the reactions of isolated populations of neurons
or glia to specific signals or environmental alterations—e.g., oxidative stress, excitotoxins, neurotransmitters, hormones, and drugs.
Some responses may be of a global nature—increases or decreases in energy metabolism or protein biosynthesis—while others may be
quite specific to the cell studied or the agent administered.

The availability of transcript maps and collections of “expression motifs” should help us interpret some of the changes observed in
human or mouse brain samples. For example, if it were possible to examine the responses of isolated raphe neurons to SSRI
treatment in vitro, it might be easier to recognize similar responses in tissue samples. Bear in mind, however, that the majority of
arrayed genes discovered by sequencing the human genome and large collections of cDNAs are of unknown function. Structural
motifs may hint at the function of some gene products (58 ), but the role of most will remain an enigma. Expression studies may
help solve this problem because genes with similar expression profiles often have related jobs. Functional proteomic work will be
useful as well. The combination of
 P.296

two-dimensional gel electrophoresis, ultrasensitive detection methods, and mass spectroscopic analyses will permit researchers to map
protein species to specific organelles or macromolecular complexes (59 ). It is important to remember that most proteins in a cell do not
exist or act in isolation. Components of metabolic pathways and regulatory cascades reside in protein communities. Interactions between
members of such communities can be detected with yeast two-hybrid methods, and researchers have already begun to examine protein–
protein interactions in simple organisms on a genome-wide basis (60 ,61 and 62 ). Furthermore, yeast “n-hybrid” methods have been
developed that permit one to look at protein–DNA and protein–RNA interactions (63 ). Finally, it is worth mentioning that large collections
of mice are being produced with random mutations in their genomes. The goal of “saturation mutagenesis” projects is to use multiple
screens to identify animals with interesting phenotypes. The goal of investigators who are making insertional mutations in embryonic stem
(ES) cell lines, on the other hand, is to determine the insertion site of each cell produced so that knockout animals can be made on
demand.
In the future, in analyzing the results of array experiments, the field will benefit from work on animals, proteomics, and earlier expression
studies too—but only if everyone adheres to standard formats in archiving and annotating data.

ANNOTATING EXPERIMENTS
Part of "23 - Applying Functional Genomics to Neuropsychopharmacology "
Presently, there are no standards for annotation, but efforts are under way to solve this problem. To create useful and searchable archives,
all features of each experiment will have to be described in a standard way using an explicit and unambiguous, “controlled” vocabulary.
Some of the required vocabulary already exists. For example, DNAs spotted onto an array can be given and linked to identifiers in public
databases. (Unfortunately, different databases sometimes use different identifiers for the same gene. Consequently, the sequence of each
DNA on an array should be specified.) Drugs used in array experiments can be referred to by Merck Index number (64 ), organisms can be
described using names in the taxonomy database (65 ), and mouse strains and mutants can be named according to established rules and
guidelines (66 ). Much of the language needed to describe array experiments has not been standardized, however, and for now databases
will have to contain many free-form text fields.
For studies of autopsy samples from psychiatric patients, a good deal of specialized information should be provided. The patient's age at
death, gender, diagnosis, genotype (if available), cause of death, postmortem interval, pathology, toxicology screening results, and
medication/drug abuse history should be given. The brain region dissected, dissection method used, side of the brain sampled, specimen
weight, microdissection procedure and cells selected, RNA extraction method and quality, and labeling method are all essential fields as
well. It would be useful to have quality scores for some of these—e.g., the diagnosis and sample condition. Short of a quality score for
diagnosis, a detailed description of the key elements of the patient's clinical and laboratory findings should be made available.
Annotating array experiments can be tedious and time-consuming. I am not suggesting that the recommendations above should all be
implemented immediately. This would hinder progress. On the other hand, the field would benefit from well-annotated work, and the
sooner guidelines are agreed to and implemented, the better. If this chapter serves one useful purpose, it would be to promote this agenda.

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Section III
Emerging Imaging Technologies and Their Application to Psychiatric
Research
Robert Desimone
Robert Desimone: National Institute of Mental Health, Intramural Research Program, National Institutes of Health, Bethesda, Maryland.

Emerging Imaging Technologies and Their Application to Psychiatric Research -

Introduction
In a series of chapters on advances in neuroimaging techniques, it is ironic that images per se of the brain's structures or of neural activity have
actually diminished in importance since publication of the American College of Neuropsychopharmacology's Fourth Generation of Progress. The
focus of neuroimaging in cognitive neuroscience and psychiatry is widening beyond initial questions of where normal and pathologic functions are
localized in the brain, to begin to include questions of how cognitive operations are carried out and why they sometimes fail. For these more
mechanistic questions, images become simply measurements for testing hypotheses and are not an end in themselves.

Structural imaging of the brain with magnetic resonance imaging (MRI) is a good example of a field that is no longer restricted to simple localization
of pathology in psychiatric disease. Indeed, there are few psychiatric cases that are characterized by clear pathology that is visible in MRI pictures.
By contrast, the new analytic approaches to measuring the size of structures in MRI images described by Evans in this section allow one to track
small changes in structures over time, which can potentially reveal abnormal patterns of development in either gray or white matter. Recently, for
example, these techniques have been used to track the distribution of gray and white matter during development in child-onset schizophrenia, which
is characterized by an abnormal time course of gray matter reductions in several different brain systems. These abnormal trajectories suggest possible
concomitant abnormalities in synaptic pruning, which is known to take place throughout development. New diffusion-tensor imaging techniques in
MRI, described by Makris et al. in this section, add the ability to track major fiber bundles in the white matter, which can give some insight into
cortical connectivity. Makris et al. discuss the current limitations with this technique, including the difficulty in following fiber bundles to their
termination.

Another evolutionary change in neuroimaging has been the continued shift from positron emission tomography (PET) to MRI-based techniques for
indirect measurement of neural activity. However, as described by Fujita and Innis, PET and single photon emission computed tomography (SPECT)
remain the only viable techniques for studying ligand binding in the brain, and the resolution of PET is continuing to increase as new detectors are
developed. Fujita and Innis review the status of radiotracer development in PET and SPECT and describe new tracers for measuring postreceptor
signal transduction and even gene expression. Another relatively new approach to mapping the distribution and concentration of specific molecules in
the brain is magnetic resonance spectroscopy (MRS), described by Rothman et al. in this section. The focus of this chapter is on measurements of
metabolites involved in neuroenergetics and amino acid neurotransmission, especially the flux through glutamate/glutamine andγ-aminobutyric
acid (GABA)/glutamine cycles during neural activity. GABA metabolism, in particular, appears to be sensitive to both psychiatric disorders, such as
depression, and to pharmacologic treatment.
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Some of the biggest advances in functional MRI (fMRI) methodology for the indirect measurement of neural activity have been in the time domain.
Bandettini describes new methods for improving the temporal resolution of fMRI, in particular event-related designs. In more traditional blocked-
trial designs, the BOLD signal is averaged for many seconds, typically for several trials of a behavioral task. However, with event-related designs,
one can measure BOLD changes for events lasting less than 2 seconds, which allows one to distinguish activity changes in one part of a trial from
another, e.g., from the encoding to the retrieval phase of a trial in a memory task.

The specific application of both fMRI and brain-lesion analyses to studies of mood and emotion is the focus of the chapter by Davidson. The brain
systems important for the regulation and expression of mood and emotion are highly distributed, and thus it is essential to take a systems approach to
imaging and lesion data in this field, rather than a &ldquo;function per brain structure&rdquo; approach. Davidson also describes how basic
behavioral research lays the necessary groundwork for studying mood and anxiety disorders, and he gives specific examples of basic research into
fear and anxiety and its implications for understanding disorders such as social phobia.

Despite improvements in the temporal resolution of fMRI, the technique will never approach the temporal resolution of event-related potential (ERP)
methods, which is at the millisecond level. As described by Hillyard and Kutas, new analytic techniques have improved the spatial resolution of
ERPs, and there has been considerable progress in combining the spatial resolution of fMRI with the temporal resolution of ERPs. Perhaps even more
important than these technologic advances, there have been conceptual advances in understanding the functional components of ERP signals through
the application of cognitive theory to ERP paradigms. For example, there are characteristic signals found in visual tasks for the arrival of visual
information in a cortical region, for the modulation of this signal by attention, and for the decoding of the visual information into semantic
information. With the appropriate task design and with the large base of information acquired on the timing of these cognitive operations in normal
subjects, one can then begin to ask how these operations differ in schizophrenia, for example.

Finally, the techniques described in the chapters in this section allow one to create and test models of the functional interactions between different
brain structures, but they provide no direct evidence of functional connectivity. One new, direct approach to functional connectivity is an analytic
technique known as effective connectivity mapping, described by Büchel and Friston. Here, one makes use of the moment-to-moment relationships
between fMRI signals in different brain regions to create structural equations, which can quantify the contribution of activity in one brain structure to
the activity in another. An even more direct approach to measuring connectivity is through the combined use of transcranial magnetic stimulation
(TMS) and fMRI, described by George and Bohning. They describe studies in which brain structures are directly stimulated with TMS, and the
resulting effects on far-removed brain structures, including in the opposite hemisphere, are measured using fMRI.

The chapters in this section describe an impressive armamentarium of techniques now available to brain imaging researchers, and they outline some
promising new directions in the application of these techniques to mental illness. Together, the chapters show that the key to progess in brain imaging
studies of pathophysiology will be to see beyond the images.
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24
Automated 3D Analysis of Large Brain Mri Databases

Alan C. Evans

Alan C. Evans: Department of Neurology, McGill University, Montreal Neurological Institute, Montreal, Quebec, Canada.

In recent years, the study of gross neuroanatomy and its relationship to behavior and brain function has been reenergized by the
advent of imaging techniques and the powerful computational tools with which to analyze high-resolution three-dimensional (3D)
brain images (10 ,11 ,22 ,52 ,53 ). However, such high technology tools often demand that scientific questions be restated and made
more amenable to quantitative analysis. Questions such as “How much normal variation is there in the size, shape, or location of an
individual brain structure?” or “To what extent does functional architecture of the cortex depend on the anatomic boundaries
between anatomic regions?” carry with them the assumption that the borders of individual structures can be specified accurately in
any brain. In the past, basic questions of functional neuroanatomy were difficult to address in a systematic way in the living brain.
We have learned much from anecdotal reporting of individual patients with various forms of brain lesion or from direct cortical
stimulation during brain surgery, but the generalization of individual observation to the wider population has been confounded by
the normal variation in brain structure itself. There is then a fundamental interest in understanding the nature of anatomic
variability in the population, both for its relationship to functional variability and for the potential of using structural abnormality as
a measure of development, normal aging, and disease. For instance, in some degenerative diseases like Huntington's disease and
Alzheimer's disease, the sulci become more open and the ventricles become enlarged. Magnetic resonance imaging (MRI)-based
measurements of these changes can lead to early diagnosis and treatment, but we need to understand the variation among normal
brains first.

Although the study of postmortem neuroanatomy is a long-established science, the ability to accumulate the numbers of brains
necessary to make statistically meaningful conclusions about cerebral anatomy is a relatively recent phenomenon. It is still difficult
to identify reliably in any single brain the anatomic landmarks, boundaries, and other delimiting features necessary for any
subsequent analysis. Thus, we face a new problem posed by this newfound technology and its inflexible demand that anatomic
questions be posed in numerical rather than descriptive terms. The tools exist to image large numbers of brains noninvasively with
MRI, but we are still struggling with how to extract the anatomic measurements necessary to answer the questions posed above. It is
relatively easy to identify the precentral gyrus, but few researchers attempt to define its “top” and “bottom.” Where does the
inferior frontal sulcus end? Traditional brain atlases identify brain regions only by pointing to the middle of the region or surface
feature, leaving the interfaces between regions unspecified. Neuroanatomists debate the exact boundary of even relatively simple
structures such as the thalamus or caudate nucleus. With this context, new initiatives at various laboratories are attempting to
standardize and codify the partitioning of the human brain into named regions, not without controversy. Traditional
neuroanatomists debate among themselves about what parcellation scheme and nomenclature to use. Computer scientists argue
among themselves about whether to use hierarchical, relational, object-oriented, or some other form of database structure to
organize the brain parcellation. Both groups tend to misunderstand the importance of the other's concerns. Neurobiologists or
physicians are not used to thinking in terms of inclusive sets where, for instance, every structure at one level is wholly included
within a higher level organization, where all 3D pixels, i.e., voxels, within the brain space must be labeled as one of the structures
in the partitioning scheme, or that the cerebrospinal fluid (CSF) ventricular spaces may be declared as being outside of “brain.”
Computer scientists tend to ignore the realities that many cortical sulcal features do not exist in every brain, and may be
fragmented or have multiple occurrences. Some sophisticated analytic approaches for quantifying anatomic variability assume that a
particular landmark can be perfectly identified in any brain when the reality is that errors of 5 to 10 mm typically occur, an error
that is about
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the same magnitude as the true spatial variation being sought.

Despite heroic efforts in the recent past (2 ,29 ,58 ,60 ,62 ,75 ,79 ,80 ), manual labeling of many individual MRI data sets in 3D is a
labor-intensive effort that is not likely to be widely adopted. Fully automated techniques that produce accurate neuroanatomic
segmentation in large numbers of MRI data sets are essential if questions of normal cross-sectional variability, normal longitudinal
development, and detection of abnormality in single subjects or in groups are to be answered definitively. Many groups are now
engaged in the field of MRI-based quantitative neuroanatomy, and an exhaustive review of the field is beyond the scope of this
chapter. A representative sampling of activity by other groups in the field, categorized into the four forms of segmentation
discussed in the subsequent Methods section, include the following:

 Tissue classification/voxel morphometry: This refers to MRI intensity-based classification of images into tissue classes and
voxel-based statistical analysis of the resulting class maps. In normal brain, the tissue classes are typically gray matter,
white matter, and CSF, although there is no reason in principle to restrict to these three tissue types. In these approaches,
one or more co-registered MRI images of the same neuroanatomy, obtained using different acquisition protocols [e.g., T1-
weighted, T2-weighted, proton density (PD), magnetization transfer), provide the input data. At each voxel the MRI
intensity for each of the N input images provides an N-dimensional “feature vector.” Ideally, each tissue class is identified
by a unique feature vector. In practice, many confounding factors (e.g., tissue heterogeneity, MRI field distortions, partial
volume effects, and image noise) blur the feature space and render it difficult to distinguish accurately even three tissue
classes. Many different multivariate statistical methods exist to optimize the class labeling and, for most of them, more
independent images (features) help to disentangle overlapping class distributions in feature space.

Mapping the segmented images into stereotaxic space (69 ,70 ) allows for group analysis across a population of 3D data
sets from different individuals. All of the machinery of random field statistical analysis developed for functional imaging
then becomes available for structural analysis (1 ,5 ,30 ,31 ,35 ,54 ,56 ,57 ,81 ,82 and 83 ).

 Regional parcellation/atlas deformation: Delineation of brain regions within each tissue class (e.g., caudate nucleus in the
gray matter class) is not possible using only the information available in the MR image(s) since there is not sufficient
differentiation among these regions within the feature space. Some form of prior information on neuroanatomic
boundaries is needed, usually in the form of a computerized brain atlas, to assist in 3D brain regional labeling. Regions can
be identified by vector boundaries or by labeling of all internal voxels. The atlas or parcellation scheme can be used as a
guide to manual segmentation or as the basis for automated regional segmentation in which the atlas space is deformed to
match each new 3D brain image. The atlas template is matched to the new MRI volume through a variety of nonlinear
deformation techniques, the most successful of which use image similarity criteria to deform one image into another.

Once delineated in their native space it is possible to map the regional labels into stereotaxic space in much the same way
as tissue class maps and to conduct voxel morphometry among groups using the random field statistical analysis
(3 ,4 ,6 ,12 ,18 ,18 ,21 ,26 ,32 ,34 ,36 ,39 ,40 and 41 ,50 ,68 ).

 Surface extraction/cortical unfolding: Regional parcellation is generally quite successful at labeling relatively well-defined
3D brain regions, such as the thalamus, but is typically less successful in identifying cortical gyri. Indeed, the cortex as
such is sufficiently important to merit special analytic treatment. Techniques have been developed to “extract” the
exterior cortical surface automatically by boundary detection of the intensity interface between gray matter and
subarachnoid CSF. To overcome partial effects, some groups have targeted the internal cortical margin at the interface
between gray and white matter. Obtaining a measure of the two surfaces simultaneously allows for a measure of cortical
thickness at each location over the cortical surface.

Extraction of the cortical surface has prompted some groups to explore the potential of an “unfolded” cortical surface as
a means of studying functional neuroanatomy on a two-dimensional (2D) plane. Arguably, this device reduces the
variability of functional areas introduced by cortical folding in three dimensions. The mapping from 3D to 2D is a nontrivial
task with many issues surrounding the optimal mapping function, with direct analogies to the well-known cartographic
dilemmas of preservation of area, direction, distance etc. (7 ,8 ,20 ,27 ,28 ,38 ,49 ,55 ,77 ,78 ).

 Sulcal extraction/analysis: The cortical sulci have held a historical position of prominence in functional neuroanatomy, in
part because of their utility as approximate landmarks to functional areas. Recent interest has centered upon extracting
not just the surface trace of the sulcus as a line but rather the depth of the sulcus as a ribbon. The latter approach
provides more information on buried cortex and sulcal shape than a simple line trace, which can be related to genetic
and developmental considerations (46 ,51 ,59 ,65 ,76 ).

In the United States, the Human Brain Project has specifically set out to foster the application of computational techniques,
hardware, and algorithms to neuroscience at all spatial scales. We are involved in one of these applications operating at the gross
morphology level. The International Consortium for Brain Mapping (ICBM) (52 ), seeks to create a so-called probabilistic human brain
atlas (see below).
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This chapter provides an overview of the methods developed by the Brain Imaging Centre (BIC) at the Montreal Neurological Institute
for fully automated 3D segmentation of the ICBM database and other MRI databases like it, such as those collected for the creation
of normal pediatric development and for evaluation of new pharmaceuticals. A key concept underlying this work is that of the
analysis “pipeline,” which takes 3D MRI volumes from large numbers of subjects and generates 3D statistical maps of adult brain
morphology with no manual intervention. The pipeline concept has also been implemented for clinical trial analysis of MRI data from
multiple sites. All data sets, across patients, time points, and pulse sequences, are mapped into a standardized 3D coordinate space
for automatic segmentation and statistical analysis.
Once the MRI image has been segmented, each voxel in the 3D image space carries an anatomic label and a measure of the
confidence in that label. This information can be used in a variety of ways to detect subtle neuroanatomic or neuropathologic
changes:

 Single subject vs. group data for detection subtle of structural abnormality (e.g., misshapen corpus callosum)
 Intergroup cross-sectional comparison (e.g., Alzheimer's disease group vs. normal age-matched controls)
 Longitudinal study in a single subject (e.g., tumor growth, progressive atrophy)
 Longitudinal study in a group [e.g., early development and aging in normal populations, multiple sclerosis (MS) disease
progression].

Illustrative example applications of some of these capabilities are described at the end of the chapter.

 IMAGE SEGMENTATION METHODS

 SAMPLE APPLICATIONS
 SUMMARY AND FUTURE DIRECTIONS

IMAGE SEGMENTATION METHODS

Part of "24 - Automated 3D Analysis of Large Brain Mri Databases "
Within the BIC image analysis pipeline, MRI data are processed using a series of tools that provide measurements of volume, shape,
size, and tissue composition of selected brain regions. These are summarized below. To manage the flow of MRI data through the
pipeline, we have developed PCS (Production Control System), which allows the rapid implementation and parallel execution of
analysis pipelines for processing large MRI databases. Each processing stage in the pipeline is performed by a single command. PCS
allows the user to specify this command with its options, input and output files, and dependencies on other stages in the pipeline
using a simple script language. Efficient coarse-grain parallelism is achieved by distributing the individual jobs over a network of
workstations. PCS monitors the status of each job and submits a new job when the prerequisites for submission have been satisfied
(typically the completion of all stages on whose output data the stage depends). The major elements of this environment include
(Fig. 24.1 ):

FIGURE 24.1. Brain Imaging Centre (BIC) pipeline environment for

magnetic resonance imaging (MRI) processing: major components of
pipeline analysis of large ensembles of MRI multispectral data sets.
Each multispectral data set yields labeled maps of tissue type,
three-dimensional (3D) brain region, and cortical topology.

 Thin-slice MRI data acquisition (typically 1-mm axial sampling, with 1-mm isotropic voxels).
 Multimodal, multidimensional stereotaxic data format (MINC).
 MRI simulator for validation of segmentation algorithms (MRISIM).
 Correction for coil-dependent 3D intensity nonuniformities (N3).
 Within-subject registration of different sequence volumes (MINCTRACC).
 Cross-subject mapping into a standardized “stereotaxic” 3D coordinate space (MRITOTAL).
 Fully automated 3D classification of gray/white/CSF tissue classes (INSECT).
 Fully automated 3D regional segmentation based on prior atlas templates (ANIMAL).
 Fully automated 3D extraction of gray/CSF and gray/white cortical interfaces (MSD, ASP).
 Computer-assisted 3D labeling of individual sulci (SEAL).

Stereotaxic Image Format—MINC

A fundamental aspect of this pipeline environment and its interaction with other sites within ICBM is the MINC image format for
intersite data communication. MINC (Medical Image Net CDF), developed at the MNI by Peter Neelin, is a multidimensional,
multimodality image file format that supports stereotaxic coordinate representation.
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Image volumes can be explored in real time in 3D with continuous update of stereotaxic coordinates. Image files with different
native voxel dimensions can be compared directly without regard for the original acquisition sampling grid. This simplifies
stereotaxic analysis of MRI data ensembles collected with different voxel dimensions.

MRI Simulation—MRISIM
To assist in the evaluation of these segmentation tools, we created an average MRI data set of a single young normal male, by
repeated MRI scanning followed by linear alignment of all volumes. A total of 27 separate MRI scans were collected. The improved
signal-to-noise ratio (SNR) in the composite MRI, termed ICBM27, produces a high-definition data set (37 ), suitable for brain atlas
construction, validation of segmentation/mapping algorithms, and MRI simulation. (Note: Since it incorporates the structural
idiosyncracies of a single brain, it is not intended for use as a high-definition master data set for stereotaxic normalization.) This
data set has been segmented manually to create an accurate digital phantom (17 ) for use as the source template of an MRI
simulator, MRISIM (43 ).

MRISIM requires as input a set of “fuzzy” structure maps, one for each distinct tissue (or structure) type to be modeled, in which
each voxel value is the probability of that voxel containing that tissue (structure) type. Such maps are generated by algorithms like
INSECT or ANIMAL (see below) applied to a high-SNR data set. The MRI signal is simulated by solving the Bloch equations for the
specified pulse sequence and tissue relaxation characteristics. Noise is modeled from first principles rather than by adding some
parametric (e.g., gaussian) noise distribution to the expectation image (42 ). MRISIM has been used in validation studies for
correction of MRI intensity nonuniformity (67 ) and tissue classification (84 ). It has been used to create a database of 108 simulate
MRI images [3 slice thicknesses × 3 tissue contrasts (T1/T2/PD) × 3 noise levels × 4 levels of radiofrequency (RF) inhomogeneity],
available at Web site http://www.bic.mni.mcgill.ca/.

Correction for 3D Intensity Nonuniformity—N3

A major problem for automated MRI image segmentation is the slowly varying change in signal intensity over the image, caused
principally by nonuniformities in the radiofrequency field (Fig. 24.2 ). Apparent signal from any one tissue type is therefore different
from one brain area to another, confusing automated segmentation algorithms that assume constant signal for one tissue type. We
have developed a fully automated 3D technique for inhomogeneity correction, modeling inhomogeneity as the convolution of the
true MRI intensity histogram with a blurring kernel. This effective kernel can be estimated and deconvolved by iterative entropy
maximization. The method is applicable to any pulse sequence, field strength, and scanner (66 ,67 ).

FIGURE 24.2. N3 correction for intensity nonuniformity. MRI image before (left) and after (middle) correction for
nonuniformity field (right), estimated using N3. Note the increased uniformity of white matter regions.

Intrasubject Image Alignment—MINCTRACC

Alignment of images from the same subject, either from the same modality at different times in a longitudinal study or from
different modalities, is achieved using a linear version of ANIMAL (see below), constrained to a six-parameter (three rotation, three
translation) rigid-body transformation (15 ).

Stereotaxic Transformation—MRITOTAL
Stereotaxic transformation is achieved using a simple nine-parameter linear [three rotation, three translation, three scale, (15 )]
transformation to match the image volume to a master data set already resident in stereotaxic space. The master data set therefore
defines the gross dimensions and orientation of stereotaxic space. We have previously constructed
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a composite stereotaxic MRI data set drawn from 305 normal subjects, sampled on a 1-mm voxel grid (24 ), as that master data set.
This mean data set, now termed ICBM305, has been circulated to over 100 international sites and defines the stereotaxic space for
the SPM statistical package. That data set was derived from T1-weighted data with 2-mm-thick slice data. More recently, this has
been superseded by a composite data set derived from 1-mm-thick data collected within the ICBM project (see below). That latter
data set, while exhibiting higher contrast and more anatomic detail than the original ICBM305, was nevertheless mapped into the
space of the ICBM305 using the nine-parameter MRITOTAL and is therefore a derivative of that first data set.

Tissue Classification—INSECT
We have developed an algorithm for tissue classification, known as INSECT (Intensity-Normalized Stereotaxic Environment for
Classification of Tissue) (25 ,63 ,84 ). The algorithm operates upon multispectral (typically T1-, T2-, PD-weighted) data sets. In a
series of preprocessing steps, each MRI data set is corrected for intensity nonuniformity (67 ), interslice normalization, and
intersubject intensity normalization (Fig. 24.3 ). Stereotaxic transformation is then performed (15 ). An artificial neural network
(ANN) classifier with one hidden layer is used to assign each voxel to a tissue type (gray/white/CSF) based on its MRI intensity
feature space. The algorithm also employs tissue likelihood, based on the spatial location of the voxel in stereotaxic space, as
orthogonal prior information to constrain the feature-space assignment. For example, periorbital fat exhibits a similar feature-space
signal as white matter and, without consideration of spatial location, would be classified as white matter. Spatial masks expressing
the normal distribution of tissue classes in the population (see Fig. 24.8 ) indicate that the likelihood of finding white matter in the
periorbital stereotaxic region is small, and reduce the likelihood of misclassifiation.
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INSECT operates on an arbitrary number of input images and generates a user-selected number of output tissue maps.

FIGURE 24.3. Classification with and without correction for intensity nonuniformity: tissue classification with INSECT with and
without correction for nonuniformity using N3. An idealized 3D digital phantom was created from by segmentation of a
high=nsignal-to-noise ratio (SNR) data set (17,37). The initial phantom data (top left) contains three classes: cerebrospinal
fluid (CSF) (black), gray matter (dark gray), and white matter (light gray). This phantom was used to generate a simulated MRI
image with (top middle) and without (top right) a 20% inhomogeneity running from top left to bottom right of the image. The
INSECT-classified image without prior N3 correction (bottom left) exhibits artifactually thicker cortex at bottom right and
thinner cortex at top left of the image, respectively, a consequence of the field inhomogeneity gradient. This artifact is
removed in the N3-corrected classification (bottom right).

FIGURE 24.8. Tissue probability maps. Left: Cuts through INSECT-generated 3D tissue class maps for gray matter, white
matter, and cerebrospinal fluid (CSF). Right: Serial sagittal sections through Talairach atlas with ANIMAL-generated
probabilistic frontal cortex SPAM (statistical probability anatomy map) overlaid. In both cases 100 subjects were used to
generate the SPAMs.

Regional Parcellation—ANIMAL
Manual labeling of brain voxels is both time-consuming and subjective. We have previously developed an automated algorithm to
perform this labeling in 3D (13 ). The ANIMAL algorithm (Automated Nonlinear Image Matching and Anatomical Labeling), deforms
one MRI volume to match another, previously labeled, MRI volume. It builds up the 3D nonlinear deformation field in a piecewise
linear fashion, fitting cubical neighborhoods in sequence using a mutual information residual for parameter optimization (Fig. 24.4 ).
The algorithm is applied iteratively in a multiscale hierarchy. At each step, image volumes are convolved with a 3D gaussian blurring
kernel of successively smaller width [32-, 16-, 8-, 4-, and 2-mm full-width at half-maximum (FWHM)]. Anatomic labels are defined in
the new volume by interpolation from the original labels, via the spatial mapping of the 3D deformation field. Originally, ANIMAL
used 3D gradient magnitude as the image property to be matched. The ridge-tracking Lvv operator is now used to extract additional
topologic information on brain shape in each image. Furthermore, the surface trace of major sulci, represented as 3D line segments,
can be used as local constraints on image deformation (14 ,16 ). Both steps increase the correspondence of cortical anatomy across
brains.

FIGURE 24.4. ANIMAL warping. Slice through a 3D ANIMAL deformation. The left image was warped to match the right, with
the result in the middle.

Cortical Surface Segmentation and Unfolding—ASP

We have previously developed a fully automated procedure for unfolding the entire human cortex, using an algorithm that
automatically fits a 3D mesh model to the cortical surface extracted from MRI (47 ). This algorithm, MSD, uses an iterative
minimization of a cost function that balances the distance of the deforming surface from (a) the target surface, and (b) the previous
iteration surface (Fig. 24.5 ). Specification of the relative weight of these competing forces allows MSD to range from unconstrained
(data-driven) deformation to tightly constrained (model-preserving) deformation. Further shape-preserving constraints to penalize
excessive local stretching and bending of the model surface are also employed. The initial mesh surface can be chosen arbitrarily to
be a simple geometric object, such as a sphere, an ellipsoid, or two independently fitted hemispheres. The MSD algorithm has
formed the basis of cortical analysis at both MNI and UCLA within the ICBM project (71 ,72 and 73 ). Recently, the algorithm has
been extended to allow multiple concentric surfaces to be mapped simultaneously. The new algorithm, Automatic Segmentation
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using Proximities (ASP), has the following refinements and capabilities (48 ), compared with the earlier MSD version:

FIGURE 24.5. Average cortical surface. Average of 150 normal cortical surfaces. Note the prominence of the major gyral and
sulcal features common to all brains.

 A boundary search along the normal local surface is used to increase the range of attraction of edges.
 The use of proximity constraints with appropriate weights excludes the potential for impossible self-intersecting surface
configurations.
 Some arbitrary weights are replaced by more intuitive geometric constraints.
 Multiple surfaces, models, and data sets may be combined into a single objective function.
 Automatic identification of the total cerebral cortical surface from MR images is achieved in a robust way with respect to
partial volume effects.
 A preliminary map of cortical gray matter thickness has been produced and related to previous studies.
 A higher resolution average brain surface has been created using the deeper sulcal penetration of ASP compared to earlier
versions of this algorithm (47 ).

As an alternative form of stereotaxy applicable to cortical analysis, ASP also provides a fully automated mapping from 3D to an
unfolded surface space. Since ASP iteratively deforms a starting 3D polygonal mesh onto the 3D cortical surface, the inverse
mapping projects this fitted surface and topologic feature at each surface vertex back to the model space (47 ,48 ). Individual
anatomic features such as gyral ridges and sulcal valleys are converted to measures of topology, e.g., curvature, mapped on to the
model surface. These can be analyzed in terms of 2D variability on the surface of the starting model using a 2D surface coordinate
space (Fig. 24.6 ).

FIGURE 24.6. Cortical thickness. Mean cortical thickness in 150 normal adult brains, color-coded and texture-mapped onto the
average cortical surface obtained from the same population.

Sulcal Extraction and Labeling—SEAL

We have implemented an automated sulcal extraction and labeling algorithm (SEAL) (45 ). At every voxel on the ASP-generated
exterior cortical isosurface, SEAL calculates the two principal curvatures: k1, the mean curvature, and k2, the gaussian curvature (g
= k1 * k2). Voxels with negative
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mean curvature, belonging to sulci, are extracted and pruned to obtain a set of sulcal traces on the cortical surface. SEAL extracts
the buried sulcus with an “active ribbon” that evolves in 3D from a superficial trace to the bottom of a sulcus by optimizing an
energy function. We have defined a relational graph structure that stores, for each sulcus, its length, depth, and orientation, as well
as attributes, e.g., hemisphere, lobe, sulcus type, connecting sulci, etc. Sulcal labeling is performed semiautomatically by tagging a
sulcal trace in the 3D graph and selecting from a menu of candidate labels. The menu is restricted to most likely candidates by the
use of sulcal probabilistic maps. SEAL identifies the sulci maps that overlap with each selected sulcus with highest likelihood (44 ,45 )
(Fig. 24.7 ).

FIGURE 24.7. Use of spatial priors for automatic sulcus labeling within the sulcal extraction and labeling algorithm (SEAL). 3D
representation of labeled sulcal folds occurs either automatically with SEAL, using prior probabilities (left), or manually
labeled by a neuroanatomist (right). Different colors represent different sulcal labels, e.g., central sulcus is colored magenta
(the smooth object is an average MRI surface, reduced in scale, included only to provide context for the sulcal maps). The
automated and manual labeling of the sulci are in broad agreement, although some differences are apparent.

SAMPLE APPLICATIONS
Part of "24 - Automated 3D Analysis of Large Brain Mri Databases "

ICBM: Multicenter Consortium on Statistical Neuroanatomy

The International Consortium for Brain Mapping (ICBM) multicenter initiative was launched in 1993 as part of the Human Brain
Project (52 ). Its overall goal is to create a 3D probabilistic brain atlas, based on MRI volumes from 450 normal adult brains. Within
the ICBM project, all scans at all sites were collected with a strictly defined protocol, specifying three MRI volumes per subject (a 1-
mm-thick, 1-mm-spaced gradient echo sequence for T1-weighted data and a 2-mm-thick, 1-mm-spaced double-echo sequence for PD
and T2-weighted volumes). This database as been segmented using the pipeline environment described above and the variability
captured in the form of probability maps as follows. Neuroanatomic variability can be conveniently represented in the form of 3D
stereotaxic maps where each voxel expresses the likelihood of finding a particular structure at that location. By labeling one
structure, e.g., caudate nucleus, in an ensemble of stereotaxic MRI volumes, a continuous 3D probability field for that structure (0%
to 100% at each voxel), termed a statistical probability anatomy map (SPAM), can be constructed and used to test for group
difference, e.g., pediatric versus adult brains, or outliers. For visualization purposes, these statistical maps can the thresholded at
any level of structural probability to create probability isosurfaces suitable for surface-rendering and 3D display. Example SPAMs are
shown for (a) gray/white/CSF tissue classes (INSECT, Fig. 24.8 ); (b) all major cortical gyri, cerebellum, and deep nuclei (ANIMAL,
Fig. 24.8 and Fig. 24.9 ); and (c) cortical surface (ASP) (23 ).

FIGURE 24.9. Rendered probabilistic atlas. Volume rendering (top left) and surface renderings (all others) of the 3D
probabilistic atlas (N 100). For the surface renderings, the SPAMs were thresholded at the 40% level to generate regional
probability isosurfaces.

Multicenter Clinical Trial Image Analysis

The principles of pipeline analysis described above for large databases of normal brain MRI data are equally applicable for
population analysis of neuropathology or for tracking structural change over time, such as the progressive tissue atrophy, which
occurs in some degenerative diseases. Indeed, the MRI analysis employed within the ICBM project was originally developed for a
multicenter phase III clinical trial of a new pharmaceutical for treatment of multiple sclerosis. In this trial, 14 centers in the U.S.
and Canada collected a total of 1,850 data sets, each data set composed of T1, T2, and PD volumes, from 514 subjects. All data
collection was coordinated by the BIC clinical trials group, which performed quality control before trial launch and for all data
shipped to the BIC for processing. Pipeline analysis of the database was used to generate 3D statistical maps of normal tissues and of
MS lesions. In validation studies, the
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results obtained with this automated approach for a subset of images were compared with those obtained by totally manual
methods at seven established MRI/MS sites in Europe and North America. The results of the comparison indicated no significant
differences between the BIC approach and the mean result obtained across the seven sites. They also indicate considerable
variability among the sites themselves when analyzing the same data, which emphasizes the importance of the reproducibility of
results obtained with a fully automated approach.

After correction for MRI intensity inhomogeneity, interslice and intervolume intensity normalization, and stereotaxic transformation,
the multispectral data were tissue classified to identify MS lesion voxels for each patient time point. Figure 24.10 shows a 3D
rendering of a probability map for lesion distribution obtained from all data sets. It shows the most likely locations for MS lesions
within a population and is a convenient way to distill a large amount of population data into a single entity. Tests of drug effect are
reduced to testing for a significant group difference in the overall volume of this distribution above a given threshold when
partitioned into drug and placebo groups.

FIGURE 24.10. Multiple sclerosis (MS) lesion probability map. 3D renderings of probability maps for MS lesion (light region) and
ventricle (dark region), obtained from 460 patients.

NIMH Intramural Pediatric Database

As part of an ongoing collaboration with Drs. Jay Giedd and Judy Rapoport at the National Institute of Mental Health (NIMH) Child
Psychiatry Branch, the BIC image analysis pipeline has been used to process a large pediatric MRI database collected at the NIMH.
Subjects were scanned on a General Electric 1.5 tesla Signa scanner using a 3D SPGR protocol. Approximately 1,800 T1-weighted
images with slice thickness of 1.5 to 2.0 mm in the axial plane have
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been obtained in approximately 600 children aged 3 to 18 from a number of subgroups:

 Normal development: A subset of this database, 111 normal children aged 4 to 17, was processed using the INSECT
algorithm. All data were resampled into stereotaxic space using a simple nine-parameter linear transformation prior to
image segmentation. Regression of population mean white matter intensity at each stereotaxic voxel against age yielded a
regression map with significant correlation in the left arcuate fasciculus and the bilaterally in the internal capsule (33 ,61 ).
The former tract links the anterior and posterior speech regions, while the latter is part of the corticospinal motor tract.
These areas are continuously developing during maturation and it is tempting to interpret the results as increased
myelination in these areas during development.

A subset of the intramural NIMH database has also been analyzed by the ICBM group at UCLA under the direction of Arthur
Toga (74 ). Using MSD-generated surfaces and tensor field analysis, they produced fourdimensional quantitative maps of
growth patterns in the developing brain. Serial scanning in children aged 3 to 15 years across time spans of up to 4 years
revealed a rostrocaudal wave of growth in the corpus callosum, a fiber system that relays information between brain
hemispheres (Fig. 24.11 ). Peak growth rates, in fibers innervating association and language cortices, were attenuated after
puberty, and contrasted sharply with a severe, spatially localized loss of subcortical gray matter. Conversely, at ages 3 to 6
years, the fastest growth rates occurred in frontal networks that regulate the planning of new actions.

 Child-onset schizophrenia: Fifteen patients with childhood-onset schizophrenia and 34 temporally yoked, healthy
adolescents, scanned twice with an interval of 4 years, were analyzed using the pipeline (64 ). Lobar gray and white matter
volumes were obtained with INSECT and ANIMAL. A significant decrease in cortical gray matter volume was seen for healthy
controls in the frontal (2.6%) and parietal (4.1%) regions. For the childhood-onset schizophrenia group, there was a
decrease in volume in these regions (10.9% and 8.5%, respectively) as well as a 7% decrease in volume in the temporal gray
matter. Thus, the childhood-onset schizophrenia group showed a distinctive disease-specific pattern, with the frontal and
temporal regions showing the greatest between-group differences. Changes in white matter volume did not differ
significantly between the two groups. Patients with very early onset schizophrenia exhibit a fourfold greater decrease in
cortical gray matter volume during adolescence and a disease-specific pattern of change.
 Attention-deficit/hyperactivity disorder (ADHD): Anatomic studies of boys with ADHD have previously detected volumetric
differences in basal ganglia, prefrontal regions, and the cerebellar vermis. This study sought to
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replicate those findings in young girls. MRI data from 53 girls with ADHD and 44 healthy matched female controls, ages 5 to
15, were analyzed using ANIMAL. Significantly smaller volumes were observed in prefrontal brain regions, caudate nucleus,
globus pallidus, and amygdala bilaterally. The posterior-inferior cerebellar vermis volume and the rostrum of the corpus
callosum were also significantly smaller in the ADHD group. Significantly smaller volumes were seen in the same brain regions
as previously reported in boys with ADHD. As in boys, ADHD in girls is associated with anatomic deviations in corticostriatal-
pallidal-thalamic circuits and in the posterior-inferior cerebellar vermis (9 ).

FIGURE 24.11. White matter density changes during pediatric

development. Regression maps of white matter density changes over
the age range from 4 to 17 (61). These maps show increased white
matter density, possibly myelination, in the left arcuate fasciculus
(left) and internal capsule (right), white matter tracts implicated in
the development of language and motor skills, respectively.

NIH Extramural Pediatric MRI Database

The NIMH intramural database above has been acquired with only T1-weighted information and sparse behavioral information from a
variety of subgroups, including approximately 200 normal children aged 3 to 8. While this database will provide much valuable
information on pediatric development, there remains a need to create a more complete database of MRI information from a larger
cohort of normal children, well-characterized by behavioral batteries. Therefore, a recent joint initiative by three National
Institutes of Health (NIH) agencies (NIMH, NICHD, NINDS) has been launched to create such an MRI database of normal pediatric
development in 550 children. This project, drawing upon a clinical trial model, will collect identical imaging and behavioral data at
seven U.S. sites. The data will be consolidated into a single database at the BIC for pipeline analysis and eventual dissemination to
the community. Each child in the age range of 5 to 18 will be scanned three times over a 6-year period. Behavioral batteries
covering the major performance criteria will be collected at each time point. A younger cohort of approximately 100 children, aged
0 to 5, will undergo a more frequent scanning protocol and an age-appropriate behavioral battery. Magnetic resonance spectroscopy
(MRS) and diffusion tensor imaging (DTI) information will also be collected at three of the sites to provide information on
developmental neurochemistry, myelination, and fiber tract development.

SUMMARY AND FUTURE DIRECTIONS

Part of "24 - Automated 3D Analysis of Large Brain Mri Databases "
This chapter has presented an overview and sample applications of the MRI analysis pipeline environment at the Brain Imaging
Centre (BIC) of the Montreal Neurological Institute. The key conceptual elements of this environment are as follows:

1. The use of stereotaxic space for consolidation of large ensembles of MRI data into a common spatial frame for analysis
of gross neuroanatomy;
2. Fully automated 3D image preprocessing and segmentation;
3. Statistical analysis using voxel-bases random field theory and general linear models;
4. Incorporation of nonimaging parameters such as behavioral variables, demographic information, and genetic data into
the statistical models.

The pipeline is highly modular, allowing for separate development and continued upgrading of the individual elements making up
the pipeline. Processing is distributed across the BIC computing infrastructure using the PCS control scripts to optimize the
utilization of resources. It has application in a variety of settings from basic neuroscience through clinical research to clinical trials.
However, the current environment is focused on gross morphology. Conventional MRI allows us to collect gross anatomic information
from a large sample of brains and develop population statistics. Unfortunately, this level of analysis provides no information about
the cellular and molecular organization of the brain at a finer scale. A full understanding of functional neuroanatomy links function
to macroscopic anatomy via these ultrastructural segregations. High-field MRI offers new possibilities, providing resolution of a few
hundred microns over limited volumes. Sectioning, staining, and optical digitization of cadaver brains allow even finer spatial and
chemical resolution in limited numbers of brains. A number of sites are bringing together these new acquisition technologies with
the concepts of 3D stereotaxic mapping to create probabilistic maps at this finer scale. The advantage of the stereotaxic approach is
that information from these many techniques operating at different spatial scales can be consolidated over many years into a
systematic description of the whole brain structure and function. Such a rich database of information on both cerebral structure and
function, accessible to sophisticated computational and statistical exploration, offers exciting possibilities for future brain research
and clinical practice. Quite apart from direct hypothesis testing, such an environment may allow for the detection of hitherto
unsuspected patterns of interaction among normal brain elements and the isolation of constellations of measurements that
characterize specific disease states.

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25
In Vivo Magnetic Resonance Spectroscopy Studies of the Glutamate
and Gaba Neurotransmitter Cycles and Functional Neuroenergetics

Douglas L. Rothman

Fahmeed Hyder

Nicola Sibson

Kevin L. Behar

Graeme F. Mason

Jun Shen

Ognen A. C. Petroff

Robert G. Shulman

Douglas L. Rothman, Fahmeed Hyder, Kevin L. Behar, Graeme F. Mason, Ognen A. C. Petroff, Robert G. Shulman: Yale
University School of Medicine, New Haven, Connecticut.

Nicola Sibson: University of Oxford, Oxford, United Kingdom.

Jun Shen: Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York.

In the last 5 years there has been a renewed interest in the role of metabolism in supporting brain function. Much of this interest is
based on the development of functional positron emission tomography (PET) and magnetic resonance imaging (MRI). Although often
incorrectly described as directly mapping neuronal activity, both functional PET and MRI actually measure changes in either glucose
metabolism or physiologic parameters coupled to glucose metabolism such as blood flow and volume (1 ). A major limitation in
interpreting functional imaging is that the relationship between neuronal activity and the neuroenergetic processes supported by
glucose metabolism is poorly defined (2 ,3 ). The term neuronal activity applies to a spectrum of energy-requiring processes
including action potential propagation, neurotransmitter release and uptake, vesicular recycling, and maintenance of membrane
potentials (4 ). All of these processes are involved in short-term neuronal information transfer, and the relative distribution of
energy among them remains an open question. There is also uncertainty as to how the different classes of neurons in a region
contribute to the overall energy consumption. While an increase in the imaging signal is usually assigned to an increase in neuronal
excitation, this interpretation is confounded by both inhibitory and excitatory neuronal function requiring energy. Observation of a
regional increase or decrease of the functional imaging signal is not sufficient to distinguish these possibilities. Glia also requires
energy, and the relationship between its energy demands and neuronal activity remains to be established. Given these uncertainties
about the meaning of the signal at a neuronal level, the validity of functional imaging as a tool for studying mental processes has
been largely established based on agreement with prior expectation from psychological paradigms (3 ,5 ).

An alternative approach for imaging brain function, which has the potential of directly measuring metabolic pathways involved in
excitatory and inhibitory neurotransmission, is in vivo magnetic resonance spectroscopy (MRS). MRS uses technology similar to that
of the more familiar MRI. It differs by allowing the measurement of the concentrations and synthesis rates of individual chemical
compounds within precisely defined regions in the brain. The basis of its chemical specificity is that the resonance frequency of an
MRS active nucleus depends not only on the local magnetic field strength, but also on its chemical environment, a phenomenon
referred to as chemical shift. MRS measurements of the 1H nucleus are the most commonly used for in vivo studies due to 1H being
the most sensitive nucleus present in biological systems. Metabolites that can be measured by 1H MRS include aspartate, γ-
aminobutyric acid (GABA), glucose, glutamate, glutamine, and lactate. These metabolites play critical roles in neuroenergetics,
amino acid neurotransmission, and neuromodulation. Another nucleus of importance for in vivo MRS studies is the
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13
C nucleus. The natural abundance of the 13C isotope is 1.1% so that in conjunction with the infusion of 13Cenriched substrates the rates of isotopic incorporation into brain metabolites
can be measured. Substrates labeled with the nonradioactive, stable, 13C isotope have been employed in vivo to study metabolic flux, enzyme activity, and metabolic regulation in the
living brain of animals and humans (6 ,7 ,8 ,9 ,10 ,11 ,12 ,13 ,14 ,15 ,16 ,17 ,18 ,19 ,20 ,21 ,22 ,23 ,24 ,25 ,26 ,27 ,28 ,29 ,30 ,31 ,32 ,33 ,34 ,35 ,36 ,37 ,38 and 39 ). Enhanced
sensitivity may be achieved by measuring the 13C enrichment of a molecule through indirect detection through 1H MRS. From these measurements the flux through specific metabolic
pathways may be calculated (17 ,18 ).

This chapter covers the recent development of in vivo MRS to study neuronal glutamate and GABA metabolism and the relationship of amino acid metabolism to functional
neuroenergetics. The brain pools of GABA, glutamate, and glutamine have been shown to be localized within glutamatergic neurons, GABAergic neurons, and glia, respectively (under
nonpathologic conditions). Under nonfasting conditions glucose is the almost exclusive source of energy for the brain. By following the flow of 13C label from glucose into these
metabolites, MRS has been used to determine the separate rates of glucose oxidation in these cell types. The metabolism of glutamatergic neurons, GABAergic neurons, and glia is
coupled by neurotransmitter cycles. In the glutamate/glutamine cycle, glutamate released from nerve terminals (by either vesicular release or transport reversal) is transported into
surrounding glial cells, and converted to glutamine. Glutamine in then transported out of the glia and into the neurons, where it is converted back to glutamate, thereby completing
the cycle (Fig. 25.1 ). By following the flow of 13C label from glutamate into glutamine, the rate of the glutamate/glutamine cycle may be determined using MRS. Through a similar
strategy the GABA/glutamine cycle may be measured.

FIGURE 25.1. Schematic representations of the glutamate/glutamine cycle between neurons and astrocytes and
the detoxification pathway of glutamine synthesis. A: The glutamatine/glutamate cycle between neurons and
astrocytes. Released neurotransmitter glutamate is transported from the synaptic cleft by surrounding astrocytic
end processes. Once in the astrocyte, glutamate is converted to glutamine by glutamine synthetase. Glutamine is
released by the astrocyte, transported into the neuron, and converted to glutamate by phosphate-activated
glutaminase (PAG), which completes the cycle. B: Including the ammonia detoxification (or anaplerotic) pathway
of glutamine synthesis. The net rate of glutamine synthesis reflects both neurotransmitter cycling (Vcycle) and
anaplerosis (Vana). The stoichiometric relationships required by mass balance between the net balance of
ammonia and glutamine and Vana are given in Eq. 2. C: An alternative model for neuronal glutamate repletion in
which the astrocyte repletes the lost neuronal glutamate by providing the neuron with α-ketoglutarate [or
equivalently other tricarboxylic acid cycle (TCA) intermediates] (32,33 and 34). α-Ketoglutarate is converted
back to glutamate by neuronal glutamate dehydrogenase. Glc, glucose; a-KG, α-ketoglutarate: Vtrans, net rate of
net ammonia transport into the brain (VNH4 in the text); Vefflux, rate of glutamine efflux from the brain; Vana,
anaplerotic flux; Vcycle, rate of the glutamate/glutamine cycle; Vgln, rate of glutamine synthesis. Using [2-13C]
glucose (27) and [2-13C] acetate precursors these pathways may now be distinguished.

The application of MRS to study brain glutamate and GABA metabolism and the coupling of neurotransmitter cycling to neuroenergetics have provided several new and controversial
insights into the relationship of brain metabolism and function. Contrary to the previous view of a separate metabolic and neurotransmitter pool of glutamate, glutamate release and
recycling have been shown to be a major metabolic pathway. MRS studies of GABA metabolism in the rodent and human brain have suggested that there is also an important role of the
metabolic pool of GABA in inhibitory function. Another key finding is that the glutamate/glutamine cycle in the cerebral cortex is coupled in a close to 1:1 ratio to neuronal (primarily
glutamatergic) glucose oxidation above isoelectricity. This finding, in combination with cellular studies, has led to a model for the coupling between functional neuroenergetics and
glutamate neurotransmission. The coupling between neurotransmission and neuroenergetics provides a linkage between the functional imaging signal and specific neuronal processes.
This chapter reviews these findings and discusses some of their implications for functional imaging.

MRS is a low spatial resolution method, with a resolution for studying neurotransmitter systems of approximately 1 to 4 mm3 in animal models and 7 to 40 mm3 in human brain. Even in
the best case the MRS signal is the sum of the signal from a large number of neurons and glia including many different subtypes. Fortunately, nature has localized key enzymes and
metabolites involved in neurotransmitter cycling in specific cell types, which greatly simplifies the interpretation of the MRS measurements. The evidence of the cellular
compartmentalization of metabolism largely derives from invasive methods with cellular and subcellular resolution, which are reviewed here. As with any new technique there are still
uncertainties due to methodologic issues. Studies performed to validate the MRS measurements will be reviewed, and present limits in measurement accuracy and interpretation
delineated.

 IN VIVO 13C MRS MEASUREMENTS OF THE PATHWAYS OF GLUCOSE OXIDATION: FINDINGS AND VALIDATION
 IN VIVO MRS MEASUREMENTS OF THE RATE OF THE GLUTAMATE/GLUTAMINE CYCLE: FINDINGS AND VALIDATION
 DETERMINATION OF THE IN VIVO COUPLING BETWEEN THE RATE OF THE GLUTAMATE/GLUTAMINE NEUROTRANSMITTER CYCLE AND NEURONAL GLUCOSE OXIDATION
 IN VIVO MRS STUDIES OF GABA METABOLISM AND THE EFFECTS OF DISEASE AND PHARMACOLOGIC TREATMENT ON HUMAN GABA METABOLISM
 IN VIVO MRS MEASUREMENTS OF NEUROENERGETICS DURING FUNCTIONAL ACTIVATION
 IMPLICATIONS OF MRS STUDIES FOR UNDERSTANDING BRAIN FUNCTION
 SUMMARY AND CONCLUSIONS
 ACKNOWLEDGMENTS

IN VIVO C MRS MEASUREMENTS OF THE PATHWAYS OF GLUCOSE

13

OXIDATION: FINDINGS AND VALIDATION

Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional Neuroenergetics "

This section reviews studies in which MRS was used to measure the pathways of glucose oxidation in the cerebral cortex. Glucose oxidation under nonfasting conditions is almost the
exclusive source of energy for the brain. The localization of key enzymes involved in GABA and glutamate metabolism in specific cell types provides the capability for MRS to study
their separate neuroenergetic requirements. As shown in Fig. 25.2 , which is a 13C MRS spectrum obtained by Gruetter and co-workers (35 ) at 4 T, the chemical specificity of MRS
allows the flow of 13C label from glucose to be followed into several metabolites in the brain coupled to energy metabolism including aspartate, GABA, glutamate, and glutamine. The
major finding of these studies is that in normal conditions in nonactivated human cerebral cortex and in rodent models, glucose oxidation in glutamatergic neurons accounts for
between 60% and 80% of cerebral cortex energy consumption. The remaining 20% to 40% is primarily distributed between GABAergic neurons and glia.

FIGURE 25.2. 13C magnetic resonance spectroscopy (MRS) spectrum in the occipital/parietal lobe at 4 T.
The figure shows a 50-minute accumulation 13C MRS spectrum obtained at 4 T approximately 60 minutes
after the start of a 1-13C glucose infusion. The spectrum was obtained from a 72-mL volume centered on
the midline in the occipital/parietal lobe. The top trace is an expansion of regions of the bottom trace.
Labeled resonances include the C2, C3, and C4 positions of glutamate, glutamine, aspartate, and γ-
aminobutyric acid (GABA) and the C3 position of lactate. As described in the text (see In Vivo 13C MRS
Measurements of the Pathways of Glucose Oxidation: Findings and Validation), the localization of the
synthetic enzymes and pools of GABA and glutamine to GABAergic neurons and glia, respectively, and the
localization of the majority of the glutamate pool to glutamatergic neurons, allows the relative rates of
glucose oxidation in these cell types to be determined from the flow of 13C label into these pools. (From
Gruetter R, Seaquist ER, Kim S, et al. Localized in vivo 13C-NMR of glutamate metabolism in the human
brain: initial results at 4 tesla. Dev Neurosci 1999;20:380–388, with permission.)

MRS Measurement of the Rate of Glucose Oxidation in Glutamatergic Neurons

The initial use of MRS to study brain metabolism was to measure glucose oxidation by following the flow of 13C isotope from [1-13C] glucose into the C4 position of glutamate (2 ,6 ).
Figure 25.3 diagrams the flow of 13C label from a [1-13C] glucose precursor to C4-glutamate and subsequently C4-glutamine. Glucose is metabolized to pyruvate by the glycolytic
pathway, which labels C3-pyruvate. The
 P.318

label is then transferred to the tricarboxylic acid cycle (TCA) by the actions of pyruvate dehydrogenase (PDH) and citrate synthase.
When the label reaches C4-α-ketoglutarate it is transferred to the large neuronal glutamate pool by the high activity exchange
reactions of the amino acid transaminases and mitochondrial/cytosolic transporters. The large glutamate pool was first identified in
14
C tracer studies (40 ). Based on kinetic and immunohistochemical staining studies, it is believed to correspond to the glutamate
pool of glutamatergic neurons (18 ,41 ,42 ). Due to the rate of these exchange reactions being many times faster than the TCA cycle
the glutamate pool acts as a label trap for isotope that enters the neuronal TCA cycle via pyruvate dehydrogenase (17 ,18 ). 13C MRS
may be used to measure the accumulation of 13C label into the trapping glutamate pool, and the kinetic curves analyzed by
metabolic modeling to calculate the rate of the neuronal TCA cycle (18 ). The trapping pool assumption is not essential to determine
the rate of the TCA cycle because subsequent labeling in the C3 position of glutamate can be measured to allow calculation of the
label exchange rate (17 ,18 ). Because glucose is the primary fuel for neuronal oxidation, the measurements of the TCA cycle may
be converted to measurements of glucose oxidation using known stoichiometries (17 ,18 ).

FIGURE 25.3. Isotopic labeling of C4-glutamine by the glutamate/glutamine cycle from a [1-13C] glucose precursor. Infused [1-
13
C] glucose labels neuronal C3-pyruvate. This label is then incorporated via the combined action of pyruvate dehydrogenase
and the TCA cycle into α-ketoglutarate, which is in rapid exchange with glutamate due to the action of several transaminases.
The large glutamate pool in the neuron acts as a label trap with [4-13C]-glutamate accumulating at the rate of the neuronal
TCA cycle. Released [4-13C] glutamate from the nerve terminal is taken up by glial transport and the 13C label is transferred to
[4-13C] glutamine through the action of glutamine synthetase at the rate of the glutamate/glutamine cycle. Interpretation of
glutamine labeling is complicated by 13C label entering by the astrocyte pyruvate dehydrogenase reaction. MRS studies using 15N
ammonia, [2-13C] glucose, and [2-13C] acetate, as well as comparison with traditional measurements of the uptake of net
glutamine precursors, have shown that the majority of labeling in glutamine from [1-13C] glucose is from the
glutamate/glutamine cycle (27,36,37,38 and 39).

The rate of neuronal glucose oxidation has been determined in several studies from 13C MRS and 1H-13C MRS measurements of cerebral
cortex glutamate turnover from a [1-13C] glucose precursor in animal models (2 ,14 ,15 ,16 and 17 ,21 ,22 ,25 ,26 and 27 ) and
humans (12 ,13 ,18 ,19 ,29 ,31 ,35 ,43 ,44 ). Comparison of the rates of neuronal glucose oxidation measured in these studies with
conventional arteriovenous (AV) difference and PET measurements of total glucose consumption found that the majority (between
70% and 90%) of total glucose oxidation in the rat and human brain is associated with the large glutamate pool, believed to reflect
glutamatergic neurons, measured by MRS. In two recent 13C MRS studies of resting awake human occipital parietal cortex, in which
other pathways of glucose metabolism were directly measured, a similar range of between 60% (35 ) and 80% (29 ) of total glucose
oxidation was calculated for the large glutamate pool. The large percentage of cortical synapses that are glutamatergic and the high
electrical activity of glutamatergic pyramidal cells (4 ,45 ) may explain why such a large fraction of total glucose oxidation is
associated with glutamatergic neurons.

A caveat to the interpretation of the glutamate turnover measurement is that glutamate is present in all brain cells. Based on the
sensitivity limitations of staining methods and kinetic studies in measuring glutamate levels in glia and other neuron types,
particularly GABAergic, the assignment of the fraction of glucose oxidation occurring in glutamatergic neurons may be overestimated
by up to 20%. In the future, the fraction of glutamate in glia may be measured more accurately through dynamic 13C MRS
measurements of glutamate and glutamine labeling during the infusion of labeled acetate that is incorporated into the brain
selectively in the glia (28 ,38 ,39 ).

MRS Measurements of the Rate Glucose Oxidation in GABAergic Neurons

GABA is the major inhibitory neurotransmitter and may represent over 30% of the synapses in the cerebral cortex (4 ,46 ,47 ). GABA
is synthesized from glutamate in GABAergic neurons by the enzyme glutamic acid decarboxylase (GAD). GABA may then be returned
to the TCA cycle through successive action of the GABA shunt enzymes, GABA-transaminase, and succinic semialdehyde
dehydrogenase, or released from the neuron. Almost all of the brain GABA pool is localized to GABAergic neurons under normal
conditions. The labeling of the GABA pool from [1-13C] glucose provides a minimum estimate of the rate of glucose oxidation in the
GABAergic neuron. The estimate is a minimum because label may bypass GABA and continue from α-ketoglutarate/glutamate into
the TCA cycle directly. In vitro MRS analysis of cerebral cortex from extracts of rats infused with [1-13C] glucose has been used to
measure the time course of labeling in the GABA and glutamate pools (24 ,48 ). The isotopic labeling results of the Manor et al. (24 )
 P.319

study were analyzed with a metabolic model to determine the relative rates of glucose oxidation in the glutamate and GABA pool.
Under conditions of α-chloralose anesthesia the rate of glucose oxidation in GABAergic neurons was estimated to be between 10%
and 20% of total neuronal glucose oxidation. This value is similar to previous estimates obtained using isotopic methods and by
inhibiting the degradative enzyme GABA transaminase (24 ). It should be noted that determination of the rate of GABA synthesis
from isotopic methods depends on the assumption that the glutamate precursor pool for GABA is severalfold lower in concentration
than GABA, an assumption that is consistent with findings using cellular staining (41 ,42 ). In the recent studies of human cerebral
cortex (13 ,29 ,35 ), the rate of GABA synthesis, and by inference glucose oxidation in the GABAergic pool, was estimated to be on
the order of 10% of total glucose oxidation, although no rates were given. In the future, with the higher sensitivity available using
inverse MRS methods in combination with the development of ultrahigh field magnets for human studies, measurements of the rate
of glucose oxidation in GABAergic neurons should be possible in humans.
 P.320

In Vivo MRS Measurements of the Rate of Glucose Oxidation in Glia

A long-term controversy in brain metabolism studies has been the rate of glucose oxidation in glial cells. Early estimates range from
10% to over 50% of glucose oxidation (49 ). MRS may be used to measure the rate of glial glucose oxidation based on the localization
of the enzyme glutamine synthetase in the glia (50 ). This localization allows the rate of the glial TCA cycle to be calculated from
the labeling of glutamine from glial glutamate. The most quantitative early findings were by Van den Berg and co-workers (40 ), who,
using 14C isotopic labeling strategies, assigned a rate to glial pyruvate dehydrogenase, which they referred to as the small glutamate
pool, of 15% to 25% of total pyruvate dehydrogenase (neuronal + glial) activity. The pyruvate dehydrogenase rate is equal to the rate
of complete glucose oxidation by the TCA cycle plus the rate of net glial anaplerosis. These measurements were performed using
extract analysis of whole brains. Two recent 13C MRS measurements of humans have measured glial pyruvate dehydrogenase as
accounting for between 8% (29 ) and 15% (35 ) of total pyruvate dehydrogenase activity in the occipital parietal lobe. A limitation of
these studies is that they did not measure the rate of the glial TCA cycle, only the pyruvate dehydrogenase step, and therefore the
total oxidative energy produced in the glia was not calculated. In a preliminary study in human cerebral cortex using [2-13C] acetate,
which is exclusively incorporated into the astrocyte (28 ), as a tracer Lebon et al. (38 ) found that the glial TCA cycle accounts for
approximately 15% of total glucose oxidation.

Summary and Remaining Questions

In vivo MRS measurements of nonactivated cerebral cortex in rats and humans have found that from 60% to over 80% of glucose
oxidation is associated with the large glutamate pool, reflecting primarily glutamatergic neurons. The remainder is primarily
distributed between GABAergic neurons and glia. The development of new labeling strategies such as [2-13C] acetate and higher
sensitivity MRS measurements should allow the contributions of these cell types to be more accurately determined. Within the error
of the MRS measurements, and the contribution of glutamate pools in other neuronal classes to the glucose oxidation measurement,
there may be up to 10% of total glucose oxidation available for other cell types such as dopaminergic and serotoninergic nerve
terminals. An objection that has been raised to these findings is the possibility that small highly metabolically active pools may be
missed by the MRS method. Under nonstimulated conditions the good agreement of the MRS measurement with AV difference and
PET measurements of total glucose consumption indicate that the contribution of these small pools is not large. MRS measurements
of glucose metabolism during cortical activation will be reviewed below (see In Vivo MRS Measurements of Neuroenergetics During
Functional Activation ).

IN VIVO MRS MEASUREMENTS OF THE RATE OF THE

GLUTAMATE/GLUTAMINE CYCLE: FINDINGS AND VALIDATION
Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "

The function of the glutamate/glutamine cycle is to prevent depletion of the nerve terminal glutamate pool by synaptic release.
Glial cells have a high capacity for transporting glutamate from the synaptic cleft in order to maintain a low ECF (extracellular fluid)
concentration of glutamate (50 ,51 ). In vivo and in vitro studies indicate that glutamate released by the neuron is taken up by the
glia and converted to glutamine by glutamine synthetase (53 ,54 ), an enzyme found exclusively in glia (52 ). Glutamine is
transported from the glia into the ECF where it is taken up by neurons and converted back to glutamate through the action of
phosphate-activated glutaminase (PAG) (55 ). Based on extensive data from isotopic labeling studies, immunohistochemical staining
of cortical cells for specific enzymes, isolated cell, and tissue fractionation studies, it has been proposed that glutamate (as well as
GABA) taken up by the glia from the synaptic cleft may be returned to the neuron in the form of glutamine (40 ,56 ,57 and 58 ). The
generally accepted model of the glutamate/glutamine neurotransmitter cycle is shown in Fig. 25.1A .

Despite a wealth of evidence from enzyme localization and isolated cell studies, the rate of the glutamate/glutamine cycle and its
importance for brain function have been controversial due to difficulties in performing measurements in the living brain. Because
the neurotransmitter glutamate is packaged in vesicles (59 ,60 ), controversy has arisen about the fraction of glutamate actually
involved in the cycle, leading to the concept of a small “transmitter” versus a large “metabolic” glutamate pool. Supporting the
concept that glutamate neurotransmitter flux is a small fraction of total glucose metabolism are findings in isolated cells and
nonactivated brain slices of a low rate of label incorporation from [1-13C] glucose (61 ). The concept of a metabolically inactive
neurotransmitter pool was brought into question in 1995, when, using 13C nuclear magnetic resonance (NMR), we measured a high
rate of glutamine labeling from [1-13C] glucose in the occipital/parietal lobe of human subjects (12 ). A high rate of glutamine
synthesis was calculated from these data (18 ). At the time of the initial 13C NMR study, the rate of the glutamate/glutamine cycle
could not be calculated due to the lack of a model for distinguishing isotopic labeling from this cycle from other sources of
glutamine labeling, most significantly removal of cytosolic ammonia produced by metabolism and uptake of plasma ammonia (62 ).
Net ammonia removal requires the de novo glutamine synthesis via the anaplerotic pathway in the glia. In addition, several other
pathways, including the glial TCA cycle, have been proposed as providing significant precursors for glutamine
 P.321

synthesis (61 ,62 ). To calculate the rate of the glutamate/glutamine cycle, Sibson et al. (25 ) developed a metabolic model for
separating the pathways of glutamine synthesis.

This section reviews 13C MRS measurements of the glutamate/glutamine cycle, emphasizing studies performed to validate the MRS
technique. The important and surprising result of these studies is that the glutamate/glutamine cycle is a major metabolic flux, far
exceeding de novo glutamine synthesis. The rate of the glutamate/glutamine cycle in the awake resting human cerebral cortex is
between 60% and 80% of total glucose oxidation.

Development of a Two-Compartment Metabolic Model of Glutamine Metabolism to

Separately Determine the Rate of the Glutamate/Glutamine Cycle and Anaplerotic
Glutamine Synthesis
The rate of the glutamate/glutamine cycle is calculated from the time course 13C labeling of glutamine relative to the labeling of its
precursor neuronal glutamate. If neuronal glutamate were the only precursor of glutamine, the calculation would be straightforward.
Unfortunately, the calculation is complicated by label from [1-13C] glucose entering both the neuronal and glial TCA cycles via
pyruvate dehydrogenase. Glutamate in both cell types will be labeled in the C4 position by exchange with α-ketoglutarate in the
TCA cycle (Fig. 25.3 ). The flow of label from C4-glutamate into C4-glutamine is proportional to the total rate of glutamine synthesis.
However, unless the relative flow of 13C label into the glial glutamate pool from the glial pyruvate dehydrogenase and neuronal
glutamate are distinguished, the fraction of glutamine synthesis due to the glutamate/glutamine cycle cannot be calculated. To
determine the rate of the glutamate/glutamine cycle from a [1-13C] glucose precursor, we developed a metabolic model to constrain
the rate of glutamine labeling from glial pyruvate dehydrogenase.

Glutamine production via glutamine synthetase requires two substrates, glutamate and ammonia. As shown in the flow diagram of
Fig. 25.1A , glutamine synthesis receives precursor glutamate from both glial uptake of released neurotransmitter glutamate and
glial anaplerosis. A mathematical model was developed to interpret isotopic data in order to separate these pathways
(25 ,27 ,29 ,36 ). The model extends previous formulations by imposing mass balance constraints on the brain glutamate and
glutamine pools that relate the rate of de novo glutamine synthesis to the net uptake of anaplerotic precursors from the blood.
Glutamine efflux is the primary source of nitrogen removal from the brain (49 ,62 ). Nitrogen must be removed from the brain in
order to maintain low concentrations of ammonia, which when elevated will interfere with brain function (62 ). Because at steady
state the concentration of glutamine remains constant, loss of glutamine by efflux (Vefflux) must be compensated for by de novo
synthesis of glutamine by anaplerosis (Vana). For de novo synthesis by anaplerosis, pyruvate derived from glucose is converted by CO2
fixation (VCO2) to oxaloacetate by the enzyme pyruvate carboxylase, which is active only in the glia (54 ). Through the action of the
TCA cycle oxaloacetate is converted to α-ketoglutarate, which may be converted to glutamate either by ammonia fixation via glia
glutamate dehydrogenase or alternatively through transamination with other amino acids (37 ). Glial glutamate is then converted to
glutamine by glutamine synthetase. One or two ammonia molecules are fixed per glutamine molecule synthesized through
anaplerosis, depending on the relative fluxes of NH4+ fixation versus transamination. Applying nitrogen mass balance constraints
leads to the relationship VNH4 = (1 to 2)Vefflux at steady state. The additional requirement of carbon mass balance leads to the
following relationship:

Total glutamine synthesis is then related to synthesis for ammonia detoxification (Vana) and the glutamate/glutamine cycle (Vcycle) by
the following expression:

Note that VCO2 may be higher than Vana if anaplerosis is needed to replace TCA cycle intermediates lost by oxidative processes or
pyruvate recycling (63 ,64 ).

Examination of Eq. 2 indicates that Vcycle may be derived from a measurement of Vgln from a 13C MRS experiment in combination with a
measurement of any of the rates linked by mass balance considerations to anaplerotic glutamine synthesis. A limitation of isotopic
measurements of flux is that isotopic exchange cannot be distinguished from net flux. The linkage between the labeling of glutamine
through glial pyruvate dehydrogenase and the brain anaplerosis flux allows the validation of isotopic measurements of glutamine 13C
and 15N labeling against traditional AV difference measurements.

The glutamate/glutamine cycle measurement using a [1-13C] glucose precursor also includes contributions from the GABA/glutamine
cycle (34 ,57 ,65 ). GABA is the main inhibitory neurotransmitter, and has been measured by in vivo 1H and 13C MRS in animals and
humans (12 ,13 ,24 ,29 ,35 ,66 ) (see In Vivo MRS Studies of GABA Metabolism and the Effects of Disease and Pharmacologic
Treatment on Human GABA Metabolism , below). The glutamate/glutamine and GABA/glutamine pathway may be distinguished using
[2-13C] glucose and [2-13C] acetate as precursors as described below and in the section In Vivo MRS Studies of GABA Metabolism .

13
C NMR Studies of the Glutamate/Glutamine Cycle in Rat Cerebral Cortex
To determine the rate of glutamine synthesis, rats were studied under α-chloralose anesthesia in a 7-T modified Bruker
 P.322

Biospec spectrometer. A small 13C surface coil was used for transmission and reception. The spectroscopic volume was localized
primarily to the motor and somatosensory cortices. The rats were infused with [1-13C] glucose, and the time course of label
incorporation into the C4 positions of glutamate and glutamine was measured. The time courses were fitted using differential
equations describing the proposed model of glutamate/glutamine cycle. The rate of the neuronal TCA cycle as measured from label
incorporation into the C4-glutamate was 0.46 ± 0.12 μmol/g-min [mean ± standard deviation (SD), n = 5]. The rate of glutamine
synthesis (Vgln) was 0.21 ± 0.04 μmol/g-min (n = 5), which was nearly half the rate of the TCA cycle (25 ). These results indicate that
glutamine synthesis is a major metabolic pathway in the rat cerebral cortex.

Validation of the Metabolic Model by Comparison of the Increase in the Rate of

Glutamine Synthesis During Hyperammonemia with Independent Measures of Net
Ammonia Uptake, CO Uptake, and Glutamine Efflux
2

Elevated plasma ammonia increases the rate of the anaplerotic pathway of glutamine synthesis (62 ) in order to remove ammonia
from the brain. The metabolic model predicts that under conditions of elevated plasma ammonia the increase in the rate glutamine
synthesis is stoichiometrically coupled to the increase in the uptake of the anaplerotic substrates CO2 and ammonia and the efflux of
glutamine from the brain (ΔVgln = ΔVana = ΔVefflux = ΔVCO2 = ½ΔVNH4s). To test the 13C MRS measurement, glutamine synthesis in rat
cerebral cortex was measured under normal and elevated plasma ammonia concentrations. Rats were made hyperammonemic (0.35
± 0.08 mM plasma ammonia vs. basal levels of 0.05 ± 0.01 mM) by a primed continuous infusion of ammonia and studied after 4 hours
of hyperammonemia to ensure metabolic steady state. The neuronal TCA cycle rate was not significantly increased under these
conditions relative to the control condition, which suggests that brain electrical activity and by inference the glutamate/glutamine
cycle were not substantially altered. The rate of glutamine synthesis under hyperammonemic conditions increased by 0.11 ± 0.03
μmol/g-min relative to the rate under normal plasma ammonia levels. The increase in the rate of glutamine efflux (Vefflux) measured
by AV difference under similar conditions was 0.10 μmol/g-min (67 ), in good agreement with ΔVgln. Studies that have used 14C
isotope to measure the increase in VCO2 with hyperammonemia found a rate of ˜0.15 μmol/min-g (68 ), which is slightly higher than
measured by 13C MRS, possibly due to the need for additional incorporation of CO2 to replace TCA cycle intermediates lost by
oxidation. As described below, both AV difference and direct isotope incorporation measurements of ammonia fixation into
glutamine under hyperammonemic conditions are also consistent with the predictions of the model. The agreement between the
increase in Vgln determined by 13C MRS and the increase measured by conventional methods in anaplerotic substrate utilization and
glutamine efflux predicted by Eq. 2 provides strong experimental support for the ability to determine Vcycle under normal physiologic
conditions.

N MRS Studies to Test the Relationship Between Anaplerotic Glutamine Synthesis

15

and Ammonia Detoxification

N MRS is a useful method for both in vitro and in vivo study of cerebral glutamate/glutamine metabolism under hyperammonemic
15

conditions based on the measurement of [5-15N] glutamine and [2-15N] glutamate/glutamine (69 ,70 ). Incorporation of 15N labeled
ammonia into the N5 position of glutamine may be analyzed to calculate the flux through glutamine synthetase. In the absence of
label exchange, the rate of incorporation of labeled ammonia into the N2 position of glutamate + glutamine may be analyzed to
calculate the rate of glutamate dehydrogenase.

The relationships in Eq. 2, which were used in the modeling of the 13C MRS data to deconvolute 13C labeling in C4-glutamine from
neuronal glutamate and glial PDH, are based on mass balance and previous AV difference and ammonia trapping studies. To further
test the relationship of Eq. 2 between the rate of ammonia uptake in the cerebral cortex (VNH4+) and anaplerotic glutamine synthesis
(Vana), total ammonia uptake was calculated from the time course of the sum of 15N labeled N5 glutamine and N2 glutamate +
glutamine in rat cerebral cortex during infusion of 15N ammonia. These were the only compounds into which appreciable 15N label
incorporation was observed, which agrees with previous findings that the major flows of ammonia in the brain involve these
metabolites (62 ). The calculated VNH4+ from these data was 0.13 ± 0.02 μmol/g-min (n = 6). Based on the stoichiometric relationship
of the model of ½ΔVNH4 = ΔVana, a rate of anaplerotic glutamine formation of 0.065 ± 0.01 μmol/g-min was predicted. From this
measurement an increase in the cerebral glutamine pool during the infusion of 0.065 μmol/min/g × 180 min = 11.7 μmol/g of
glutamine was predicted. This calculation is in excellent agreement with the measured increase in glutamine concentration at the
end of the study of 11.1 ± 0.4 μmol/g (36 ).

C MRS Determination of the Rate of the Glutamate/Glutamine Neurotransmitter

13

Cycle Under Normal Physiologic Conditions

To determine the rate of the glutamate/glutamine cycle from a [1-13C] glucose precursor under physiologic conditions,
 P.323

Sibson et al. (25 ) measured Vgln and calculated Vana using Eq. 2 and previously published measurements (62 ,71 ). The value of Vana
calculated in this manner ranged from 0.00 to 0.04 μmol/g-min. Comparison with the 13C MRS measurement of Vgln of 0.21 ± 0.04
μmol/g-min, yields a Vcycle that is 80% to 90% of the rate of glutamine synthesis. A similar high percentage of Vcycle was calculated
using measurements of the net incorporation of 14CO2 into the cerebral cortex (72 ). The CO2 measurement is coupled to total brain
anaplerosis, which may be higher than anaplerosis used for net glutamine synthesis, and therefore represents the maximum estimate
of this flux.

Validation of the Measurement of Glutamine Synthesis by Comparison of Rates

Calculated from N MRS and C MRS Results
15 13

To obtain an independent measurement of Vgln and Vana, 15N MRS was used to measure the rate of 15N-labeled ammonia incorporation
into the N5 position of glutamine and the unresolved resonance of N2 glutamate plus glutamine (36 ). A mathematical analysis based
on the model was used to derive Vgln from the MRS measurement of the time course of [5-15N]glutamine and [2-15N] glutamate +
glutamine. The labeling in the first hour was almost exclusively within the N5 position of glutamine, which is consistent with the
delayed onset of anaplerosis previously reported under these conditions (73 ) and previous measurements using 13N and 15N labeled
ammonia (62 ,69 ). The low initial rate of anaplerosis allows the rates determined from the 15N NMR study to be compared with the
rates measured by 13C NMR under normal physiologic conditions. The measured Vgln of 0.20 ± 0.06 μmol/g-min (mean ± SD, n = 6) from
these studies (36 ) is in excellent agreement with the results from the 13C NMR measurement of 0.21 ± 0.04 μmol/g-min (25 ).

Validation of the C MRS Measurement of the Glutamate/Glutamine Cycle, and

13

Assessment of Alternate Models of Neuronal/Glial Trafficking, Through Comparison

of Results Using [1- C] Glucose, [2- C] Glucose, NH4, and [2- C] Acetate as
13 13 15 13

Precursors
Several alternative models to the glutamate/glutamine cycle (Fig 25.1A ,Fig 25.1B ) have been proposed. In one alternative model
the 13C labeling of glutamine represents an internal glial glutamate/glutamine cycle as opposed to trafficking between the neuron
and glia. Label enters C4-glutamine from [1-13C] glucose in this model through exchange in the glial cell between glutamate and
glutamine catalyzed either through the reverse reaction of glutamine synthetase, or alternatively via glial phosphate activated
glutaminase. Released neuronal glutamate in this model is taken up directly by the nerve terminal. In another alternate model,
diagrammed in Fig. 25.1C , the glia releases α-ketoglutarate, or equivalently citrate or malate, to the neuron to replace the carbon
skeleton of released glutamate (32 ,34 ,64 ). In support of this pathway, which is referred to here as the glutamate/α-ketoglutarate
cycle, several TCA cycle intermediates including malate, α-ketoglutarate, and citrate are released from glia in cell culture and may
be taken up by synaptosomes and cultured neurons (32 ,33 and 34 ).
The two pathways of glutamate trafficking shown in Fig. 25.1 cannot be distinguished on the basis of a 13C MRS study using [1-13C]
glucose as the label source. As described above, [1-13C] glucose will label both the glial and neuronal glutamate pools directly via
pyruvate dehydrogenase. An alternative strategy is to use isotopic precursors that exclusively introduce label into the glia. Analysis
of the flow of isotope from the glia into the neuronal glutamate pool yields the rate of total neuronal/glial glutamate trafficking.
Comparison with the rate calculated using [1-13C] glucose gives the fraction of neuronal/glutamate trafficking due to the
glutamate/glutamine cycle (27 ,36 ).
The initial use with MRS of the strategy of glial selective precursors to calculate the fraction of glutamate trafficking due to the
glutamate/glutamine cycle measurement was by Shen et al. (36 ), who calculated the relative fraction of the glutamate/glutamine
cycle and glutamate/α-ketoglutarate cycle, using 15N MRS measurement of the labeling in glutamine and glutamate from 15NH4+.
Under hyperammonemic conditions the rate of 15N ammonia incorporation into the N5 and N2 position of glutamine is the same in
the glutamate/α-ketoglutarate cycle because only the anaplerotic pathway of glutamine synthesis is present. In contrast, in the
glutamate/glutamine cycle, there is additional incorporation of 15N label into the N5 position of glutamine selectively in the glia (due
to the localization of glutamine synthetase) due to the cycle. To distinguish these models, the endpoint 15N enrichment of the N2
positions of glutamate and glutamine were calculated relative to the glutamine N5 position for each model using the N5 glutamine
labeling curve as an input and compared with experimental values. As shown in Fig. 25.4 , the low 15N fractional enrichment of the
N2 position of glutamine and glutamate relative to glutamine N5 at the end of the study strongly supports the glutamate/glutamine
cycle as the primary pathway of neuronal glutamate repletion.

FIGURE 25.4. Calculated 15N2/15N5 fractional enrichment ratios of glutamine and glutamate for three models of glial glutamine
synthesis. Three models of neuronal glutamate completion were compared with experimental results in which the time course
of [5-15N] glutamine and [2-15N] glutamine and glutamate were measured by 15N nuclear magnetic resonance (NMR) in the cortex
of a rat infused with 15N-labeled ammonia at 7 T (36). The measured ratio at the end of the infusion is in excellent agreement
with the ratio predicted if the glutamate/glutamine cycle is the major pathway of astrocytic repletion of released neuronal
glutamate (model a, which is diagrammed in Fig 25.1A,Fig 25.1B). If instead the cycle was internal to the astrocyte the N2/N5
glutamine relative 15N enrichment would be two times higher than measured and no labeling would have been observed in N2
glutamate (model b). If glutamate neurotransmitter repletion took place through the astrocytes providing the neurons with α-
ketoglutarate (model c, which is diagrammed in Fig 25.1C), the rate of anaplerotic and total glutamine synthesis would be
similar and the N5/N2 ratio of glutamine would be close to 1.0 as opposed to the measured ratio of 0.25. A similar labeling
strategy has recently been used with [2-13C] glucose and [2-13C] acetate, both substrates that selectively label glutamine
through glial specific pathways, and has put an upper limit of the rate of the α-ketoglutarate/glutamate cycle at 20% of the
total rate of glutamate trafficking, the remainder being due to the glutamate/glutamine cycle (27,38).

An additional test of the glutamate/glutamine cycle model was recently performed using 2-13C] glucose as an isotopic precursor (27 ).
Label from [2-13C] glucose enters the inner positions of glutamate and glutamine only through pyruvate incorporation into the TCA
cycle by pyruvate carboxylase, which is localized to the glia (27 ,74 ). The initial flow of label from this precursor is into the glial
TCA cycle intermediates, and then glial glutamate and glutamine (27 ).
 P.324

Subsequently, neuronal/glial cycling moves the label to the neuron where it labels the large glutamate pool. The labeling measured
in the glutamate pool is the sum of all trafficking pathways from the glia. In contrast the rate of labeling of glutamine from a [1-13C]
glucose precursor is a measure of the glutamate/glutamine cycle. In vivo and in vitro 13C MRS at 7 T was recently used to measure
the labeling time course of glutamate and glutamine in the cerebral cortex of rats under hyperammonemic and normoammonemic
conditions during infusion of either [1-13C] or [2-13C] glucose (27 ). The rate calculated for the neuronal/glial glutamate cycle was
similar, with both labels indicating that the glutamate/glutamine cycle is the major pathway of neuronal/glial glutamate trafficking
accounting for between 80% and 100% of total glutamate trafficking. A similar conclusion was recently reported for human cerebral
cortex using [2-13C] acetate as a precursor (38 ), which selectively introduces label into glutamate and glutamine through glial
pyruvate dehydrogenase.

The Effect of Glutamate Oxidation on the Glutamate/Glutamine Cycle Measurement

and Estimates of Its Rate In Vivo
An alternate pathway of neuronal/glial glutamate trafficking is glial glutamate oxidation (10 ,63 ,64 ). In this pathway glutamate
taken up by the glial cell is transaminated into α-ketoglutarate and enters the TCA cycle. Reactions in the TCA cycle convert α-
ketoglutarate to oxaloacetate, which is then converted to pyruvate by the action of malic enzyme. The pyruvate formed from
glutamate is oxidized in the TCA cycle through the action of pyruvate dehydrogenase. Glutamate lost to the brain by this pathway is
then replaced by anaplerosis through pyruvate carboxylase. Evidence of this pathway is derived primarily from isolated cell cultures.
It has been proposed that the fraction of glutamate going through this pathway increases with brain electrical activity (64 ).

The major effect of the glutamate oxidation pathway on the MRS measurement of the glutamate/glutamine cycle is to cause the
fraction of glutamine synthesis of net anaplerosis to be overestimated and Vcycle to be consequently underestimated, because the
labeling of the internal positions of glutamine from the two pathways from [1-13C] and [2-13C] glucose is similar. The unambiguous in
vivo determination of glutamate oxidation requires the 13C label flow from glutamate to pyruvate to be measured (10 ,63 ). This
measurement is complicated by other metabolic pathways that produce similar labeling patterns, including scrambling of isotopic
labeling into other positions of glucose in the liver (23 ,27 ,63 ,75 ), and as a consequence glutamate oxidation has not been
definitively demonstrated in vivo under normal physiologic conditions. Suggestive evidence of this pathway is the finding in several
studies that the rate of anaplerosis under normal ammonia conditions calculated from labeling of glutamine by 13C labeled glucose is
approximately two to three times higher than that predicted from measurements of brain glutamine efflux (27 ). An alternate
possibility is that rather than glutamate oxidation this extra labeling reflects cycling between oxaloacetate and pyruvate to
generate reduced nicotinamide adenine dinucleotide phosphate (NADPH) reducing equivalents in the glia, a pathway that has been
shown to be highly active in the liver (75 ).

Validation of the C MRS Glutamate/Glutamine Cycle Measurement by Correlation

13

with Brain Electrical Activity

If the 13C labeling measured in glutamine by 13C MRS is due to the glutamate/glutamine cycle, then the calculated rate of this
pathway should correlate with brain electrical activity. Neuronal glutamate release is known to increase with neuronal
depolarization associated with action potentials.
 P.325

To test this prediction, 13C MRS was used to measure the rates of neuronal glucose oxidation and the glutamate/glutamine cycle in
the rat cerebral cortex at three levels of cortical electrical activity: isoelectric EEG induced by high-dose pentobarbital anesthesia,
and at two milder levels of anesthesia (26 ). During isoelectric conditions, under which minimal glutamate release takes place,
almost no glutamine synthesis was measured, consistent with the conclusion that the 13C MRS measurement of glutamine synthesis
primarily reflects the glutamate/glutamine cycle. Above isoelectricity, the rates of the glutamate/glutamine cycle and neuronal
glucose oxidation both increased with higher electrical activity. The relationship measured in this study between the rate of the
glutamate/glutamine cycle and neuronal glucose oxidation is described below (see Determination of the In Vivo Coupling Between
the Rate of the Glutamate/Glutamine Neurotransmitter Cycle and Neuronal Glucose Oxidation ).

C MRS Measurements of the Rate of the Glutamate/Glutamine Cycle in Human

13

Cerebral Cortex
In 1994 we first demonstrated that in vivo 13C NMR may be used to measure the rate of glutamine labeling (12 ,18 ) from [1-13C]
glucose in human occipital/parietal cortex. These studies showed clearly that glutamine is labeled rapidly from [1-13C] glucose in the
human cerebral cortex. However, the rate of the glutamate/glutamine cycle was not uniquely determined in the initial experiments
due to the inability to distinguish the glutamate/glutamine cycle from other sources of glutamine labeling. To determine whether
there is a similar high rate of the glutamate/glutamine cycle in human cerebral cortex as in the rat, we (29 ) and Gruetter and co-
workers (13 ,35 ) have determined this rate from 13C MRS measurements in the human occipital/parietal lobe.

A time course from the study of Shen and co-workers (29 ) showing the rapid labeling of C4-glutamine and C4-glutamate from [1-13C]
glucose in a single subject is shown in Fig. 25.5 . A best fit of the metabolic model is plotted through the data. A lag is clearly shown
in the labeling of C4-glutamine relative to C4-glutamate, which is consistent with the large neuronal glutamate pool being the main
precursor for glutamine synthesis. The combination of the metabolic model validated in the rodent and improved MRS sensitivity
allowed the rate of the glutamate/glutamine cycle, the neuronal TCA cycle, the glial TCA cycle, and anaplerotic glutamine synthesis
to be calculated from the 13C MRS data. The analysis gave a total TCA cycle rate of 0.77 ± 0.05 μmol/min/g (mean ± SD, n = 6), a
neuronal TCA cycle rate of 0.71 ± 0.02 μmol/min/g, a glial TCA cycle rate of 0.06 ± 0.02 μmol/min/g, a glutamate-glutamine cycle
rate of 0.32 ± 0.04 μmol/min/g (mean ± SD, n = 6), an anaplerotic glutamine synthesis rate of 0.04 ± 0.02, and a glucose oxidation
rate of 0.39 ± 0.03 μmol/min/g (mean ± SD, n = 6). In agreement with studies in rat cortex, the glutamate/glutamine cycle is a
major metabolic flux in the resting human brain with a rate approximately 80% of the rate of total glucose oxidation.

FIGURE 25.5. In vivo 13C NMR time course of the human occipital/parietal lobe: the time course from one subject of the
concentrations of [4-13C] glutamate and [4-13C]glutamine during a [1-13C] glucose infusion, and the best fit of the two-
compartment model to these data. At time 0 on the plot an intravenous infusion of [1-13C] glucose was started. The model is
shown to provide an excellent fit to the data. The rise of [4-13C] glutamine is clearly seen to lag the labeling of [4-13C]
glutamate, consistent with neuronal glutamate being the main precursor for glutamine synthesis via the
glutamate/GABA/glutamine cycle. *, glutamate; ○, glutamine. (From Shen J, Petersen KF, Behar KL, et al. Determination of
the rate of the glutamate-glutamine cycle in the human brain by in vivo 13C NMR. Proc Natl Acad Sci USA 1999;96:8235–8240,
with permission.)

In a study performed at 4 T, Gruetter and co-workers (13 ) measured rapid labeling of glutamate and glutamine from infused [1-13C]
glucose using 13C MRS. A high rate of the glutamate/glutamine cycle was measured using a two-compartment model, similar to the
model used by Shen and co-workers (29 ). The improved spectroscopic resolution provided by the higher field strength at 4 T
allowed the additional positions of the C2 and C3 resonances of aspartate, glutamate, and glutamine to be incorporated into the
modeling. More recently Gruetter and co-workers (35 ) studied six subjects using localized 13C MRS measurements of a 45-mL volume
in the occipital lobe. The main differences from the rates derived from the Shen et al. (29 ) study are a higher rate of anaplerosis,
approximately 25% of total glutamine synthesis as opposed to 11% in the Shen et al. study and a somewhat lower rate of the
neuronal TCA cycle of 0.62 ± 0.05 μmol/min/g. The lower calculated neuronal TCA cycle rate was due to a lower rate of neuronal
mitochondrial α-ketoglurate/glutamate exchange calculated from the data than in a previous study by Mason and co-workers (18 ).
The lower exchange rate was due to the assignment of a higher concentration of aspartate in the glutamatergic
 P.326

neuron and glutamate in the astrocyte in the metabolic model of Gruetter and co-workers. The higher anaplerosis rate also reflects
differences in which the isotopic data was modeled. In the Shen et al. study anaplerosis was calculated primarily from the labeling
kinetics of C4-glutamine and C4-glutamate, whereas in the Gruetter et al. (35 ) study it was calculated primarily from the
measurement of the time course of the differential 13C labeling of the C2, C3, and C4 glutamate and glutamine resonances. Both
approaches suffer from needing to deconvolute 13C label entering these carbon positions from pyruvate dehydrogenase from the
label entering via pyruvate carboxylase. In the future these differences should be reconcilable by using labeling strategies such as
[2-13C] glucose, which labels glutamate and glutamine internal positions only by pyruvate carboxylase. If the anaplerotic pathway is
due to glutamate oxidation (see Validation of the 13C MRS Glutamate/Glutamine Cycle Measurement by Correlation with Brain
Electrical Activity , above) as opposed to ammonia detoxification, the rate of the glutamate/glutamine cycle reported in both
studies is an underestimate by the calculated rate of anaplerosis. The differences in the anaplerotic flux calculated in these studies
should not obscure the major point of agreement—that the glutamate/glutamine cycle is major metabolic pathway with a rate
accounting for between 60% and 80% of total glucose oxidation in the cerebral cortex.

Cellular and Molecular Evidence that Astroglia have a Major Role in the Uptake of
Glutamate Released from Neurons
The high rate of the glutamate/glutamine cycle indicates that astroglial uptake of glutamate and GABA plays a key role in
maintaining the low extracellular levels of these neurotransmitters needed for proper receptor-mediated functions. There is
considerable evidence from several lines of research that support this conclusion. Overstimulation of glutamate receptors can lead
to excitotoxicity (76 ,77 ). Studies of glutamatergic synapses have shown them to be closely surrounded by glial end processes
possessing high densities of glutamate transporters (78 ). Glutamate and GABA transporters are sodium dependent and electrogenic
and are present on both neurons and glia (58 ,78 ,79 and 80 ). Glutamate transporters have an affinity, Km, of 1 to 3 μM (80 ), which
is in the range of normal estimated ECF glutamate concentrations. Immunohistochemical studies have showed that the glutamate
transporters GLT-1 and GLAST (glutamate astrocytic transporter) are localized primarily in astrocytes (48 ,81 ,82 and 83 ), whereas
EAAC1 is found on neurons (51 ). Antisense oligonucleotides directed against the astrocytic glutamate transporters GLT-1 or GLAST in
vivo results in elevated ECF glutamate in vivo and excitotoxicity (84 ,85 ). The majority of glutamate uptake after its release
appears to be either postsynaptic or astroglial (86 ,87 ), although an electrophysiologic study of the hippocampal slice suggests that
astroglial uptake dominates (88 ).

Summary and Remaining Questions

MRS allows the glutamate/glutamine cycle to be measured from the labeling of glutamate and glutamine by 13C and 15N labeled
precursors. The major complications in determining the rate of the glutamate/glutamine cycle from isotopic measurements are
separating the labeling of glutamine from the glutamate/glutamine cycle from alternate pathways of glutamine synthesis and
isotopic exchange, and distinguishing different pathways of neuronal/glial glutamate trafficking. To overcome these obstacles the
metabolic modeling of the glutamate/glutamine cycle has been extended to include ammonia detoxification, alternate pathways of
glutamate trafficking, and glutamate oxidation (27 ). The MRS rate measurement has been validated by several strategies including
(a) comparison of the rate of glutamine synthesis measured under different ammonia levels with measurements of anaplerotic
substrates by AV difference and isotopic trapping methods (25 ); (b) comparison of the rates of glutamine synthesis and the
glutamate/glutamine cycle calculated from the isotopic labeling from [1-13C] glucose, [2-13C] glucose, 15N ammonia, and [2-13C]
acetate (27 ,35 ,36 ,39 ); and (c) measurement of the rate of the glutamate/glutamine cycle as a function of brain electrical activity
(26 ,37 ). The results of these studies indicate that the glutamate/glutamine cycle is the major pathway of glutamine synthesis and
neuronal/glial glutamate trafficking under normal conditions, with a rate similar to the rate of neuronal glucose oxidation under
conditions of high electrical activity. Measurements in awake nonstimulated human cerebral cortex have found that the rate of the
glutamate/glutamine cycle is between 60% and 80% of total glucose oxidative metabolism (29 ,35 ).

Objections have been raised to the MRS measurement of the glutamate/glutamine cycle for having neglected alternate pathways of
glutamate trafficking and the need for comparison with direct measurements of neuronal glutamate release. As described above,
isotopic strategies have been developed to assess these pathways and under physiologic conditions they were found to account for
less than 20% of glutamate trafficking. However, under pathologic conditions such as seizure the rate of these pathways may be
much higher. Glutamate oxidation may have a significant contribution to total neuronal/glial glutamate trafficking. However, the
unambiguous in vivo measurement of glutamate oxidation will require strategies for eliminating isotopic labeling from other
pathways. Although direct measurement of bulk neuronal release of glutamate for comparison with 13C MRS is presently not possible,
advances in molecular and cellular methods for studying glutamate transport indicate that neurotransmission is the major, if not
exclusive, pathway of glutamate release from glutamatergic neurons and the vast
 P.327

majority of this flux is taken up by astroglia in the cerebral cortex. Correlation of the MRS glutamate/glutamine cycle with indirect
measures of neuronal glutamate release such as microdialysis and nerve terminal labeling would be highly desirable, as would
further studies better defining the relevant pool sizes and enzyme distribution in glia and glutamatergic neurons, particularly in
regions other than the cerebral cortex.

DETERMINATION OF THE IN VIVO COUPLING BETWEEN THE RATE OF

THE GLUTAMATE/GLUTAMINE NEUROTRANSMITTER CYCLE AND
NEURONAL GLUCOSE OXIDATION
Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "

This section presents evidence from MRS and other studies for a model of the coupling between the glutamate/glutamine cycle and
glial glucose uptake and subsequent neuronal oxidation. The model is based on work in cellular systems primarily by Magistretti and
co-workers (90 ) and recent findings, using 13C MRS in rat cortex, that the glutamate/glutamine cycle (a) increases in rate with
increasing brain electrical activity in a near 1:1 stoichiometry with neuronal glucose oxidation (26 ), and (b) is 60% to 80% of the rate
of total glucose oxidation in the awake nonstimulated cerebral cortex (13 ,26 ,29 ,35 ,37 ). Several comprehensive reviews of the
evidence from molecular and cellular studies supporting glial localization of glucose uptake related to functional neuroenergetics
have been published by Magistretti and co-workers (52 ,89 ) and are not duplicated here. The focus of this section is on the evidence
from in vivo studies that support the model and key tests that remain to be performed.

Determination by C MRS of the Relationship Between the Glutamate/Glutamine

13

Cycle and Neuronal Oxidative Glucose Consumption

To determine the relationship between the glutamate/glutamine cycle and cerebral cortex neuroenergetics, 13C MRS was used to
measure the rate of neuronal glucose oxidation and the glutamate/glutamine cycle in rat cortex under conditions of isoelectric EEG
induced by high-dose pentobarbital anesthesia, and at two milder levels of anesthesia (26 ). The rate of neuronal glucose oxidation
and the glutamate/glutamine cycle was calculated using a two-compartment metabolic model from the isotopic turnover of C4-
glutamate and C4-glutamine. Under isoelectric conditions, at which minimal glutamate release takes place, almost no glutamine
synthesis was measured, consistent with the conclusion that the 13C MRS measurement of glutamine synthesis primarily reflects the
glutamate/glutamine cycle. Above isoelectricity, the rates of the glutamate/glutamine cycle and neuronal glucose oxidation both
increased with increasing electrical activity. The results, shown in Fig 25.6 , indicate an approximately 1:1 relationship between the
increase in the rates of the glutamate/glutamine cycle and neuronal glucose oxidation with brain activity. Under the highest cortical
activity studied, the glutamate/glutamine cycle rate was approximately 80% of the rate of neuronal glucose oxidation. A similar
ratio of the rate of the glutamate/glutamine cycle to the rate of neuronal glucose oxidation has been reported for measurements of
awake nonstimulated human cerebral cortex (13 ,29 ,35 ).

FIGURE 25.6. An approximately 1:1 correlation between the rate of oxidative glucose consumption and the rate of the
glutamate glutamine cycle. The rate of neuronal glucose oxidation (CMRglc(ox)) and the glutamate/glutamine cycle (Vcycle) was
measured by 13C MRS at 7 T in the rat somatosensory cortex at different levels of cortical activity induced by anesthesia. A
significant positive correlation (p <.001) was found between CMRglc(ox) and Vcycle. The regression line shown is y = 1.04x + 0.10
with a Pearson product-moment correlation coefficient, r, of 0.94. (From Sibson NR, Dhankhar A, Mason GF, et al.
Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity. Proc Natl Acad Sci USA 1998;95:316–
321, with permission.)

A Model for the Stoichiometric Coupling of the Glutamate/Glutamine Cycle to

Neuronal Glucose Oxidation
Figure 25.7 shows a model that provides a mechanistic explanation for the observed ratio of the rates of the glutamate/glutamine
cycle to neuronal glucose oxidation (26 ,37 ,90 ). The model is an extension of the model proposed by Magistretti and co-workers
that nonoxidative glial glycolysis is coupled to glutamate uptake due to the preference of the glia to use glycolytic adenosine
triphosphate (ATP) to pump out the cotransported three Na+ ions (52 ,90 ,91 ). The pyruvate and lactate formed by glial glycolysis
would then be transported to the neuron where it is oxidized. Prior to the
 P.328

in vivo 13C MRS studies, the evidence for the model was primarily from enzyme localization studies and isolated cell studies (see refs.
89 and 90 for reviews of these studies). Both lines of evidence of localization have been criticized based on the presence of the
enzymes required for glucose transport and glycolysis in the neurons, and the strong dependence of glutamate-stimulated glial
glucose metabolism on cell culture conditions (92 ).

FIGURE 25.7. A metabolic model coupling the glutamate/glutamine cycle to oxidative glucose consumption. In this model the
two molecules of adenosine triphosphate (ATP) required by the astrocyte to take up one molecule of glutamate (Glu) and
convert it through glutamine synthetase to glutamine (Gln) are provided by nonoxidative glycolysis of one molecule of glucose
(Glc). The lactate produced by nonoxidative glycolysis is then released from the astrocyte and taken up by the neuron for
oxidative glycolysis. Glc, glucose; Lac, lactate; Vgln, rate of glutamine synthesis; Vcycle, rate of the glutamate/glutamine cycle.
(From Sibson NR, Dhankhar A, Mason GF, et al. Stoichiometric coupling of brain glucose metabolism and glutamatergic
neuronal activity. Proc Natl Acad Sci USA 1998;95:316–321, with permission.)

Comparison of the In Vivo C MRS Results with the Stoichiometry Predicted by the
13

Model
The ambiguities in the determination of the relative rates of metabolic pathways from enzymatic localization and measurements of
isolated cells are not unexpected. Metabolic control analysis has shown that the total activity of an enzyme within a metabolic
pathway does not determine the flux through the pathway (93 ). Extrapolation to in vivo rates from studies of cell cultures is
complicated by the difficulty of reproducing the complex cellular interactions that occur in vivo (64 ). To compare the results of the
in vivo measurement with the predictions of the model, Sibson et al. (26 ) calculated the stoichiometric relationship between the
glutamate/glutamine cycle and neuronal oxidative glucose consumption. Glutamate is cotransported into the glia with two to three
Na+ ions, with one K+ ion countertransported (60 ,78 ,94 ). Transport of three Na+ ions out of the glia by the Na+/K+ adenosine
triphosphatase (ATPase) on the glial end process membrane requires approximately one ATP molecule (91 ). Synthesis of glutamine
from glutamate through glutamine synthetase requires one ATP molecule per glutamine molecule synthesized (53 ). If the ATP for
this process were derived entirely from glycolysis, then a 1:1 stoichiometry is predicted between glial nonoxidative glucose
consumption and the glutamate/glutamine neurotransmitter cycle. Provided that the lactate formed is released to the neurons for
oxidation, then this predicted stoichiometry is in excellent agreement with the in vivo 13C MRS findings.

If the model is correct, it may account for a substantial fraction of total glucose consumption in the awake nonstimulated cerebral
cortex. Based on the measurements of the rate of the glutamate/glutamine cycle and total glucose oxidation in human cerebral
cortex (13 ,29 ), between 60% and 80% of total brain glucose oxidation may be accounted for by this mechanism.

Additional In Vivo Evidence of the Model

The model (90 ) has been criticized for not leaving room for other energy consuming processes in the cerebral cortex. Within the
error of the measurements, approximately 10% of total glucose oxidation is available for other neuronal systems such as
dopaminergic or serotoninergic. The finding of the large majority of glucose oxidation in the cerebral cortex being associated with
glutamatergic and GABAergic neurons and their surrounding astrocytes is not surprising, because cell staining studies have shown
that the vast majority of synapses and neurons in the cerebral cortex are either glutamatergic or GABAergic (4 ).

A prediction of the model is that a large fraction of glucose uptake and phosphorylation is localized in the cerebral cortex to the
glial end sheaths surrounding the synapses of glutamatergic neurons. In agreement with this prediction studies using 14C-
deoxyglucose autoradiography indicate that the majority of brain glucose uptake is used to support synaptic activity. Increased
glucose uptake in response to functional stimulation in peripheral neurons and in cortex is primarily localized in dendritic and nerve
terminal cortical layers (where there are associated glial end processes) and not in layers associated with cell bodies (1 ,95 ,96 and
97 ).

The rapid incorporation of 13C label into glutamine by the glutamate/glutamine cycle indicates that the vesicular glutamate pool is
rapidly turning over and is in dynamic equilibrium with cytosolic glutamate. This conclusion is in contradiction to the traditional
view that the small vesicular pool is metabolically isolated from cellular glutamate metabolism (60 ,61 ). However, these studies
were performed in cellular and tissue preparations, which have a low rate of synaptic metabolism relative to intact cerebral cortex.
In support of this conclusion Conti and Minelli (42 ) showed that inhibition of PAG, which is enriched in nerve terminals (55 )
 P.329

and has been proposed to primarily replete the vesicular pool of glutamate (34 ), results in a similar rapid depletion of both synaptic
and whole cell glutamate in the rat cerebral cortex.

Further support for the coupling between glial glycolysis and the glutamate/glutamine cycle is provided from studies performed
looking at glutamine synthesis in mice in which the astrocytic TCA cycle was inhibited by injection with fluoroacetate (28 ). In these
studies mice were given fluoroacetate and injected with a combination of [1,2-13C] acetate and [1-13C] glucose. From measurements
of the isotopomer distribution in glutamate and glutamine, the labeling from glucose and acetate was distinguished. The labeling
from acetate in glutamate and glutamine was greatly reduced by fluoroacetate administration, which the authors interpreted as
resulting from the near-complete inhibition of the glial TCA cycle. Despite this inhibition, there was still a substantial amount of
glutamine labeling from [1-13C] glucose, approximately one-third to one-half the labeling found in the control mice. The only
mechanism by which this labeling of glutamine from glucose could occur is the glutamate/glutamine cycle, because glutamate
labeling in the astrocyte from glucose was completely blocked. The ability to maintain a high glutamate/glutamine cycle flux,
despite the near-complete inhibition of glial mitochondrial ATP generation, has been interpreted by Bachelard (98 ) as supporting
the importance of the glutamate/glutamine cycle as well as the potential coupling to glycolytic ATP production: “This singlet
labeling of the C4 of glutamine, which can only be derived from [1-13C] glucose metabolism in the neurones, also quite clearly
demonstrates that even though the glial TCA cycle is blocked by the toxin, the glia are still capable of participating in the
glutamate-glutamine cycle, taking up glutamate from the neurones and converting it to glutamine.”

Another testable prediction of the model is that if another substrate is supplied for neuronal oxidation, the decrease in glucose
oxidation will be greater than the decrease in glucose consumption, due to the remaining need for glycolytic ATP to fuel the
clearance of glutamate. Consistent with this prediction, an AV difference study of the anesthetized rat brain found that infusion of
β-hydroxybutyrate, which is an alternate fuel for brain oxidative ATP production, led to a two- to threefold greater decrease in
glucose oxidation than in glucose consumption, with the difference accounted for by a large increase in the efflux of lactate from
the brain (99 ). Consistent with this finding, Pan et al. (100 ) measured an increase in brain lactate in 3-day-fasted human subjects
with elevated plasma ketone concentrations,

Summary and Remaining Questions

The linear relationship and stoichiometry found using 13C MRS of the rates of the glutamate/glutamine cycle and neuronal glucose
oxidation support a direct mechanistic coupling between the glutamate/glutamine cycle and glial glucose uptake. This mechanism
may account for between 60% and 80% of the rate of total glucose oxidation in awake nonstimulated human cerebral cortex, and
possibly an even larger fraction of the increment in glucose oxidation with stimulation (5 ,90 ). However, there are alternate
potential explanations for the in vivo results that need to be tested. Most importantly, it will be necessary to devise strategies for
directly distinguishing glial glucose uptake from neuronal glucose uptake and phosphorylation in the intact cerebral cortex. In
addition, the stoichiometry between neuronal glucose oxidation and the glutamate/glutamine cycle remains to be measured under
conditions of sensory stimulation, and in different brain regions.

IN VIVO MRS STUDIES OF GABA METABOLISM AND THE EFFECTS OF

DISEASE AND PHARMACOLOGIC TREATMENT ON HUMAN GABA
METABOLISM
Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "

GABA is the major inhibitory neurotransmitter in the cerebral cortex (46 ,47 ). It is synthesized from glutamate in specialized cells
called GABAergic neurons. The release of GABA by a GABAergic neuron inhibits the electrical activity of adjacent neurons. Extensive
studies in animals and isolated brain cells and slices have shown that GABAergic function is altered in a variety of models of
neurologic and psychiatric disease (46 ,101 ,102 ). Several antiepileptic and psychiatric drugs are targeted at the GABAergic system.
GABA is overlapped in the in vivo 1H MRS spectrum by the more intense resonances of macromolecules (103 ), glutathione, and
creatine. The development of 1H MRS spectral editing of GABA in animals and humans (6 ,66 ,104 ,105 ) has provided a new window
on studying GABA metabolism and GABAergic function in animals and humans. Several of the main findings using MRS to study
alterations in GABA metabolism in disease and the effect of pharmacologic treatment on GABA metabolism are reviewed below.

MRS Studies of the Effect of the Antiepileptic Drug Vigabatrin on GABA Metabolism
Vigabatrin irreversibly inhibits the enzyme GABA transaminase (GABA-T). GABA-T catalyzes the breakdown of GABA in GABAergic
neurons and in astrocytes. By inhibiting GABA-T, the drug leads to an elevation in GABA concentration. The ability of 1H MRS editing
to measure GABA elevated by GABA-T inhibitors was first demonstrated in the rat brain (106 ,107 ). Subsequent MRS editing studies
of vigabatrin action on patients have made several new observations relevant to optimum administration of the drug including (a)
chronic dosing above 3 g per day
 P.330

fails to additionally increase GABA concentration (108 ), (b) GABA concentration reaches a maximum level within 2 hours of initial
drug administration (109 ), (c) the effectiveness of vigabatrin in controlling seizures depends on elevating GABA concentration above
the mean level found in nonepileptic subjects (108 ), (d) GABA concentration is increased by over two times the predrug
concentration within 2 hours of an acute dose of 3 g of vigabatrin and remains elevated over 48 hours after a single dose of
vigabatrin (109 ), and (5 ) there is no down-regulation of GABA-A receptors during chronic dosing with vigabatrin (see refs. 110 and
111 for a review of these studies). Figure 25.8 shows two edited spectra of total GABA obtained from the visual cortex before and
after chronic vigabatrin treatment of a patient of epilepsy (108 ,112 ).

FIGURE 25.8. In vivo 1H MRS spectra of total edited GABA from the
occipital lobe of a patient with epilepsy before and after treatment with
vigabatrin. The 1H MRS spectra were obtained using spectral editing (112)
from a 14-cm3 volume centered on the midline in the visual cortex. Chronic
treatment with vigabatrin led to an over twofold increase in the
concentration of total edited GABA, which is the sum of GABA and
homocarnosine.

Cortical GABA Synthesis Is Reduced Following Prolonged GABA-Transaminase

Inhibition
An initial finding in 1H MRS studies of vigabatrin action in epilepsy patients was that the drug failed to raise GABA above a dose of 3
g/day (108 ). The enzymatic mechanisms controlling GABA levels in vivo are complex, involving short-term regulation of GAD by
modulators (e.g., ATP, Pi) that affects the binding of cofactor pyridoxal phosphate, longer-term regulation involving enzyme protein
levels, availability of glutamate precursors and their pathways (e.g., GABA-glutamine cycling), and postsynaptic and astroglial
catabolic pathways. GAD exists as two major isoforms (GAD67 and GAD65) in the brain; each is the product of separate genes
(113 ,114 ) and each has distinct kinetic properties (114 ,115 ). GAD67 is distributed throughout the cytoplasm of GABAergic neurons,
whereas GAD65 is associated with synaptic terminals. Recently, it was shown that the 67-kd isoform of GAD protein is reduced in
response to elevated levels of GABA in vitro and in vivo (116 ,117 ). Differential control of the GAD isoforms suggests that they may
mediate different fluxes. To investigate the effects of elevated GABA on GABA synthesis and quantitatively assess the role of the
GAD isoforms in GABA synthesis, rates of turnover of cortical glutamate and GABA were determined in anesthetized rats during an
infusion of [1-13C] glucose after administration of the GABA-transaminase inhibitor vigabatrin (500 mg/kg, i.p.), to increase GABA
levels (24 ). GABA concentration was increased twofold at 24 hours. Tricarboxylic acid cycle flux was not affected by vigabatrin
treatment compared to nontreated rats despite the increased GABA level. An analysis of the turnover data revealed a ˜70% decrease
in the rate of GABA synthesis following vigabatrin-treatment (control, 0.14 μmol/g/min; vigabatrin-treated, 0.04 μmol/g/min). The
reduction in GABA synthesis concomitant with the selective inhibition of GAD67 suggests that GAD67 accounts for the major fraction of
GABA synthesis in the rat cerebral cortex under anesthetized nonstimulated conditions. This conclusion is supported by studies of
GAD67 and GAD65 in knockout mice (118 ,119 ), which have found an order of magnitude greater reduction in GABA concentration in
the mice with a GAD67 knockout. The isoform composition of human brain is presently unknown.

Elevation of GABA by Other Antiepileptic Drugs

1
H MRS studies have found that several antiepileptic drugs, with no known metabolic mechanisms of action on the GABAergic system,
also lead to a rapid elevation of GABA concentration. These drugs include GABApentin, topirimate, and lamotragine
(120 ,121 ,122 ,123 and 124 ). The elevation of GABA may be an important mechanism in the effectiveness of these medications as
antiepileptic compounds. In addition, these findings provide evidence that the regulation of GABA metabolism is tightly integrated
with the regulation of GABAergic function. Figure 25.9 shows a time course of the elevation of GABA after administration of an acute
dosage of topirimate. As with vigabatrin, the concentration of GABA reaches a maximum of two times the predrug concentration
within 2 hours of drug administration. A similar rapid elevation of GABA was measured after an acute dosage of GABApentin
(122 ,124 ). The ability of 1H MRS to track the response of the GABAergic system to pharmacologic therapy may potentially be useful
for developing optimum dosing strategies.

FIGURE 25.9. A time course of GABA plus homocarnosine concentration after administration of topiramate. Topiramate at 3
mg/kg was administered to six volunteers without epilepsy. 1H MRS spectral editing measurements were performed at 4 T (123).
GABA levels were measured to peak within 3 hours after administration of topiramate at approximately two times the predrug
levels. A study looking at the acute effect of topiramate on GABA levels of epileptic patients found that the increase was
almost entirely due to GABA (121). The GABA plus homocarnosine concentrations are normalized to a creatine concentration of
9 μmol/g for comparison with measurements by Petroff and co-workers (111), and the predrug concentrations are in excellent
agreement. (Redrawn from Kuzniecky RI, Hetherington HP, Ho S, et al. Topiramate increases cerebral GABA in healthy humans.
Neurology 1998;51:627–629.)
 P.331

Effects of Neurologic and Psychiatric Disease on GABA Concentration

Impaired GABAergic function has been implicated in the etiology of epilepsy (101 ). Consistent with this proposal, 1H MRS editing
studies have found decreased GABA in adult epilepsy (111 ,125 ) and pediatric epilepsy (126 ). In epilepsy the release of cytosolic
GABA has been proposed as an important mechanisms for seizure suppression (127 ,128 ). The finding that a low GABA concentration
was strongly associated with poor seizure control in epilepsy supports this proposal. Further support for a role of cytosolic GABA
concentration in inhibiting cortical excitability comes from the finding that the improvement in seizure control of subjects
administered vigabatrin chronically depended on the elevation of cerebral cortex GABA concentration (108 ).

In addition to epilepsy, reduced GABA concentration has been found in unipolar depression (129 ), alcohol withdrawal, and hepatic
encephalopathy (130 ). These disorders are associated with an alteration in inhibitory GABAergic function. The finding of low GABA
associated with these disorders is additional evidence that the brain metabolic GABA pool has an important role in GABAergic
function. The finding in unipolar depression appears paradoxical because the condition is not associated with enhanced cortical
excitability. A potential explanation of this finding is that the low GABA concentration in this disease is a compensation for the
reduction in excitatory glutamatergic activity (129 ).

Parsing the Edited GABA Resonance

The edited GABA resonance consists of GABA and the GABA derivative homocarnosine, which is a condensation product of GABA and
histidine. Homocarnosine is a neuromodulator present in a specific subclass of GABAergic neurons in the primate brain. Short TE 1H
MRS with macromolecule suppression may be used to measure the homocarnosine histidine proton resonances in the downfield
region of the short TE spectrum (103 ,112 ). Combining the homocarnosine measurement and the total GABA editing measurements
has allowed the separate measurement of homocarnosine and GABA. Through modification of the editing selectivity, the GABA
derivative pyrolidinone may also be measured in the edited spectrum (131 ). It was recently shown that GABA, homocarnosine, and
pyrolidinone have different time courses in response to a first-time challenge with vigabatrin (109 ).

Estimate of the GABA/Glutamine Cycle in Nonstimulated Human Cerebral Cortex

The in vivo 13C MRS measurement may potentially be extended to study the rate of GABA synthesis and the GABA/glutamine cycle
(34 ,65 ). However, the ability to directly measure GABA synthesis at 2.1 T is limited by the [2-13C] GABA resonance being overlapped
at 2.1 T by the isotopomer sideband of the [4,3-13C] glutamate resonance. Due to the entry of GABA into the glial TCA cycle at the
level of succinate, the labeling kinetics of C4-glutamine derived from GABA are indistinguishable from label entering through
anaplerosis. From the results of Shen et al. (29 ), the maximum estimate of the rate of the GABA/glutamine cycle, obtained by
assuming that Vana, is entirely due to GABA, would be approximately 10% of the rate of glutamine synthesis (29 ). In the 4-T studies of
Gruetter et al. (13 ,35 ), the C2 resonance of GABA was resolved (Fig. 25.2 ), which indicates that direct quantitation of the rate of
GABA synthesis and the GABA/glutamine cycle will be possible in human cerebral cortex at higher field strengths.

Summary and Remaining Questions

The ability of 1H MRS to measure regional levels of GABA and GABA derivatives has provided a new window on the GABAergic system
in neurologic and psychiatric disease. Reduced levels of cerebral cortex GABA have been found in patients with adult and pediatric
epilepsy, depression, and alcohol withdrawal. Studies have found that several of the new generation of antiepileptic drugs raise
GABA levels,
 P.332

and GABA elevation may be related to their effectiveness in seizure depression. The recently demonstrated ability to perform GABA
spectroscopic imaging (105 ) opens up the potential for using regional variations in GABA level diagnostically and to track the
effectiveness of drugs targeted at the GABAergic system.
Two important questions are raised by these findings: What is the relationship between GABA levels and the rate of the
GABA/glutamine cycle? What is the relationship between the GABA/glutamine cycle and cortical excitability? A preliminary study has
recently demonstrated the ability to use isotopic labeling strategies, similar to those developed to measure the
glutamate/glutamine cycle, to measure the rate of the GABA/glutamate cycle (39 ). This strategy, in combination with the
manipulation of GABA levels either pharmacologically or through transgenic methods, may provide significant insight into how the
regulation of GABA concentration affects GABAergic function.

IN VIVO MRS MEASUREMENTS OF NEUROENERGETICS DURING

FUNCTIONAL ACTIVATION
Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "
Under physiologic conditions brain oxygen and glucose consumption are tightly coupled (49 ), with between 90% and 95% of glucose
uptake being completely oxidized. The tightness of this coupling during brain activation was questioned when Fox and co-workers
(132 ) measured by PET a mean increase of 51% in CMRglc in the primary visual cortex of humans during stimulation by a flashing
checkerboard pattern accompanied by only a 5% increase in oxygen consumption (CMRO2). This finding was surprising because of the
16- to 18-fold lower ATP production from nonoxidative glycolysis compared with the complete oxidative consumption of glucose. It
was concluded from these results that the energy for supporting electrical activity derives primarily from nonoxidative glycolysis as
opposed to glucose oxidation (132 ,133 ). More recently, the greater increase in cerebral blood flow than oxygen consumption that
leads to the BOLD (blood oxygenation level dependent) effect has been taken as evidence of the hypothesis of stimulated neuronal
activity requiring little energy (134 ).
The apparently minimal need for energy from glucose oxidation to support stimulated neuronal activity is paradoxical because of the
considerable evidence that the majority of energy consumption in the nonstimulated brain, which primarily uses glucose oxidation,
is to support neuronal electrical activity. This evidence includes the critical dependence of brain function on oxygen delivery, the
50% to 70% reduction of brain energy requirements under isoelectric conditions (133 ), and the 13C MRS findings of a high activity of
the glutamate/glutamine cycle in the resting awake brain and the linear coupling of this rate to neuronal glucose oxidation (see the
above sections In Vivo MRS Measurements of the Rate of the Glutamate/Glutamine Cycle: Findings and Validation , and
Determination of the In Vivo Coupling Between the Rate of the Glutamate/Glutamine Neurotransmitter Cycle and Neuronal Glucose
Oxidation ). Furthermore, there is no cellular evidence that stimulated and nonstimulated neuronal activity have different energetic
requirements. As discussed below (see Implications of MRS Studies for Understanding Brain Function ), the ambiguity created by the
variable degree of uncoupling between glucose consumption and oxidation hinders the interpretation of the functional imaging
signal quantitatively in terms of changes in neuronal activity.
MRS provides a powerful tool for studying the question of metabolic coupling between glucose and oxygen by allowing measurements
of the rates of nonoxidative glycolysis and glucose oxidation. MRS experiments have shown that under stimulated conditions the
majority of energy for functional activity is from glucose oxidation (14 ,15 ,135 ). They have also confirmed the presence of
metabolic uncoupling at high levels of brain activity (136 ,137 and 138 ). A model has been proposed to explain the uncoupling of
glucose consumption and oxidation during certain types of stimulation as an extension of the normal energetic processes used to
support the glutamate/glutamine cycle (139 ).

MRS Studies of Lactate Generation and Glucose Oxidation During Sensory

Stimulation
A prediction of the presence of uncoupling of the increase of glucose consumption and oxidation during visual activation is that
there will be an elevation of lactate in the visual cortex. Several laboratories have found an increase in lactate concentration
(136 ,137 and 138 ) during visual stimulation of the human visual cortex by 1H MRS of approximately 0.2 to 4 μmol/g-min within 2 to
6 minutes of activation. The small increase in lactate is consistent with earlier findings in animal models (40 ).
The degree of mismatch between the increase in glucose consumption and oxidation during sensory stimulation was studied by Hyder
and co-workers (14 ,15 ) using forepaw stimulation of an anesthetized rat measured the rate of increase in neuronal glucose
oxidation from the 13C isotopic turnover of glutamate using POCE (proton observe carbon edit) heteronuclear editing. These studies
found a large increase in the rate of glucose oxidation during sensory stimulation (Fig. 25.10 ). This increase was in good agreement
with previous measurements of the increase in total glucose consumption (140 ). While within the accuracy of the measurements
there was room for a significant rate of nonoxidative glycolysis, the contribution of nonoxidative glycolysis to total cerebral ATP
production during activation would be minor due to the much greater number of ATP molecules produced by the complete oxidation
of glucose (32 ,33 ,34 ,35 and 36 ) than by nonoxidative glycolysis to lactate (2 ).

FIGURE 25.10. Time course of C4 glutamate labeling in the ipsi- and contralateral somatosensory cortex of a rat during single
forepaw electrical stimulation. In vivo 1H-13C MRS spectra were obtained from two 24-μL volumes, positioned in the ipsi- and
contralateral somatosensory cortex of rats at 7 T. The spectroscopic volumes are superimposed on a coronal image. The time
courses on the right shows the labeling of C4-glutamate during the infusion of a control rat. The time courses on the left were
obtained during single forepaw electrical stimulation. The lighter region on the image obtained during stimulation is the
superposition of the blood oxygenation level dependent (BOLD) functional MRI (fMRI) obtained during the study. The rate of
labeling on the contralateral side to the stimulation is observed to increase to approximately two times the rate of the
ipsilateral side. The rates on the figure are the calculated rates of the TCA cycle from the group of rats studied in the contra-
and ipsilateral volumes. (Redrawn with data from Hyder F, Rothman DL, Mason GF, et al. Oxidative glucose metabolism in rat
brain during single forepaw stimulation: a spatially localized 1H[13C] NMR study. J Cereb Blood Flow Metab 1999;17:1040–1047.)
 P.333

Calculation of the Relative Contributions of Glucose Oxidation and Nonoxidative

Glycolysis to the Incremental Energy Production During Brain Activation
Two recent papers reviewed the increase in glucose oxidation during cognitive and sensory reported in a large number of studies,
and concluded that in almost all reports the majority of incremental energy production is from glucose oxidation (131 ,141 ). Table
25.1 presents the measured fractional change in the rate of total glucose consumption (cerebral metabolic rate of glucose
metabolism, CMRglc) and oxidation (cerebral metabolic rate of oxygen, CMRO2) in the human visual cortex during visual stimulation.
The studies tabulated used either PET or quantitative functional MRI (fMRI) (142 ,143 ,144 ,145 ,146 ,147 ,148 and 149 ). In most
cases the increase in CMRO2 is greater than reported by Fox et al. (132 ), and the increase in CMRglc is less. These differences have
been attributed to differences in stimulation paradigms, with greater uncoupling from simple stimuli and almost complete coupling
from complex stimuli (142 ,149 ). As shown in the table, even in the most extreme reports of uncoupling the fraction of the
increment in total ATP production supplied by glucose oxidation is larger than that supplied by nonoxidative glycolysis.

TABLE 25.1. NEUROENERGETIC YIELD WITH STIMULATION

A limitation of both the PET and quantitative fMRI measurements of oxygen consumption is that they depend on assumptions about
blood flow/oxygen delivery coupling in order to derive rates (131 ). The measurement of glucose oxidation by MRS gets around the
requirement of detailed knowledge, or calibration, of this parameter by directly measuring the flow of labeled glucose into the TCA
cycle. The recent demonstration of high spatial resolution POCE measurements of glucose oxidation in human visual cortex at 4 T
indicate that this method is ready to address this question in the human visual and other sensory systems (31 ,44 ).

The Glycogen Shunt, a Model of the Mismatch Between Glucose Consumption and
Oxidation During Stimulated Neuronal Activity
The results tabulated in Table 25.1 show that the degree of mismatch between the increment in glucose consumption and glucose
oxidation is the greatest for stroboscopic stimuli, which require alternate periods of intense activation followed by a quiescent
period. For example, in visual stimulation the greatest mismatch was reported for a flashing red dot matrix (132 ), while no
mismatch was found for
 P.334

the increase from a colored alternating radial checkerboard, which produces a continuous level of stimulation (142 ,149 ). The
largest sustained mismatch between glucose consumption and oxidation occurs in bicuculline-induced status epilepticus where total
glucose consumption increases to fourfold the prestatus value, whereas oxidation is increased twofold (49 ,148 ). In bicuculline-
induced status epilepticus, brain cerebral cortex electrical activity is characterized by a burst of intense firing followed by a
suppressed period of little electrical activity.

We have proposed a model to explain these observations based on the requirement for ATP from glial glycolysis to supply the ATP
needed for glial glutamate clearance and glutamine synthesis (see Determination of the In Vivo Coupling Between the Rate of the
Glutamate/Glutamine Neurotransmitter Cycle and Neuronal Glucose Oxidation , above). In this model the majority of glucose
required to fuel the pumping of glutamate from the synaptic cleft during the intense bursts of neuronal firing induced by sensory
stimulation is provided by brain glycogen (150 ,151 ). Glycogen phosphorylase is kinetically well suited for rapid increases in activity
through its regulation by signaling pathways and phosphorylation. There is in vivo evidence that brain glycogen may be rapidly
mobilized to support function including in status epilepticus (49 ) and in physiologic brain activation (152 ,153 and 154 ). In the
glycogen shunt model, after an initial period of glycogen depletion during intense stimulation, a steady state is reached in which the
glycogen used to rapidly generate ATP for the transport of glutamate into the glial cell and conversion to glutamate during bursts of
intense activity is resynthesized during the interim quiescent periods. Only one ATP molecule is produced per glucose moiety used
by this pathway, instead of the two produced by glycolysis, and therefore the stoichiometry between the glutamate/glutamine cycle
and glial glucose uptake changes to 1:1 from 1:2. The 1:2 ratio is approximately the ratio measured during status epilepticus, in
which almost all cortical electrical activity is involved in a burst and suppress pattern. The presence of simultaneous synthesis and
breakdown of glycogen has been demonstrated in the exercising muscle (155 ), and more recently in the cerebral cortex of the
stimulated and anesthetized rat (152 ). Figure 25.11 describes the model schematically.

FIGURE 25.11. A schematic diagram of the glycogen shunt model. Glucose taken up by the glia can flow through two pathways
after phosphorylation to glucose 6-phosphate. In the standard pathway (left arrow), which occurs during normal electrical
activity, glucose 6-phosphate is directly converted to lactate by glycolysis producing two ATP molecules per glucose molecule.
The stoichiometry between glucose uptake and glutamate transport and conversion to glutamine is 1:1 in this pathway. The
majority of lactate is subsequently oxidized in the neuron. In the glycogen shunt pathway (right arrow), which occurs during
intense repeated bursts of electrical activity, such as in seizures or intense sensory stimulation, glucose is synthesized into
glycogen first before being converted to lactate. An ATP molecule is used in the synthesis of glycogen, resulting in a reduction
in the energetic yield from the pathway to one ATP molecule per glucose molecule. The stoichiometry between glucose uptake
and glutamate transport is increased to 2:1 in this pathway. The extra lactate produced in the shunt pathway is not required
for neuronal energy metabolism and eventually leaves the brain.

Summary and Remaining Questions

In vivo MRS studies have made significant contributions to the understanding of functional neuroenergetics. The measurement of the
glutamate/glutamine cycle under different levels of electrical activity (see the above sections In Vivo MRS Measurements of the Rate
of the Glutamate/Glutamine Cycle: Findings and Validation , and Determination of the In Vivo Coupling Between the Rate of the
Glutamate/Glutamine Neurotransmitter Cycle and Neuronal Glucose Oxidation ) has shown that the majority of brain energy
production in even the nonstimulated state supports neuronal activity. Several MRS studies have provided insight into the mismatch
between glucose consumption and oxidation during sensory stimulation. Lactate elevation during visual stimulation provided direct
validation of the findings using PET (132 ) of the mismatch between oxygen consumption and glucose consumption (136 ,137 and
138 ). Although there is a significantly greater increase in glucose consumption than oxidation in certain stimulated states, studies of
 P.335

the rate of neuronal glucose oxidation during sensory stimulation in the rat cerebral cortex have shown that neuronal glucose
oxidation provides the majority of energy production in the stimulated state (14 ,15 ). The conclusion derived from the MRS studies
is consistent with PET measurements of the mismatch when the much greater efficiency of ATP production from glucose oxidation is
taken into account (135 ), as shown in Table 25.1 . A potential explanation of the mismatch has been proposed based on the
requirement for rapid glycolytic energy generation to clear glutamate from the synaptic cleft during the bursts of intense neuronal
firing associated with stimulated neuronal activity (151 ). In this glycogen shunt model, the power required is provided by rapid
glycogen breakdown. The glycogen is resynthesized during the inter-burst periods resulting in a reduction in the normal
stoichiometry between glutamate transport into the glia and glial glucose uptake from 1:1 to 1:2.

Several major questions remain to be addressed on the neuroenergetic support of functional activity. Paramount among these is the
need for a measurement of the glutamate/glutamine cycle during sensory stimulation. In addition, although the majority of the
increase in energy consumption during stimulation is associated with glutamatergic neurons (15 ,156 ), the relative contributions of
glia and GABAergic neurons are not known. At present there are only minimal data from brain studies supporting the glycogen shunt
model of the mismatch between glucose consumption and oxidation. Studies measuring glycogen turnover directly under these
conditions (30 ) may be able to directly test this hypothesis, and better establish the role of glycogen in functional neuroenergetics.

IMPLICATIONS OF MRS STUDIES FOR UNDERSTANDING BRAIN

FUNCTION
Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "

The stoichiometry of the rate of the glutamate/glutamine cycle and oxidative glucose metabolism has implications for connecting
models of brain function at the macroscopic level, as studied by functional imaging, with neurobiological studies at the level of
synapses and networks of neurons. This section reviews work in which this relationship was used to calibrate the PET and fMRI
signals and neuroenergetic signals, which are either indirectly or directly measures of functional glucose metabolism, with
neurotransmitter cycling (5 ,139 ,157 ). Some implications of this calibration for the interpretation of brain functional imaging are
explored.

Calibration of the Relationship Between the Glutamate/Glutamine Cycle and the

PET and BOLD MRI Functional Imaging Signal
At present, functional PET and fMRI are not quantitated in terms of specific neuronal processes involved in information transfer. The
functional imaging signal in PET and fMRI is either directly or indirectly coupled to the change in glucose oxidation with activation
(1 ,2 ,134 ). The tight coupling between the glutamate/glutamine cycle and neuronal glucose oxidation in the rat cerebral cortex
(26 ,90 ) provides a relationship for calibrating the functional imaging signal to the specific neuronal process of glutamate release
and recycling (5 ).

The similarity of the ratio of the rates of the glutamate/glutamine cycle to neuronal glucose oxidation in human cerebral cortex and
the rat cerebral cortex supports a similar relationship holding for the awake nonstimulated human cerebral cortex. Although studies
are needed to establish the exact stoichiometry during sensory or other external stimulation, it is reasonable to extrapolate a
positive (and possibly stoichiometric) relationship between changes in the rate of neuronal glucose oxidation and the
glutamate/glutamine cycle during activation. Using this relationship, the functional imaging signal may be converted to a first order
to changes in the rate of glutamate/glutamine neurotransmitter cycle (5 ). The advantage of performing this calibration over the
direct MRS measurement of the glutamate/glutamine cycle is that several orders of magnitude of higher spatial and temporal
resolution is possible with the MRI measurement.
 P.336

Estimate of the Total Neuroenergetics Used to Support Functional Neuronal

Activity During Sensory Stimulation
An assumption in the conventional interpretation of functional imaging is that the increment in neuronal activity during brain
activation is sufficient to support the incremental mental processes in a region during sensory stimulation or a cognitive task (5 ).
This interpretation is trivially valid if there is insignificant neuronal activity in the nonstimulated state. The high rate of the
glutamate/glutamine neurotransmitter cycle found by 13C MRS in the nonstimulated brain raises the question of whether the
incremental neuronal activity is sufficient for mental processing or if the total regional neuronal activity is needed. This question
was addressed by analyzing previous studies that measured the change in glucose consumption during stimulation in the sensory
cortices of animals stimulated under anesthetized and awake conditions. The studies chosen used anesthetics that interfere
minimally with the electrical response to sensory stimulation (157 ). During anesthesia, the baseline glucose consumption was
reduced by as much as two- to threefold. Based on the standard paradigm, a constant increment of neuronal activity, and by
inference glucose consumption, during stimulation would be expected regardless of whether the animal was anesthetized or awake.
In contrast, if the majority of regional neuronal activity was required for sensory processing, then the glucose consumption required
during stimulation would be similar whether the animal was awake or anesthetized. The prediction of these two models is
diagrammed in Fig. 25.12 . Results from a number of studies indicated that a similar level of cortical activity was reached during
stimulation, independent of the degree of suppression of resting glucose consumption by the anesthesia (139 ,157 ). These results
were supported by the MRS studies that found a large increment in glucose oxidation with somatosensory stimulation under α-
chloralose anesthesia (15 ,156 ). This finding supports the view that during stimulation the total neuronal activity in sensory regions
is required to support brain function. Results of this literature survey have recently been reinforced by similar results using
quantitative MRI to measure changes in oxygen consumption in the same animal at two different levels of anesthesia (158 ).

FIGURE 25.12. Schematic representation of possible increase in the functional imaging signal, as measured by
neuroenergetics, upon sensory stimulation for an animal that is nonanesthetized (A) and anesthetized (B,C). The difference in
the magnitude of the functional imaging signal, as quantified by glucose oxidation, between stimulated and nonstimulated
states is represented by the shaded rectangles. In functional imaging these increments are commonly used to identify focally
activated regions. The remaining signal, which is represented by white rectangles, is removed by fMRI analysis methods. 13C
MRS studies have shown that a large fraction of the total signal is from neuroenergetic processes coupled to neuronal activity.
If the neuronal activity needed to perform the task was the same as the increment from the awake state, then the increase in
the signal upon stimulation would be the same for the anesthetized or awake states (compare A and C). If instead a large
fraction of the total neuronal activity in the region supports the sensory processing, then the incremental signal from
anesthesia would be much larger (compare A and B). A survey of results in the literature showed that in most cases, when
anesthetics such as α-chloralose that do not block stimulated electrical activity are used, the total glucose consumption and
oxidation rises to the same absolute level during stimulation independent of the initial awake state. These results support the
magnitude of the neuronal activity required to support a task (B) being substantially larger than the increment in neuronal
activity over the resting awake state (C). (From Shulman RG, Rothman DL, Hyder F. Stimulated changes in localized cerebral
energy consumption under anesthesia. Proc Natl Acad Sci USA 1999;96:3245–3250, with permission.)

Implications of the Calibration of the Functional Imaging Signal on the Standard

Interpretations of Functional Imaging Studies
The goal of many functional imaging studies is to determine the anatomic location of brain regions involved in performing mental
processes. To achieve this goal, subjects are given cognitive or motor tasks to perform, or exposed to sensory stimulation, while
being scanned. The degree of involvement of a region in the performance of a task is determined by the increment in the magnitude
of the imaging signal relative to the signal when the subject is in a resting state, or performing some other task. An implicit
assumption in this analysis often used is that the size of the increment of the signal is proportional to the total neuronal activity
recruited by these mental processes (3 ,5 ,159 ).

As described above, MRS studies have shown that the total neuronal activity in a region, as quantified by the glutamate/glutamine
cycle, is much larger than the incremental increase with functional activation. The impact on interpretation of knowing the total
magnitude of, as opposed to the incremental, neuronal activity associated with the processing of a stimulus or task may be
illustrated through a simple example. Consider a hypothetical experiment in which two subjects perform a cognitive task. In one
subject the regional increment in the functional imaging signal in the frontal lobe, quantified as the change in the rate of glucose
oxidation, is 1% of the resting rate of total glucose oxidation. In the second subject the same task induces a signal/rate increment of
2%. In the standard interpretation, the second subject recruited twice the neuronal activity to perform the task as the first subject.
If instead these increments are calibrated as increments in the glutamate/glutamine cycle the relative difference in neuronal
activity is only
 P.337

a few percent. This example shows that knowing the total size of the signal associated with neuronal activity is important in cases
where inferences are being made about differences in the level of neuronal activity, such as when functional imaging is used to
study the effects of drugs or disease on brain function (139 ,160 ). It is also important in the interpretation of functional imaging
data to locate a mental process or function (5 ).

Implications for Studies of Brain Function

The prevailing theory used to interpret functional imaging studies, particularly of cognitive processes, is based on cognitive
psychology (3 ,139 ,159 ,161 ). In the cognitive psychology model of the brain, complex mental processes are broken down using
information theory into component processes, sometimes called modules. Functional imaging is used to locate these postulated
modules. The search for specialized functional areas has been facilitated by analysis methods such as statistical parametric mapping.
These methods identify regional changes in the imaging signal based on the statistical significance of the temporal correlation of the
signal changes with the mental processes postulated by the investigator (which are expressed in the design matrix) involved in
performing a task (162 ). In the statistical representation of brain activity, the magnitude of the regional neuronal activity is often
ignored in favor of a binary representation as “active” and “inactive.” Statistical methods have been widely adopted to analyze fMRI
data. A key assumption often used in the statistical representation of brain function is that the regional neuronal activity used for
performing a mental process is independent and separable from the activity of other regions (162 ,163 ), as well as the neuronal
activity recruited by other processes being supported within the same region.

The use of MRS to calibrate neuroimaging provides the potential for examining complex regional brain interactions that do not fulfill
the strict modular criteria of independence. Several lines of experiments have shown that parallel regional brain functions interact,
and alter the magnitude of the neuronal activity used in processing a stimulus or task (163 ,164 and 165 ). An example are the
studies by Desimone's group (164 ) on the effect of attention upon the neuronal activity, as measured by the distribution of single
neuron firing rates in a region, associated with visual perception. An illustrative set of experiments used two closely spaced visual
signals within the same receptor field of a particular region of the striate cortex. The change in neuronal firing rate obtained from
one stimulus was found to depend critically on the degree of attention paid to the nearby stimulus. Consistent with this result, an
effect of attention on the magnitude fMRI BOLD signal in the human extrastriate cortex has been reported (165 ,166 ). The
calibration of the functional imaging signal in terms of the glutamate/glutamine cycle will extend these studies by allowing these
interactions between regions to be described quantitatively in terms of neuronal activity changes, as is presently is done only in
electrophysiology studies of animal cerebral cortex. This quantitation should allow the exploration of these complex interactions in
much finer detail in humans than is presently possible.

In addition to providing enhanced capability to understand horizontal interactions between brain regions, the calibration of
neuroimaging by MRS also allows a vertical dimension of neuronal activity to be explored. The MRS finding of a high rate of the
glutamate/glutamine cycle even under nonstimulated conditions is consistent with recent experimentally based proposals that
maintaining a constant high level of neuronal activity is critical for brain function. Two recent experiments support this hypothesis.
The need for substantial unfocused neuronal activity for the service of even sensory responses was suggested by a recent experiment
of Grinvald's group (167 ). Starting with the recognition that “cortical neurons are spontaneously active in the absence of external
input even in primary sensory areas,” the authors studied the correlation between single-unit recordings and real-time optical
imaging, which provided a measure of total neuronal activity in the region. They concluded by suggesting that “in the absence of
stimulation the cortical network wanders through various states represented by coherent firing of different neuronal assemblies,”
and that a stimulus pushes the network into a state representing the stimulus. Analogously, Singer (168 ) measured the temporal
synchronization of neuronal responses and concluded, “Of the many responses of V1 those that become synchronized best will be
particularly effective in influencing neurons in higher areas.” These studies both support the presence of a large amount of neuronal
activity in the unstimulated state and suggest a role for this activity in brain function. The results from MRS studies provide
quantitative measures of the total amount of stimulated and unstimulated activity in a region, and thereby can provide a
quantitative basis for analysis.

SUMMARY AND CONCLUSIONS

Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "

Below is a summary of the major findings of MRS studies of the glutamate/glutamine cycle, GABA/glutamine cycle, and functional
neuroenergetics, and some of the implications of these findings for understanding brain function.

Approximately 60% to 80% of total glucose oxidation (and energy consumption) in the nonstimulated cerebral cortex is by
glutamatergic neurons, with most of the remainder in GABAergic neurons and glia (13 ,18 ,24 ,26 ,27 ,29 ,35 ,38 ).

The energetic needs of glutamatergic and GABAergic neurons and glia dominate cerebral cortex energy requirements.

In the awake nonstimulated cerebral cortex of humans

 P.338

and rats, the rate of the glutamate/glutamine cycle is 60% to 80% of total glucose oxidation (13 ,26 ,29 ,35 ):

1. Glutamate release and recycling is a major metabolic pathway.

2. Glutamate metabolism and neurotransmission can no longer be conceptually separated.
3. The nonstimulated awake brain has a high level of neuronal activity, most likely greater than the increment in activity
with external stimulation.

The rate of the glutamate/glutamine cycle increases linearly with neuronal glucose oxidation in a close to 1:1 stoichiometry (26 ):

1. Energy metabolism in cortical glutamatergic neurons is tightly coupled to glutamate release and recycling.
2. The stoichiometry supports a model in which astrocyte glucose uptake is coupled mechanistically to the
glutamate/glutamine cycle (90 ) through the need for glycolytic ATP to transport glutamate into the astrocyte and
synthesize glutamine.
3. The increase in glucose consumption measured during functional activation may be directly coupled to the
glutamate/glutamine cycle, providing a calibration for the functional imaging signal.

The GABA level in human cerebral cortex is reduced in epilepsy, alcohol withdrawal, and depression and is raised by several
pharmacologic treatments (111 ,129 ):

1. The concentration of the metabolic pool of brain GABA may play a critical role in inhibitory GABAergic function.
2. Measuring cerebral cortex GABA level provides a useful index of brain GABAergic function and the effectiveness of
certain antiepileptic drugs.

Reduction in the activity of GAD67 by elevation of GABA leads to a major reduction in the rate of GABA synthesis under nonstimulated
conditions (24 ):

1. GAD67 is the major enzyme controlling nonstimulated GABA synthesis in the rate cerebral cortex.
2. Through regulation of GABA concentration GAD67 may play a key role in the etiology and pharmacology of epilepsy and
other neurologic and psychiatric disorders.
3. The ability of 13C MRS to measure the rate of GABA synthesis in combination with GABA/glutamine neurotransmitter
cycling (39 ) may allow the functional roles of GAD65 and GAD67 isoforms to be distinguished quantitatively.

The glycogen shunt model provides a mechanistic explanation for the apparent uncoupling of glucose consumption and oxidation
during sensory stimulation (151 ).
The majority of energy to support incremental and total neuronal activity during sensory stimulation is provided by neuronal
oxidative glucose metabolism (14 ,131 ,156 ):

1. The total as opposed to incremental neuronal activity is required to support brain function during sensory stimulation
(143 ).
2. A large amount of unfocused neuronal activity in the nonstimulated state is required for brain function.

ACKNOWLEDGMENTS
Part of "25 - Magnetic Resonance Spectroscopy Studies of the Glutamate and Gaba Neurotransmitter Cycles and Functional
Neuroenergetics "
We gratefully acknowledge grant support from the National Institute of Health—DK-27121 (R.G.S.), NS-32126 (D.L.R.), HD-32573
(K.L.B.), NS-34813 (K.L.B.), NS-37203 (F.H.), RO1-NS032518, PO1-NS06208 (O.A.C.P.)—and the National Science Foundation—DBI-
9730892 (F.H.). We benefited greatly from discussions with Dr. James Lai on the localization and kinetics of key enzymes in
glycolysis and the glutamate/glutamine cycle and the careful reading of the manuscript by Dr. Vincent Lebon.

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26
The Spatial, Temporal, and Interpretive Limits of Functional MRI

Peter A. Bandettini

Peter A. Bandettini: Unit on Functional Imaging Methods, Laboratory of Brain and Cognition, National Institute of Mental Health,
Bethesda, Maryland.

Since the inception of functional magnetic resonance imaging (fMRI) in 1991, an explosive growth in the number of users has been
accompanied by steady widening of its range of applications. A recent search of the National Library of Medicine database for
articles with fMRI or BOLD (blood oxygenation-dependent) in the title revealed more than 1,000 citations. Improvements continue in
pulse sequence design, data processing, data interpretation, and the tailoring of cognitive paradigms to the unique advantages and
limits of the technique. This chapter describes the receding limits of fMRI. Specifically, the limits of spatial resolution, temporal
resolution, interpretability, and implementation are discussed. The goal is to give the reader a perspective of the evolution of fMRI
in the past 9 years and a sense of excitement regarding its ultimate potential.

A user of fMRI primarily is interested in extracting at least one of three types of neuronal information: where neuronal activity is
happening, when it is happening, and the degree to which it is happening. To extract this information optimally, an understanding of
the basics of some of the more esoteric details is necessary, which are presented in this chapter. First, the basics of fMRI contrast
are discussed. Second, the key of fMRI interpretation, the neuronal–hemodynamic transfer function, is described. Third, an
overview of methods by which neuronal activation is played out in fMRI subjects and subsequently measured is provided. In this
section, the popular technique of event-related fMRI (ER-fMRI) is described in detail, along with emerging methods of neuronal
information extraction. Fourth, the issues of temporal and spatial resolution are discussed. Fifth, the limits of interpretation and the
potential for further neuronal–hemodynamic information extraction are discussed. Lastly, some implementation limits are finally
given as a practical guideline.

 CONTRAST IN fMRI
 HEMODYNAMIC TRANSFER FUNCTION
 SCANNER-RELATED ISSUES
 BEST RESULTS SO FAR
 NEURONAL ACTIVATION INPUT STRATEGIES
 CONCLUSION

CONTRAST IN fMRI
Part of "26 - The Spatial, Temporal, and Interpretive Limits of Functional MRI "

Several types of physiologic information can be mapped with fMRI. This information includes baseline cerebral blood volume (1 ,2
and 3 ), changes in blood volume (4 ), baseline and changes in cerebral perfusion (5 ,6 ,7 ,8 ,9 and 10 ), and changes in blood
oxygenation (11 ,12 ,13 ,14 ,15 ,16 and 17 ). Recent advances in fMRI pulse sequence and experimental manipulation have allowed
quantitative measures of cerebral metabolic rate of oxygen (CMRO ) changes and dynamic, noninvasive measures of blood volume
2

with activation to be extracted from fMRI data (18 ,19 and 20 ).

Blood Volume
In the late 1980s, the use of rapid MRI allowed tracking of transient signal intensity changes over time. One application of this utility
was to follow the T2*- or T2-weighted signal intensity as a bolus of an intravascular paramagnetic contrast agent passed through the
tissue of interest (2 ). As it passed through, susceptibility-related dephasing increased then decreased as the bolus washed out. The
area under these signal attenuation curves is proportional to the relative blood volume. In 1990, Belliveau and colleagues (4 ) took
this technique one step further and mapped blood volume during rest and during activation. The first maps of brain activation
obtained with fMRI were demonstrated with this technique. As soon as the technique was demonstrated, it was rendered obsolete
(for brain activation imaging) by the use of an endogenous and oxygen-sensitive contrast agent—hemoglobin.

Blood Oxygenation
As early as the 1930s, it was known that hemoglobin is paramagnetic and deoxyhemoglobin is diamagnetic (21 ). In 1982, it was
discovered that changes in blood oxygenation change the T2 of blood, but it was not until 1989 that
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this knowledge was used to image in vivo changes in blood oxygenation (22 ). Blood oxygenation-dependent contrast, coined BOLD contrast by Ogawa
et al. (23 ), was used to image the activated brain for the first time in 1991. Interestingly, Ogawa et al. predicted its utility for functional brain
imaging; however, they predicted a signal decrease rather than a signal increase, as implied by some earlier positron emission tomography (PET)
results by Fox and Raichle (24 ) suggesting that the oxygen extraction fraction decreased during activation. The first results of the use of BOLD
contrast were published in 1992 (13 ,15 ,23 ). Because of its sensitivity and ease of implementation, the contrast used to observe susceptibility
changes with changes in blood oxygenation is the most commonly used functional brain imaging contrast, and this is the technique primarily
discussed in this chapter. The cascade of events that follow brain activation and lead to BOLD signal changes is shown in Fig. 26.1 .

FIGURE 26.1. The cascade of hemodynamic and magnetic resonance

imaging events that occur following brain activation.

Blood Perfusion
An array of new techniques exist for mapping cerebral blood perfusion in humans. Arterial spin labeling-based perfusion mapping MRI techniques are
similar to those applied in other modalities, such as PET and single-photon emission computed tomography (SPECT); in-flowing blood is tagged and
then allowed to flow into the imaging plane. The radiofrequency (RF) tagging pulse is usually a 180-degree pulse that “inverts” the magnetization.

Generally, these techniques can be subdivided into those that use continuous arterial spin labeling, which involves continuously inverting blood
flowing into the slice, and those that use pulsed arterial spin labeling, which periodically inverts a block of arterial blood and measures the arrival of
that blood into the imaging slice. Examples of these techniques are echo-planar imaging with signal targeting and alternating RF (EPISTAR) (25 ) and
flow-sensitive alternating inversion recovery (FAIR) (10 ,26 ). Recently, a pulsed arterial spin-labeling technique known as quantitative imaging of
perfusion using a single subtraction (QUIPSS) has been introduced (27 ).

Hemodynamic Specificity
With each of the above-mentioned techniques for imaging volume, oxygenation, and perfusion changes, the precise type of observable
cerebrovascular information can be more finely delineated. Although this information is typically more than the cognitive neuroscientist requires, it
is useful to give an abbreviated summary of how specific MRI can actually be. Regarding susceptibility contrast imaging, spin-echo sequences are
more sensitive to small susceptibility compartments (capillaries and red blood cells), and gradient-echo sequences are sensitive to susceptibility
compartments of all sizes (28 ,29 ,30 and 31 ). Outer-volume RF saturation removes in-flowing spins (32 ), thereby reducing non–susceptibility-
related inflow changes when short-repetition time (with high flip angle) sequences are used. Diffusion weighting or “velocity nulling,” involving the
use of b > 50 s/mm2, reduces the intravascular signal (33 ), thereby reducing, but not eliminating, large-vessel effects (intravascular effects are
removed but extravascular effects remain) in gradient-echo fMRI and all large-vessel effects in spin-echo fMRI. Performing BOLD contrast fMRI at high
field strengths has the same effect as diffusion weighting in the context of susceptibility-based contrast because the T2* and T2 of venous blood
becomes increasingly shorter than the T2* and T2 of gray matter as field strength increases; therefore less signal arises from venous blood at higher
field strengths. (34 ). Figure 26.2 is a schematic diagram summarizing the pulse sequence selectivity of the specific aspects of the vasculature.

FIGURE 26.2. The vascular tree, including arteries (left) and arterioles,
capillaries, and veins (right). If the inside of the vessel drawing is filling
in, the signal has an intravascular contribution. Arterial spin labeling
(ASL) is differentially sensitive to the arterial–capillary region of the
vasculature, depending on the inversion time (TI) used and whether or
not velocity nulling (otherwise called diffusion weighting) gradients are
used. A small amount of velocity nulling and a TI of about 1 s make ASL
techniques selectively sensitive to capillaries. Susceptibility-based
techniques, including gradient-echo and spin-echo, are also
differentially sensitive to specific aspects of the vasculature. Gradient-
echo techniques are sensitive to susceptibility perturbers of all sizes;
therefore, they are sensitive to all intravascular and extravascular
effects. Spin-echo techniques are sensitive to susceptibility perturbers
about the size of a red blood cell or capillary, so that they are sensitive
to intravascular effects in vessels of all sizes and to extravascular
capillary effects. Velocity nulling makes gradient-echo sequences
sensitive to extravascular capillary-to-vein effects, and makes spin-echo
sequences selectively sensitive only to capillary effects. See color
version of figure.

Cerebral Metabolic Rate of Oxygen

Recently, advances in mapping activation-induced changes in the CMRO2 with fMRI have been developed (18 ,20 ,35 ,36 and 37 ). The basis for such
measurement is that BOLD and perfusion contrast can be explained by the combination of a handful of parameters. The key, then, is either to
constrain the contrast or manipulate the physiologic state such that the number of parameters reduces to one or two. Normalization by means of a
hypercapnia has evolved as a method for mapping changes in CMRO2 (18 ). The basic idea is that when the brain is activated, increases in flow,
volume, and oxygenation are accompanied by an increase in CMRO2. When a subject at rest is undergoing a hypercapnic stress (5% CO2), the cerebral
flow, volume, and oxygenation increase without an accompanying increase in CMRO2; therefore,
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less oxygen is extracted from the blood stream, so that the blood oxygenation change, relative to the perfusion change, is greater
than with brain activation. By comparing the ratio of the (simultaneously measured) perfusion and BOLD signal changes during
hypercapnia and during brain activation, CMRO2 information can be derived.

HEMODYNAMIC TRANSFER FUNCTION

Part of "26 - The Spatial, Temporal, and Interpretive Limits of Functional MRI "

The hemodynamic transfer function is referred to here as the combined effect on the fMRI signal change by the spatial and temporal
variation in neuronal–vascular coupling, blood volume, blood flow, blood oxygenation, hematocrit, and vascular geometry, among
other things. A goal of fMRI method development is to characterize this transfer function completely (i.e., its spatial, temporal,
pulse sequence, subject, physiologic, and pharmacologic dependencies), so that more precise inferences can be made about
underlying neuronal activation location, magnitude, and timing. The ultimate limits of fMRI depend on this characterization. This
goal is particularly relevant in the context of understanding pharmacologic effects on brain function.

After the onset of activation, or rather after the neuronal firing rate has passed an integrated temporal–spatial threshold, either
direct neuronal, metabolic, or neurotransmitter-mediated signals reach arteriole sphincters and cause dilation. The time for this
initial process to occur is likely to be less than 100 ms. After vessel dilation, the blood flow rate increases by 10% to 200%. The time
for blood to travel from arterial sphincters through the capillary bed to pial veins is about 2 to 3 s. This transit time determines how
rapidly the blood oxygenation saturation increases in each part of the vascular tree. As shown in Fig. 26.2 , depending on the pulse
sequence used, different aspects of the hemodynamics are manifested.

Location
In resting state, hemoglobin oxygen saturation is about 95% in arteries and 60% in veins. The increase in hemoglobin saturation with
activation is largest in veins, changing from about 60% to 90%. Likewise, capillary blood saturation changes from about 80% to 90%.
Arterial blood, already saturated, shows no change. This large change in saturation is one reason why the strongest BOLD effect is
usually seen in draining veins.

The second reason why the strongest BOLD effect is seen in draining veins is that activation-induced BOLD contrast is highly
weighted by blood volume in each voxel. Because capillaries are much smaller than a typical imaging voxel, most voxels, regardless
of size, likely contain about 2% to 4% capillary blood volume. In contrast, because the size and spacing of draining veins are on the
same scale as most imaging voxels, it is likely that veins dominate the relative blood volume in any voxel that they pass through.
Voxels that pial veins pass through can have 100% blood volume, whereas voxels that contain no pial veins may have only 2% blood
volume. This stratification in blood volume distribution strongly determines the magnitude of the BOLD signal.

As suggested in Fig. 26.2 , different pulse sequence weightings can give different locations of activation. For example, Fig. 26.3
shows the activation in the motor cortex with two different functional MRI contrast weightings collected in the same plane—
perfusion and BOLD. Although much overlap is seen, the hot spots vary by as much as 10 mm. The perfusion change map is sensitive
primarily to capillary perfusion changes, whereas the BOLD contrast activation map is weighted mostly by veins. A potential worry
regarding fMRI location is that venous blood, flowing away from the activated area, may maintain its elevated oxygen
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saturation as far as a centimeter away. When brain activation is observed on a scale of centimeters, this has not been a major
concern. Nevertheless, this issue is discussed in detail later in the chapter.

FIGURE 26.3. Comparison of activation-induced signal changes in perfusion and BOLD (blood oxygenation-dependent)
measurements. Both measurements were obtained at 3 T. Perfusion measurements were obtained by using FAIR-EPI (flow-
sensitive alternating inversion recovery echo-planar imaging) with an inversion time of 1,400 ms. BOLD (blood oxygenation-
dependent) measurements were obtained by using gradient-echo EPI with an echo time of 30 ms.

Latency
One of the first observations made regarding fMRI signal changes is that after activation, the BOLD signal takes about 2 to 3 s to
begin to deviate from baseline (16 ,38 ). Because the BOLD signal is highly weighted toward venous oxygenation changes, with a flow
increase, the time for venous oxygenation to begin to increase is about the time that it takes blood to travel from arteries to
capillaries and draining veins, which is 2 to 3 s. The hemodynamic “impulse response” function has been effectively used to
characterize much of the BOLD signal change dynamics (39 ,40 and 41 ). This function has been derived empirically by performing
very brief and well-controlled stimuli. In addition, it can be derived by deconvolving the neuronal input from the measured
hemodynamic response (42 ,43 ). This type of analysis assumes that the BOLD response behaves in a manner that can be completely
described by linear systems analysis, which is still an open issue. Regardless, observed hemodynamic response to any neuronal
activation can be predicted with a reasonable degree of accuracy by convolving expected neuronal activity timing with the BOLD
“impulse response” function. This function has typically been mathematically described by a γ variate function (39 ).

If a task onset or duration is modulated, the accuracy to which one can correlate the modulated input parameters to the measured
output signal depends on the variability of the signal within a voxel or region of interest. In a study by Savoy et al. (44 ) addressing
this issue, variability of several temporal sections of an activation-induced response was determined. Six subjects were studied, and
for each subject, 10 activation-induced response curves were analyzed. The relative onsets were determined by finding the latency
with which the correlation coefficient was maximized with each of three reference functions representing three parts of the
response curve: the entire curve, the rising section, and the falling section. The standard deviations of the whole curve, rising phase,
and falling phase were found to be 650, 1,250, and 450 ms, respectively.

Across-region differences in the onset and return to baseline of the BOLD signal during cognitive tasks have been observed. For
example, during a visually presented event-related word stem completion task, Buckner et al. (45 ) reported that the signal in visual
cortex increased about 1 second before the signal in the left anterior prefrontal cortex. One might argue that this observation
makes sense from a cognitive perspective because the subject first observes the word stem and then, after about a second,
generates a word to complete this task. Others would argue that the neuronal onset latencies should not be more than about 200 ms.
Can inferences of the cascade of brain activation be made on this time scale from fMRI data? Without a method to constrain or work
around the intrinsic variability of the onset of BOLD signal over space, such inferences should not be made in temporal latency
differences below about 4 s.

Lee et al. (46 ) were the first to observe that the fMRI signal change onset within the visual cortex during simple visual stimulation
varies from 6 to 12 s. These latencies were also shown to correlate with the underlying vascular structure. The earliest onset of the
signal change appeared to be in gray matter, and the latest onset appeared to occur in the largest draining veins. Similar latency
dispersions in motor cortex have been observed. In one study, latency differences, detected in visual cortex with the Hilbert
transform, did not show a clear correlation of latency with evidence for draining veins (47 ).

Figure 26.4 is a summary of the sources of temporal variability. Figure 26.4A shows a plot of the average time course from the motor
cortex as a result of 2-second finger tapping. As mentioned, the first source of variability is the intrinsic noise in the time series
signal. The standard deviation of the signal is on the order of 1%. The second source of variability is that of the hemodynamic
response. As mentioned, this ranges from 450 to 1,250 ms, depending on whether one is observing the rising phase of the signal or
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the falling phase. The third source of variability is the latency spread over space.

FIGURE 26.4. Demonstration of several of the limits of functional magnetic resonance imaging temporal resolution. Echo-
planar imaging was performed at 3 T by using a Bruker Biospec 3T/60 equipped with a local head gradient coil. A time course
series of axial images (matrix size = 96 × 96, field of view = 20 cm, echo time = 40 ms, repetition time = 500 ms, flip angle = 80
degrees) through the motor cortex was obtained. Bilateral finger tapping was performed for 2 s, followed by 18 s of rest.
These figures demonstrate that the upper temporal resolution is determined by the variability of the signal change in time and
space. A: Time course of the signal elicited by tapping fingers for 2 s. The standard deviation at each point is in the range of
1% to 2%. The standard deviation of the hemodynamic change, in time, is in the range of 450 to 650 ms. B: Map of the dot
product (a measure of the activation-induced signal change magnitude) and the relative latencies or delays of the reference
function (the plot in A was used as the reference function) at which the correlation coefficient was maximized. The spatial
distribution of hemodynamic delays has a standard deviation of about 900 ms. The longest delays approximately match the
regions that show the highest dot product and the area where veins are shown as dark lines in the T2*-weighted anatomic
image. C: Histogram of relative hemodynamic latencies. This was created from the latency map in (B).

The plot in Fig. 26.4A was used a reference function for correlation analysis and allowed to shift ± 2 s. Figure 26.4B is a histogram of
a number of voxels in an activated region that demonstrated a maximum correlation with the reference function at each latency
(relative to the average latency) to which the reference function was shifted. The spread in latencies is more than 4 s. Figure 26.4C
includes a map of dot product (measure of signal change magnitude) and latency; the regions showing the longest latency roughly
correspond to the regions that show the largest signal changes. Although these largest signal changes are likely downstream draining
veins, it is important to note that this approximate correlation between latency and magnitude is extremely weak. Many very small
signal changes show very long latencies. It is also interesting to note that the inverse, that many large signal changes show short
latencies, is typically not true. This implies that many downstream vessels may be almost fully diluted back to resting state
oxygenation, therefore showing only a small signal change but still a large latency. Again, work is ongoing to characterize this effect
better.

Magnitude
As previously discussed, the magnitude of the fMRI signal change is influenced by many variables across subjects, neuronal systems,
neuronal systems, and voxels. Making a complete and direct correlation between neuronal activity and fMRI signal change magnitude
in a single experiment will remain impossible until all the variables can be characterized on a voxel-related basis. Because of these
primarily physiologic variables, the magnitude of BOLD signal changes on brain activation maps typically ranges from 1% to 5% [at,
say, 1.5 tesla (T), gradient-echo sequence, echo time of 40 ms]. The picture is not that bleak, though. In the past several years,
considerable progress has been made in characterizing
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the magnitude of the fMRI signal changes with underlying neuronal activity.

The progressive series of studies was as follows: First, as mentioned previously, it was clear that areas that showed significant BOLD
signal change were in the appropriate neuronal area corresponding to specific, well-characterized tasks. Second, inferred neuronal
modulation was carried out by systematically varying some aspect of the task. Clear correlations between BOLD signal change
magnitude and visual flicker rate, contrast, word presentation rate, and finger tapping rate were observed (13 ,48 ,49 and 50 ).
This parametric experimental design represented a significant advance in the manner in which fMRI experiments were performed,
enabling more precise inferences, not about the BOLD signal change with task modulation. Nevertheless, of course, the degree of
neuronal activation (i.e., integrated neuronal firing over a specified area) was still inferred.

Recently, several more intriguing studies have emerged correlating measured neuronal firing rate with well-known stimuli in animals
(51 ) and humans (52 ,53 ) and demonstrating a remarkably high correlation between BOLD signal change and electrophysiologic
measures.

Linearity
Related to the topic of signal change magnitude is that of BOLD signal change linearity. It has been found that, with very brief
stimulus durations, the BOLD response shows a larger signal change magnitude than expected if one assumes that it behaves as a
linear system (54 ,55 ). This greater than expected BOLD signal change is generally specific to stimulus durations below 3 s. Reasons
for nonlinearities in the event-related response can be neuronal, hemodynamic, or metabolic in nature. The neuronal input may not
be a simple boxcar function. Instead, an increased neuronal firing rate at the onset of stimulation (neuronal “bursting”) may cause a
slightly larger amount of vasodilation that later plateaus at a lower steady-state level. The amount of neuronal bursting necessary to
change the hemodynamic response significantly, if a linear neuronal–hemodynamic coupling is assumed, is quite large. For example,
to account for the almost double functional contrast for the experimental relative to the linear convolution-derived single-event
responses, the integrated neuronal response greater than 2 s must double. If it is assumed that neuronal firing is at a higher rate
only for about the first 50 ms of brain activation, the neuronal firing rate must be 40 times greater than steady state for this
duration.

BOLD contrast is highly sensitive to the interplay of blood flow, blood volume, and oxidative metabolic rate. If, with activation, any
one of these variables changes with a different time constant, the fMRI signal may show fluctuations until a steady state is reached
(56 ,57 ). For instance, an activation-induced increase in blood volume would slightly reduce the fMRI signal because more
deoxyhemoglobin would be present in the voxel. If the time constant for blood volume changes were slightly longer than that of flow
changes, then the activation-induced fMRI signal would first increase, then be reduced as blood volume later increased. The same
could apply if the time constant of the oxidative metabolic rate were slightly slower than that of flow and volume changes. Evidence
for an increased oxidative metabolic rate after 2 min of activation is given by Frahm et al. (57 ), but no evidence suggests that the
time constant of the increase in oxidative metabolic rate is only seconds longer than the flow increase time constant—as would be
required to be applicable only to relatively high-amplitude single-event responses. These hemodynamics, which may also differ on a
voxel-related basis, remain to be characterized fully.

SCANNER-RELATED ISSUES
Part of "26 - The Spatial, Temporal, and Interpretive Limits of Functional MRI "

A complete discussion of all scanner-related issues and potential solutions is beyond the scope of this chapter. A rudimentary yet
necessary description of the most basic problems and solutions is presented in the following sections. Most practitioners of
functional MRI typically undergo a painful, frustrating, and prolonged period of learning about all scanner-related limitations and
issues. Some give up hope completely. Those who are determined usually emerge hopeful again, and with a much better “feel” for
what can and cannot be done in regard to brain imaging. This learning process also applies to understanding the physiology of the
signal, but typically the greatest anguish arises in the context of MRI pulse sequences and hardware.

In the first place, all the categories listed below are more or less linked. In this section, an attempt is made to walk the reader
informally through the trade-offs and issues involved in performing an fMRI experiment.

Acquisition Rate
Image acquisition rate is ultimately limited by how fast the signal can be digitized and by how rapidly the imaging gradients can be
switched. MRI can be logically divided into single-shot and multishot techniques. In single-shot imaging, spins are excited with a
single excitation pulse and all the data necessary to create an image are collected at once. Echo-planar imaging (EPI) is the most
common single-shot technique; with one “echo,” a single “plane” is acquired. Multi-shot techniques are most commonly used for
high-resolution anatomic imaging. In clinical scanning (with multi-shot imaging), a single “line” of raw data is acquired with each RF
excitation pulse. Because of the relatively long time it takes for the longitudinal magnetization to return to equilibrium
(characterized by the T1 of the tissue), a certain amount of time, between 50 and 500 ms, is spent waiting between shots;
otherwise, soon no signal would be
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left. It would be “saturated.” Because of this necessary waiting time, multishot techniques are typically slower than single-shot
techniques. For a 150-ms “waiting time,” or repetition time (TR), an image with 128 lines of raw data would take 150 ms multiplied
by 128, or 19.2 s.

In the case of EPI, the entire data set for a plane is typically acquired in about 20 to 40 ms. In the context of performing a BOLD
experiment, the echo time (TE) is about 20 to 40 ms. Along with some additional time for applying other necessary gradients, the
total time for an image to be acquired is about 60 to 100 ms, so that 10 to 16 images can be acquired in a second. Improvements in
digital sampling rates and gradient slew rates will allow small improvements, but essentially, this is about the upper limit for
imaging humans.

In the context of an fMRI experiment with EPI, the typical image acquisition rate is determined by how many slices can temporally
fit into a TR. For whole-brain imaging, approximately 20 slices (5-mm thickness) are required to cover the entire brain. This allows a
TR of about 1.25 to 2 s at minimum. This image collection rate is more than adequate to capture most of the details of the slow and
dispersed hemodynamic response.

Spatial Resolution
The spatial resolution is also primarily determined by gradient strength, digitizing rate, and time available. For multishot imaging,
as high a resolution as desired can be achieved if one is willing to wait. One can keep on collecting lines of data with more RF pulses.
For EPI, the signal decay rate (described by T2* with gradient-echo EPI and by T2 with spin-echo EPI) plays a significant role in
determining the resolution. One can sample for only so long before the signal has completely decayed away. For this reason, the
resolution of EPIs is generally much lower than that of multishot images. To get around this problem, two further strategies are
commonly used. The first is multishot EPI, in which the full EPI acquisition is used multiple times (but many fewer times than in
typical clinical multishot imaging) and interleaved to increase the resolution. The second is to perform an operation called
conjugate synthesis, which basically makes use of the fact that, in raw data space, half of the data is redundant. This allows at most
twice the resolution, with some cost in signal to noise and image quality. An example of multishot EPI is shown in Fig. 26.5 .

FIGURE 26.5. An example of multishot echo-planar imaging. Number of excitations ranged from 1 to 8. The image resolution
increases but the signal to noise and functional contrast to noise decrease. In addition, instabilities are introduced into the
time course by the use of multishot imaging.

Signal to Noise
The signal to noise and the functional contrast to noise are influenced by many variables. These include, among others, voxel
volume, TE, TR, flip angle, receiver bandwidth, field strength, and RF coil used. Not considering fMRI for a moment, the image
signal to noise is increased with larger voxel volume, shorter ET, longer RT, larger flip angle (to 90 degrees), narrow receiver
bandwidth, higher field strength, and smaller RF coil. That said, in the context of fMRI, the functional contrast to noise is optimized
with a voxel volume equaling the size of the activated area, TE ≈ gray matter T2*, short TR (optimizing samples per unit time), flip
angle = Ernst angle = Cos (-TR/T1), narrow receiver bandwidth, high field strength, and smaller RF coil. Of course, all of these
variables come at some expense to others.

Stability
Theoretically, the noise, if purely thermal in nature, should propagate similarly over space and across time. In fMRI, this is not at all
the case because each image is essentially captured in 40 ms and the time series is collected in minutes. Stability is much more of
an issue on the longer time scale.
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Flow and motion are correlated in many areas with cardiac and respiratory cycles. Subject movement and scanner instabilities also
contribute. Single-shot techniques have generally better temporal stability than multishot techniques because, with multishot
techniques, the image is collected over a larger time scale; instabilities on a longer time scale enter into the image creation itself.
This leads to nonrepeatable ghosting patterns that generally decrease temporal signal to noise ratio. Work is ongoing to characterize
and reduce temporal instabilities for both single-shot and multishot imaging techniques (58 ,59 ). Correction techniques include
cardiac gating (60 ), navigator pulses (61 ,62 ), and raw data reordering (63 ,64 ).

Image Quality
Image quality issues that are the most prevalent are image warping and signal dropout. Although books can be written on this
subject, the description here is limited to the bare essentials.

Image warping is fundamentally caused by either or both of two factors, Bo-field inhomogeneities and gradient nonlinearities. A
nonlinear gradient causes nonlinearities in spatial encoding, so that the image is distorted. This is primarily a problem when local,
small-gradient coils are used that have a small region of linearity that drops off rapidly at the base and top of the brain. With the
growing prevalence of whole-body gradient coils for performing EPI, this problem is no longer a major issue. If the Bo field is
inhomogeneous, as is typically the case with imperfect shimming procedures, particularly at higher field strengths, the protons are
processing at frequencies different from those expected in their particular location. This causes image deformation in the areas of
poor shim, particularly with the long readout window or acquisition time of EPI. A solution is either to shim better (65 ,66 ) or map
the Bo field and perform a correction based on this map (67 ).

Signal dropout is related to the problem described above in that it is also caused by localized Bo-field inhomogeneities, typically at
interfaces of tissues having different susceptibilities. If within a voxel, because of the Bo inhomogeneities, spins are of different
frequencies, their signals cancel each other out. Several strategies exist for reducing this problem. One is, again, to shim as well as
possible at the desired area. Because of still imperfect shimming procedures, this is usually not satisfactory. The other is to reduce
the voxel size (increase the resolution), so that stratification of different frequencies is reduced within a voxel. The third is to
choose the slice orientation such that the smallest voxel dimension (in many studies, the slice thickness is greater than the in-plane
voxel dimension) is perpendicular to the largest Bo gradient. For this reason, many studies are performed with the use of sagittal or
coronal slice orientations.

As with many of the topics discussed, much more can be said, but the goal here is simply to provide an introduction and references
to additional reading material.

BEST RESULTS SO FAR

Part of "26 - The Spatial, Temporal, and Interpretive Limits of Functional MRI "

The primary discussion up to this point has focused on the limits imposed by the scanner and the hemodynamics. In this section,
some of the most successful, thought-provoking, and innovative fMRI studies, from a methodologic perspective, performed as of
September 2000 are discussed. The best results in temporal and spatial resolution are presented.

Temporal Resolution
As explained in previous sections, MR images can be acquired at an extremely rapid rate; therefore, scanner-related limits are not
the prime determinant of the upper limits of temporal resolution in fMRI. The key to increasing the temporal resolution in fMRI is
either to characterize the hemodynamic response better or to work around its limits. The results described here are examples of
this work during the past few years.

To obtain information about relative onsets of cascaded neuronal activity from hemodynamic latency maps, it is possible to
determine relative latency changes on modulation of the task timing. In a study of Savoy et al. (68 ), activation onset latencies of
500 ms were discernible when they used a visual stimulation paradigm in which the left and right hemifields were stimulated at
relative delays of 500 ms. First, the subject viewed a fixation point for 10 s. Then, the subject's left visual hemifield was activated
500 ms before the right. Both hemifields were activated for 10 s, then the left hemifield stimulus was turned off 500 ms before the
right.

Although with careful choice of region of interest, from which the time course plot is made, these onset differences can be shown,
maps of latency cannot reveal the onset differences because, as mentioned, the variability over space, which is about 4 s,
dominates the inserted 500-ms variability from left to right hemifield.

To map the relative latency differences between hemifields, it is necessary to modulate the relative stimulation timing. As an
extension of their results, the left—right onset order was switched so that, in the second run, the right hemifield was activated and
turned off 500 ms before the left. Latency maps were made for each onset order and subtracted from each other to reveal a clear
delineation between the right and left hemifields that was not apparent in each of the individual maps. This operation and the
resulting relative latency map is shown in Fig. 26.6 . These maps are of the change in onset of one area relative to another, not of
absolute latency. It is also useful to note that the standard deviation of these maps is reduced simply to the standard
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deviation of the latencies in each voxel, not the standard deviation of the latencies over space. Maps such as these can be
extremely useful in determining which regions of activation are modulated relative to other areas with a specific task timing
modulation.

FIGURE 26.6. The use of latency maps and task modulation to extract relative latencies. Activation within a region of visual
cortex is shown. In one condition (left), the right visual hemifield stimulation precedes the left by 500 ms. In the other
condition (middle), the left visual hemifield precedes the right hemifield stimulation by 500 ms. Latency maps from both these
conditions show an across-voxel spread of ± 2.5 s, which is too large to identify clearly the relative latencies across hemifields.
However, once the data are normalized for this intrinsic variance by directly comparing the hemodynamic response from the
two different lags within individual voxels, the offset between the left and right hemifields can be observed (right). This
demonstration suggests that normalization of the hemodynamic lag can allow small relative temporal offsets to be identified.
These normalized offsets can then be compared across regions to make inferences about neuronal delay. For this experiment,
the repetition time was 400 ms. See color version of figure.

Published work by Menon et al. (69 ), Kim et al. (70 ), Richter et al. (71 ), and Bandettini (72 ) has explored the temporal resolution
limits of fMRI. The results of Menon et al. (69 ), similar to those mentioned above, indicate that relative brain activation timings on
the order of 50 ms can be discerned.
In the study of Richter et al. (71 ), a parametrically varied event-related mental rotation task was used. Each mental rotation task
was presented individually. A high correlation between task duration and event-related width was demonstrated. The longer the
task took to accomplish (larger rotation angle), the wider the event-related response was shown to be in specific parietal locations.

Spatial Resolution
The hemodynamic point spread function was first considered and characterized by Engel et al. (73 ,74 and 75 ). Localization to 1.1
mm was determined.
The first successful mapping of ocular dominance columns in humans was published by Menon et al. (76 ). Their intriguing results
show that the optimal way to pull out differences in activation across closely spaced units is to perform very brief stimuli so as not
to reduce the dynamic range of the oxygenated blood that is flowing away beyond the unit of activation.
In regard to MRI pulse sequence, it is important to note that mapping cortical columns multishot imaging is required (76 ).
Performance of multishot imaging requires either navigator echoes or shot-to-shot phase-correction schemes. If these are not
performed, temporal stability is seriously compromised.
Many have argued that some aspects of the BOLD signal change dynamics are more spatially localized to neuronal activity.
Specifically, the evasive “pre-undershoot” has been indicated as such (77 ,78 ). The rationale is that this transient “dip” in the
signal that occurs 0.5 to 2 s after the onset of activation and quickly gives way to the much larger signal increase is secondary to
direct extraction of oxygen from the blood by adjacent activated tissue. Recently published work has demonstrated the utility of
such an approach for mapping cortical columns in animals (79 ,80 and 81 ).
The highest-resolution fMRI performed with single-shot EPI was obtained by Jesmanowicz et al. (82 ). Here, a partial k-space
strategy was used to obtain a presumed 256 × 256 resolution. The actual resolution achieved is debatable because T2* effects
reduced the resolution below that implied by the matrix size.

NEURONAL ACTIVATION INPUT STRATEGIES

Part of "26 - The Spatial, Temporal, and Interpretive Limits of Functional MRI "
Much of this chapter has been devoted to the basics and some esoteric issues regarding fMRI. This section provides an overview of
the types of neuronal input strategies with which fMRI has been used to extract information about what the brain is doing. Given a
question of brain function, what are the available strategies that one can use to design their paradigm? A schematic summary of
these strategies appears in Fig. 26.7 .

FIGURE 26.7. Overview and schematic depiction of types

of neuronal input strategies available in functional
magnetic resonance imaging. In addition, parametric
designs, which involve systematically varying some aspect
of the intensity of the neuronal input, can be applied to
any of the design strategies.

Block Design
A block design paradigm was the first used in fMRI and is still the most prevalent neuronal input strategy. Borrowed
 P.352

from previous PET studies, it involves having a subject alternately perform a task for at least 10 s, then a control task for a similar
time, so that the hemodynamic response reaches a steady state in each condition. This is a useful technique in that it is easy to
implement, and standard statistical tests can be used to compare each condition.

Phase and Frequency Encoding

Phase encoding of a stimulus involves varying some aspect of the stimulus in a continuous and cyclic manner. This strategy has been
most successfully used in retinotopic mapping (75 ,83 ,84 ). In this type of study, the eccentricity of a visual stimulus ring is
continuously varied; then, after the most extreme eccentricity is reached, the cycle is repeated. The data are typically subjected to
Fourier analysis, and the areas that show a signal change temporal phase that correlates with the stimulus phase are mapped. This is
a powerful technique because it makes use of the entire time series, in that there are no “off” states, and lends itself to Fourier
analysis. This method has also been used for somatotopic mapping (85 ) and tonotopic mapping (86 ).

Frequency encoding is much less common but can be achieved for certain types of stimuli. The method is to designate a specific on–
off frequency for each type of stimulus used. Again, Fourier analysis reveals the most power under a spectral peak corresponding to
the brain area specific to the particular on/off frequency. The utility of this method has been demonstrated in mapping left and
right motor cortex by cueing the subject to perform a finger-tapping task at different on–off rates for each hand (87 ).

Orthogonal Designs
Orthogonal task design is a powerful extension of block design. The basic concept is that if one designs two different task timings to
create BOLD responses that are orthogonal to each other, then these tasks can be performed simultaneously during a single time
series collection with no cross-task interference, so that comparison is much more precise. This technique was pioneered by
Courtney et al. (88 ). In their study, six orthogonal tasks were designed into a single time series. This type of design also lends itself
to event-related studies.

Parametric Designs
As mentioned in the section on magnitude, parametric designs are powerful in that more precise statements can be made about
relative neuronal activity. A parametric task design simply involves systematically varying some aspect of the task during the time
series. This may be a finger-tapping rate, stimulus contrast or flicker rate, cognitive load, or attention demand, and instead of
mapping the magnitude of the change with a single task, the slope of the change corresponding to a task is mapped. In this manner,
relative brain activation magnitude may be teased out of the time series.

Event-Related Designs
Before 1995, a critical question in event-related fMRI was whether a transient cognitive activation could elicit a significant and
usable fMRI signal change. In 1996, Buckner et al. (45 ) demonstrated that event-related fMRI in fact lends itself quite well to
cognitive neuroscience questions. In their study, a word stem completion task was performed; a block design and an event-related
strategy were used. Robust activation in the regions involved with word generation were observed in both cases.

Given the substantial number of recent reports of event-related fMRI (40 ,41 and
42 ,65 ,89 ,90 ,91 ,92 ,93 ,94 ,95 ,96 ,97 ,98 ,99 ,100 ,101 ,102 ,103 ,104 ,105 ,106 ,107 ,108 ,109 ,110 ,111 and 112 ), it can
probably be said that this is one of the more exciting developments in fMRI since its discovery.

The advantages of event-related activation strategies are many (113 ). These include the ability to randomize task types in a time
series more completely (114 ,115 and 116 ), the ability to analyze fMRI response data selectively based on measured behavioral
responses to individual trials (111 ), and the option to incorporate overt responses into a time series. Separation of motion artifact
from BOLD changes is possible by use of the temporal response differences between motion effects and the BOLD contrast-based
changes (91 ).

When a constant ISI is used, the optimal interstimulus interval (ISI) is about 10 to 12 s. Dale and Buckner (43 ) have shown that
responses to visual stimuli, presented as rapidly as once every 1 s, can be adequately separated by
 P.353

using overlap correction methods. Overlap correction methods are only possible if the ISI is varied during the time series. These
results appear to demonstrate that the hemodynamic response is sufficiently linear that deconvolution methods can be applied to
extract overlapping responses. Burock et al. (95 ) have demonstrated that remarkably clean activation maps can be created with an
average ISI of 500 ms. If one assumes that the hemodynamic response is essentially a linear system, there appears to be no obvious
minimum ISI in attempts to estimate the hemodynamic response. Dale has suggested that an exponential distribution of ISIs with a
mean as short as psychophysically possible is optimal for estimation (100 ). Of course, the speed at which one can present stimuli
depends on the study being performed. Many cognitive tasks may require a slightly longer average presentation rate.

Future work in event-related experimental optimization rests on what further information can be derived from these responses.
Between-region, between-voxel, between-subject, and stimulus-dependent variations in amplitude, latency, shape, and responsivity
of the event-related fMRI responses are still relatively uncharacterized. Reasons for these differences are also still unclear.

Free Behavior Designs

With many types of cognitive neuroscience questions, it is not possible to constrain the timing or performance of a task precisely. It
is necessary to allow the subject to perform the task “freely” and obtain a continuous measurement of the performance, then use
the measurement as a reference function for subsequent time series analysis. Examples of this type of design are emerging. As an
example, skin conductance changes are difficult to predict or control. In a study by Patterson et al. (117 ), skin conductance was
simultaneously recorded during an array of tasks and during “rest.” The skin conductance signal change was then used as a
reference function in the fMRI time series analysis. In several cortical and subcortical regions, signal changes were observed that
were highly correlated with the skin conductance changes. Without the use of this measurement, such signal changes would have
appeared as noise. It is thought that this type of design will become more prevalent as methods to monitor subject performance or
state precisely become more sophisticated.

CONCLUSION
Part of "26 - The Spatial, Temporal, and Interpretive Limits of Functional MRI "

This chapter has attempted to combine a review of the fundamentals of fMRI with a glimpse of the state of the art. Starting with the
basics of fMRI contrast, the discussion moved on to hemodynamic transfer function—the basis of understanding fMRI signal change.
Characteristics related to the hemodynamic transfer function include location, latency, magnitude, and linearity. Then, perhaps less
provocative but still important issues of working with an MRI scanner and understanding some practical limitations were discussed. A
sampling of best results, those successfully bringing into play many of the features of experimental design and analysis already
mentioned, was presented. The chapter ended with a brief overview of neuronal input strategies, or rather, ways in which one can
activate the brain in the context of an fMRI experiment.

Functional MRI is about 9 years old and apparently still at the beginning of its growth curve in terms of users and applications.
Clinical applications are just beginning, whereas cognitive neuroscience applications are in full swing. The field of fMRI continues to
develop along intertwining paths of understanding signals, creating tools, and refining the questions being asked.

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27
Diffusion Tensor Imaging

Nikos Makris

G. M. Papadimitriou

A. J. Worth

Bruce G. Jenkins

L. Garrido

A. Gregory Sorensen

V. J. Wedeen

David S. Tuch

O. Wu

Merit E. Cudkowicz

V. S. Caviness Jr.

B. R. Rosen

David N. Kennedy

Nikos Makris, G. M. Papadimitriou, A. J. Worth, Bruce G. Jenkins, L. Garrido, A. Gregory Sorensen, V. J. Wedeen, David S.
Tuch, O. Wu, Merit E. Cudkowicz, V. S. Caviness, Jr., B. R. Rosen, and David N. Kennedy: Massachusetts General Hospital,
Harvard Medical School, Boston, Massachusetts.

The central nervous system (CNS) spans across different levels of organization, covering the gamut from genes to behavior. It is the
aim of neuroscience to elucidate all these levels at a gross and an ultrastructural resolution and to define the relationships among
them. One of the highest levels of organization is the systems level, which includes the motor, sensory, and central neural systems.
Among the central systems are included those related to cognitive function, such as attention, memory, language, and executive
function. Each system can be considered as a set of interconnected processors or centers constituted by nerve cells. Their physical
connections are composed of axons of different lengths that can form fascicles as they run from origin to destination. Within the
neocortex, these connections are selective and architectonic (1 ). How cytoarchitecture, connections, and function relate within the
neocortex is a fundamental problem in neuroscience. This question addresses basic organizational principles of the nervous system
and aims to elucidate the mechanisms through which the cerebrum mediates behavior (2 ,3 ). Behavior, to a large extent the
product of cerebral function and at the same time a modulator of this function, is certainly the key to solving the problem of
meaning related to any neural system. To explore thoroughly the behavioral dimension of an organism, it appears necessary to
perform studies in vivo. If experiments were designed in such manner that information related to structure was gathered along with
behavioral information, we would have an ideal setting for structural–functional or, in the case of disease, anatomic–clinical
correlation. This can be done in the domain of neuroimaging by using currently available technology in an unprecedented way. In
the past two centuries, functional–structural correlations were derived mostly from experimental nonhuman material, whereas
anatomic–clinical correlations were derived principally from human behavioral and, eventually, postmortem lesion analyses. With
the tremendous development of magnetic resonance imaging (MRI) technology—both structural and functional MRI (fMRI) and
magnetic resonance spectroscopy (MRS)—the study of the structure, function, and metabolism of the living human is an ongoing
reality.

One of the latest advancements of MRI technology has been diffusion tensor imaging (DTI), a technique capable of measuring the
diffusivity of water molecules and rendering visible the preferential orientation of their movement. As water molecules diffuse
within the brain, their movement is constrained by the structural “fingerprint” of the tissue. If the tissue is equally distributed in all
directions, like light in a room, then a “random walk” of water molecules occurs and their diffusion is isotropic (i.e., “equally
behaving” in all directions). On the other hand, within a strongly oriented tissue such as a white matter tract, water diffusion is not
equal in all directions but is instead anisotropic, specifically predominant along the direction of the tissue. The strongly parallel
axonal arrangement within a fiber bundle creates a highly oriented and anisotropic environment for
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water molecules. Because DTI can measure the anisotropy and orientation of a tissue, and because brain white matter can be
characterized to a large extent by its orientation and anisotropy, the detection of white matter fiber pathways has become feasible in the
living brain.
Tractography (i.e., the “writing” or tracing of tracts) has been considered to be the most difficult task in neuroanatomy (4 ). To delineate
neuroanatomic connections exactly, white matter fiber tracts have been traced systematically in postmortem experimental material during
the last decades. It has emerged from studies in nonhuman primates that fiber pathways establish connections between distinctive
architectonic cortical areas, and that they constitute fundamental components of neural systems that subserve specific functions (1 ,5 ,6 ).
Extensive research during the past two centuries in higher brain function (and aphasiology in particular) has demonstrated the tight
relationship between damage of commissural or associational fiber tracts and breakdown in cognitive human behavior (7 ,8 and 9 ). In the
light of brain organization at a systems level in terms of architecture and its connections, it becomes evident how relevant the precise
knowledge of neocortical pathways is to the understanding of human behavior in normal and disease states. Although the delineation of
fiber tracts has been accomplished precisely and comprehensively in postmortem nonhuman primates, this goal has not been met in
humans satisfactorily. Moreover, the study of fiber tracts in vivo is only beginning and is largely based on MRI techniques. DTI is one avenue
that may provide solutions to the connectional structure of the CNS. Even though the identification and reconstruction of major fiber
bundles has been accomplished with the use of DTI and computational techniques, the basic problems related to the biological sources of
the DTI signal remain to be clarified. Thus, to achieve a comprehensive understanding of the sensitivity and specificity of the DTI technique,
studies addressing both the gross and ultrastructural level of the white matter are necessary.
In this chapter, we overview the connectional composition of the cerebrum, emphasizing the morphology and architectonic structure of its
pathways. Subsequently, we overview the DTI technique and illustrate its use at an ultrastructural and a gross neuroanatomic level. In this
perspective, we elaborate on three representative white matter fiber pathways of the brain and emphasize how they can be mapped in
terms of computational MRI neuroanatomy. Additionally, we discuss the utility, potential, and limitations of the DTI technique in the
context of its applications and its integration within a larger neurofunctional MRI examination. For this purpose, we present a case of
amyotrophic lateral sclerosis that we studied with MRI, DTI, MRS, and fMRI.

 OVERVIEW OF WHITE MATTER PATHWAYS

 OVERVIEW OF DIFFUSION TENSOR IMAGING
 HOW DO WE “BUILD” A DIFFUSION IMAGE?
 “ZORRO”
 APPLICATIONS AND DISCUSSION
 CONCLUSION
 ACKNOWLEDGMENTS

OVERVIEW OF WHITE MATTER PATHWAYS

Part of "27 - Diffusion Tensor Imaging "

Anatomic Connections
The white matter of the CNS contains axons serving corticocortical, commissural, cortico-subcortical, and cerebellar connections. These
connections can be categorized in three principal classes—namely, associational, commissural, and projectional. Intrahemispheric
associational corticocortical connectivity in particular is accomplished in general by (a) short U fibers that constitute the local circuitry
within a gyrus, (b) intermediate-range fibers within the extent of a lobe, and (c) long association pathways that connect different lobes
(10 ). A tract can be described at a morphologic level in terms of a set of descriptors (i.e., its stem, splay, and origins and terminations),
whereas the set or map of its architectonic connections is its principle structural descriptor.

Morphologic Descriptors of a Fiber Pathway

Conceptually, a white matter fiber pathway is a group of axons that originate from a set of neuronal bodies and end on one or more sets of
target neurons. Morphologically, we can distinguish three different components in a fiber bundle—namely (a) a compact portion or “stem”
or “stalk” (11 ), where the axons run together to form a fascicle; (b) a zone of divergence/convergence called spray (12 ) or splay, where
the axons fan out; and (c) the distal, peripheral origins and terminations, where the axons either originate or end. However, in current MRI
research, the term extreme periphery seems more practical than the terms origin and termination for a couple of reasons. First, DTI does
not allow the identification of this portion of a fiber pathway clearly enough to justify the application of such specific terminology. Second,
both the corticocortical associational and the commissural connections are generally bidirectional, meaning that within the stem of a tract
fibers are running in both directions; thus, origins and terminations pertaining to the same fiber tract occur in adjacent locations. The
differentiation of these features is well beyond the current spatial resolution capabilities of DTI. Therefore, at a morphologic level, a
comprehensive delineation of a fiber pathway should include its stem, splay, and extreme periphery. A description of the extreme
periphery of an association fiber tract is related to a cortical field of origin or termination (Fig. 27.1 ).

FIGURE 27.1. Morphologic description of a fiber pathway. In this lateral

preparation of Ludwig and Klingler (53), the sample tract is the human
superior longitudinal fasciculus, which is a long, associational,
corticocortical connection. Its three components are (a) the stem, where
axons run compactly and in a bidirectional fashion; (b) the splays or
sprays, where the axons diverge or converge; and (c) the extreme
peripheries, the cortical regions within which the axons originate or
terminate. Arrows denote the bidirectionality of axons within the stem
and the splays of the fiber tract as they run from origin to termination.

Architectonic Connections
The detailed characterization of a fiber pathway in terms of its cytoarchitectonic correlates is a fundamental step for the understanding of
cerebral structural and functional organization. These issues have been addressed at the architectonic level in experimental animals and in
human postmortem material. In particular, cerebral cytoarchitecture has been described precisely in both the human and the experimental
animal, such as the macaque. In the experimental nonhuman primate, the white matter fiber pathways have been delineated precisely and
comprehensively (i.e., in terms
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of their stems, splays, and origins and terminations). Although this is currently the case for the nonhuman primate, the status of
research in human brain anatomic connectivity is very different.

Studies in Human Postmortem Material

In human postmortem material, traditional techniques such as myelin stain, bichromate fixation, and gross anatomic dissection
allow visualization of the stems of these fiber bundles (10 ). With very few exceptions, in which dyes such as the carbocyanine dye
(DiI) were used for very short connections (13 ), histologic description of human fiber pathways is incomplete because it does not
provide a detailed understanding of their origins and terminations, and no technique is available that can identify with certainty the
origin and distal terminations of a fiber pathway satisfactorily in the human. The closest inferences at this level of description are
obtained from white matter degeneration studies of brains with specific neurologic damage. Most of these studies deal with cortico-
subcortical connections and are not specific because the cortical lesions that cause the remote degeneration are very large (10 ,14 ).

Studies in the Experimental Nonhuman Primate

Experimental approaches with available techniques have addressed the problem of origin and termination of fiber pathways in the
monkey. The injection of radioactively labeled amino acids (15 ) into nonhuman primate brain with appropriate histologic processing
techniques permits the accurate and reliable interpretation of the origins, trajectories, and terminations of the subcomponents of
the various fiber pathways of the CNS. With this technique, a number of studies of the fiber connections between the different lobes
of the cerebral cortex, between the two hemispheres, and between the cerebral cortex and subcortical regions have been carried
out in the monkey. These studies have demonstrated that the various fiber bundles are distinct and occupy unique trajectories from
origin to termination.

Extrapolation from Animal Experimental Material to Human Material

In the monkey, it is known how different pathways correlate with radioactively labeled material, so that their origin and termination
can be delineated. Because the stems of the major pathways are similar in the monkey and the human, one can extrapolate the
origins and terminations of the observed pathways in the human to correlate specific fiber pathways with cortical architectonic
fields. Drawing these inferences, we can formulate for each individual fiber bundle a specific map that characterizes the tract in
terms of its connections.

Maps of Anatomic Connectivity

A map of anatomic connectivity (MAC) is a set of neuroanatomic regions interconnected by a particular white matter fiber pathway,
and we can symbolize it as PATHWAYMAC. For instance, the MAC for cingulum bundle (CB) would be CBMAC (Fig. 27.2 ). Whereas in the
human we have precise knowledge only of the stem of this bundle, the architectonic connections in the nonhuman primate are well
documented. A comprehensive description of maps for anatomic cerebral connectivity has been formulated, derived from anatomic
studies in the human and by extrapolation from experimental material, and has been integrated in the context of a methodology for
topographic characterization and quantification of human forebrain white matter (16 ,17 ). Because of their versatility, MACs could
be relevant in the formulation of more sophisticated tractographic experiments and in interpreting neurofunctional data. Integrating
the neuroanatomic knowledge of fiber tracts within an fMRI experiment is an additional challenge, but at the same time it seems to
be key if we are to study behavior in normal and clinical conditions, which implies the study of fiber pathways in vivo.

FIGURE 27.2. Map of cortical anatomic connectivity (MAC) for the cingulum
bundle (CB), or CBMAC. The connections of the CB are represented in the mesial
(A), lateral (B), and ventral (C) views of the human brain on a cortical
parcellation system (46). The shaded area in blue within the frontal pole in the
ventral view corresponds approximately to the rostral part of the frontoorbital
cortex that is anterior to the transverse orbital sulcus. AG, angular gyrus; CALC,
intracalcarine cortex; CGa, cingulate gyrus, anterior; CGp, cingulate gyrus,
posterior; CN, cuneiform cortex; CO, central operculum; F1, superior frontal
gyrus; F2, middle frontal gyrus; F3o, inferior frontal gyrus, pars opercularis; F3t,
inferior frontal gyrus, pars triangularis; FMC, frontal medial cortex; FO, frontal
operculum; FOC, frontal orbital cortex; FP, frontal pole; H1, Heschl gyrus; INS,
insula; JPL, juxtaparacentral cortex; LG, lingual gyrus; OP, occipital pole; OF,
occipital fusiform gyrus; OLi, lateral occipital cortex, inferior; OLs, lateral
occipital cortex, superior; PAC, paracingulate cortex; PCN, precuneus; PHa,
parahippocampal gyrus, anterior; PHp, parahippocampal gyrus, posterior; PO,
parietal operculum; POG, postcentral gyrus; PP, planum polare; PRG, precentral
gyrus; PT, planum temporale; SC, subcallosal cortex; SCLC, supracalcarine cortex;
SGa, supramarginal gyrus, anterior; SGp, supramarginal gyrus, posterior; SPL,
superior parietal lobule; T1a, superior temporal gyrus, anterior; T1p, superior
temporal gyrus, posterior; T2a, middle temporal gyrus, anterior; T2p, middle
temporal gyrus, posterior; T3a, inferior temporal gyrus, anterior; T3p, inferior
temporal gyrus, posterior; TFa, temporal fusiform, anterior; TFp, temporal
fusiform, posterior; TO2, middle temporal gyrus, temporooccipital; TO3, inferior
temporal gyrus, temporooccipital; TOF, temporooccipital fusiform gyrus; TP,
temporal pole. See color version of figure.

In Vivo Analysis of Fiber Pathways

The capability of studying tracts in the living human brain opens up a new window in structural–functional and anatomic–clinical
relationships. Currently, the detection of
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fiber tracts in vivo has been addressed by MRI techniques such as DTI. DTI analysis enables us to characterize a white matter fiber
pathway in terms of its orientation, location, and size. To date, tractography has been performed in two different ways. Using
manual or model-independent methods, we can derive the trajectory of the fiber bundle and approximate its extreme peripheries
(12 ). Using mathematically driven model-based methods, we can also trace a fiber pathway (18 ,19 and 20 ). In the section on
applications, we give examples in which both methods are used and different tracts are visualized in two and three dimensions.
Although the field of DTI-based brain tractography is expanding rapidly with impressive results, it has to be pointed out that certain
basic conceptual obstacles still need to be overcome. For instance, in this stage, it has not been demonstrated that we are able to
delineate completely and precisely a fiber tract in the brain by means of any DTI analysis technique. At the most, we can identify
and characterize the stems of the major fiber tracts (12 ,21 ); however, the problem of elucidating the splays and extreme
peripheries of the bundles remains to be solved reliably. Therefore, when we use the term pathway, tract, or bundle, we currently
refer to its stem.

OVERVIEW OF DIFFUSION TENSOR IMAGING

Part of "27 - Diffusion Tensor Imaging "

Self-diffusion of molecules has been studied with magnetic resonance methodologies for several decades (22 ,23 ). For
comprehensive reviews of the use of diffusion in nuclear magnetic resonance, we refer the reader to other sources (24 ,25 and 26 ).
With respect to the physical principles underlying diffusion (also known as brownian motion), water in tissues with an oriented
structure tends to diffuse more along the orientation of the tissue structure (Fig. 27.3 ). The incoherent motion of the diffusing
water, when it occurs in the presence of a magnetic field gradient, leads to dephasing of the MR signal. This dephasing produces
signal attenuation (SA), which is related to the magnitude of diffusivity of the water along the direction and magnitude of the
applied gradient in an exponential fashion. For anisotropic gaussian diffusion, the SA is proportionate to

For isotropic diffusion, this reduces to the Stejskal–Tanner relation: SA = SA0 e-bD, where D is the diffusion coefficient and b is the
diffusion sensitivity factor. Note that b = γ2g2δ2(Δ-δ/3), where the values of g, δ, and Δ correspond to the values of the gradient
amplitude, duration, and spacing, respectively, and γ is the hydrogen gyromagnetic ratio (27 ). The diffusion process can be
parameterized by a 3 × 3 symmetric tensor, which can be represented by an ellipsoid, as shown in Fig. 27.3 .

FIGURE 27.3. Schematic representation of water diffusion in the presence of (A) a nonoriented tissue structure and (B) an
oriented tissue structure. Water mobility is highest along the direction with least interference—that is, along the direction of
the fibers. D,E: Overview of the procedural steps involved in diffusion tensor acquisition. Six images with gradient sampling
directions of g1 = {1, 1, 0}, g2 = {1, -1, 0}, g3 = {0, 1, 1}, g4 = {0, -1, 1}, g5 = {1, 0, 1}, and g6 = {-1, 0, 1}, and an additional
baseline acquisition with no diffusion gradients, g0 = {0, 0, 0}, are acquired. The six first data sets (gradients g1 through g6) are
analyzed relative to the baseline acquisition with no diffusion encoding (g0). This set of observations is sufficient to define the
symmetric diffusion tensor representation of water self-diffusion, shown in (C). See color version of figure.

HOW DO WE “BUILD” A DIFFUSION IMAGE?

Part of "27 - Diffusion Tensor Imaging "
 P.361

The acquisition of the DTI requires the acquisition of six directionally weighted samples of the effect of the diffusion process
relative to the axes of the imaging system. Specifically, the magnitude of the diffusion attenuation in MR signal along the x, y, and z
axes themselves, as well as in the xy, xz, and yz directions, must be measured. The attenuation of MR signal in the presence of
gradients in each of these directions is calculated relative to an image acquired with no diffusion encoding (baseline). Hence, seven
(six directions, one baseline) acquisitions for each slice level are required. Once the tensor is sampled, the magnitude calculated
from the trace expresses the total (no directionality) diffusivity at each voxel location (Fig. 27.4 ).

FIGURE 27.4. The tensor representation can be visualized in many ways. A: Diffusion anisotropy, defined as the relative
magnitude of the major axis of the diffusion ellipsoid in comparison with the minor axes, can be visualized; in this figure,
regions of high anisotropy are bright, yielding an observable substructure within the cerebral white matter. B: A primary
eigenvector map (PEM) can be generated to observe the orientation of the major axis of the diffusion ellipse in three-
dimensional space; red indicates medial–lateral, blue indicates superior–inferior, and green indicates anterior–posterior
orientation, respectively. C: The PEM can include anisotropy modulation if the intensity of the color is made proportional to
the degree of anisotropy present in each voxel. Regions of high anisotropy can be colored with intense color, whereas regions
of low anisotropy have a pale coloring, so that the underlying anatomic image can be viewed. See color version of figure.

The directionality of diffusion is assessed by an eigen decomposition of the diffusion tensor. The largest eigenvalue corresponds to
the major axis of the diffusion ellipsoid and so represents the major directionality of diffusion at that location.

Color Coding
A color is assigned for each voxel location by using the primary eigenvector (corresponding to the largest eigenvalue) of the diffusion
tensor. At each voxel, the absolute values of the x, y, and z components are used as the red, green, and blue color values,
respectively, such that a red voxel in the image means the vector points left–right (or right–left), green means anteroposteriorly (or
posteroanteriorly), and blue represents superoinferiorly (or inferosuperiorly). For instance, if a vector points mostly in the red
direction, then the x value of the vector will be large and the color will be pure red; otherwise, the color will be a mixture of red,
green, and blue, depending on the magnitudes of the vector components (i.e., on the direction of the vector). In Fig. 27.4 , this
color coding scheme is shown with the appropriate color painted onto a sphere, and the principal eigenvector map (PEM) is the
result of color-coding a tensor image (Fig. 27.4 ).

Modulation by Anisotropy
To further distinguish white matter fiber pathways from other regions, the color is modulated by a measure of anisotropy of the
voxel. This emphasizes the stems (i.e., the compact portions of the fiber tracts) by diminishing the brightness of everything else.
Here, anisotropy can be either lattice (21 ,28 ,29 ) or fractional (30 ) anisotropy. Figure 27.4C shows the effect of anisotropy
modulation.

“ZORRO”
Part of "27 - Diffusion Tensor Imaging "
 P.362

In the section on applications, we illustrate tractographic representations of different pathways that have been created with use of
a novel tool for DTI data analysis. This tool, which we call zorro (for its capability to create masks), is described here.

Zorro is a program for visualization and quantitative measurement of diffusion-weighted MR tensor scans. It was written with the
“visualization toolkit” (31 ). Its main purpose is to create “masks” interactively that designate regions of voxels in the three-
dimensional (3D) data. The masks are then used to make 3D visualizations and quantitative measurements, such as volume,
anisotropy, and direction. The program loads both tensor files and their registered nonattenuated baseline echo-planar imaging (T2-
EPI) files. Anisotropy values are calculated from the tensor data (e.g., fractional, lattice), or they are loaded if they were previously
calculated. A rough segmentation into brain, background (air), and cerebrospinal fluid is performed with use of the T2-EP image,
and this facilitates visualization of the data by providing an anatomic context. Zorro can display all three colorized diffusion
eigenvectors, anisotropy images, the T2-EP image, the segmented image, and all mask images. A mouse click prints all numeric data
for a given voxel (the full tensor, anisotropy values, and mask values). The brightness and contrast can be adjusted for both color
(vector) and grayscale (scaler) images, and there are options to show and hide the background and cerebrospinal fluid in the color
images and to enable and disable the anisotropy modulation of colors. Any number of different voxel “masks” can be created by
clicking on particular voxels. One click adds the voxel to the mask being edited, and another turns it off. Once chosen, various
statistics may be calculated from any value associated with every voxel in the mask. Different masks may be combined in binary
operations (and, or, xor).

Masks may be semiautomatically created by using “region growing.” For this, a mouse click starts the region at a “seed” voxel, and
then each of the seed';s neighbors in 3D is added to the region if the neighbor is similar enough to the seed voxel. A voxel is “similar
enough” if, for instance, its anisotropy and primary diffusion direction differ from the seed's by less than some given threshold. Once
a neighbor is included, it becomes a seed, and its neighbors are checked to see if they should be included in the result. In zorro, the
three kinds of region growing are direction of interest, change in angle, and flow. For all types, one can also specify an anisotropy
threshold, so that if the anisotropy of the neighbor is below this threshold, it will not be included in the result. Region growing can
also be 2D instead of 3D and can be prevented in cerebrospinal fluid regions or the background region.

Flow Region Growing

Flow region growing involves three vectors: the seed voxel's primary diffusion vector, the neighbor's primary diffusion
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vector, and the displacement vector (a vector from the seed voxel location to the neighbor voxel location). A neighbor voxel is
included if the displacement vector matches both the seed and neighbor voxels' primary diffusion vectors. This match is determined
by comparing a threshold to the product of the dot products of the displacement vector with the seed and the neighbor's primary
diffusion vectors. This means that both primary diffusion vectors are pointing in a direction similar to that of the displacement
vector.

Change-in-Angle Region Growing

Here, a neighbor is included in the mask if its direction is close enough to the seed direction. This match is determined by
comparing a threshold to the acrcosine of the dot product of the seed's and neighbor's primary diffusion vectors. As the region grows,
the angle is defined by the new seeds.

Direction of Interest
Here, an absolute direction is specified and each neighbor is included if its direction is close enough to a user-specified direction of
interest. This match is determined by comparing a threshold to the acrcosine of the dot product of the user-specified direction of
interest and the neighbor's primary diffusion vector. Again, growing can be limited in each of these region-growing methods by
providing a threshold for anisotropy and by choosing not to grow out of the 2D slice or into the background or cerebrospinal fluid.

The problem with region growing is that noisy data may cause the region to “escape” and grow out of a region where it might be
expected to remain. Besides allowing the interactive examination of tensor and anisotropy data, zorro also produces three kinds of
output: mask statistics, angular histograms, and 3D visualization with the use of “boxels.”

Mask Statistics
After all voxels in a mask have been chosen by manual editing, region growing, or a combination thereof, zorro prints the average
and variance of fractional and lattice anisotropies taken over all voxels in the given mask.

Angular Histogram
The primary diffusion direction (an angle in 3D) can be represented by two angles (q1, q2). An angular histogram then can be
presented as a 2D image in which each pixel is a bin of the histogram and the brightness of a pixel represents the height of that bin.
The size of the image represents the number of bins chosen to characterize each angle, n. Angles are calculated and then scaled to
a range of ± n/2. For a given primary diffusion vector (x,y,z):

q1 = atan(z/x) / n/2

q2 = atan(y/h) / p/2

where h = sqrt(z2 + x2).

To create the histogram, for each voxel of the mask, the bin (pixel location) indicated by these two scaled angles for the primary
diffusion vector is incremented.

3D Visualization by “Boxels”
“Boxels” are a method for visualizing DTI data in which all tensor information can be seen at the same time. Along with color coding
and modulation by anisotropy, boxes are drawn to indicate the directions and relative lengths of all three vectors. The orientation
and lengths of the three parallel faces are proportional to the eigenvectors and eigenvalues, respectively.

APPLICATIONS AND DISCUSSION

Part of "27 - Diffusion Tensor Imaging "

Diffusion tensor imaging tractography has opened up the capability to study white matter fiber pathways in the living brain in
clinical conditions and in normality, and can be integrated within more general structural–functional, clinical, and behavioral
paradigms. However, basic problems remain to be elucidated. At an anatomic level, fiber tracts can be delineated precisely only at
the level of their stems, not at their splays and extreme peripheries. This is a challenging problem that should be solved if DTI is to
enable us to delineate tracts in their entirety. At a signal analysis level, a fundamental question relates to the source of the DTI
signal. For instance, the relative contributions of the intracellular and extracellular compartments to the diffusion signal are not
known with certainty (32 ,33 ,34 and 35 ). One powerful approach to the solution of this problem is study at the ultrastructural
level.

Ultrastructural Studies
To acquire a better understanding of in vivo diffusion MR measurements in the nervous system, we investigated the diffusion of
water in excised sciatic nerve of mice. Fixed and freshly excised nerves were placed straight in a tube and immersed in Fluorinert
(Sigma, St. Louis, Missouri). The sample was positioned in a solenoid with its axis coinciding with the nerve axis and perpendicular to
B0. Also, it was possible to exploit the particular geometry of the sample to align the nerve axis with one of the field gradients (in
these experiments, the gy gradient, or 0, 1, 0). The diffusion MRI experiments were performed at 2.0 tesla (T) (proton frequency
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of 84.74 MHz) on a SISCO (Varian Associates, Palo Alto, California) system equipped with an 18-cm horizontal-bore superconducting
magnet (Nalorac, Martinez, California) and a set of coils capable of producing 120-mT/m field gradients. Proton MR diffusion-
weighted images were obtained by using a spin-echo sequence with an echo time of 50 ms and a repetition time of 1 s. The
measurements were performed at room temperature (21°C). Typically, 12 scans were averaged. The b values used for diffusion
weighting were 0, 200, 400, 800 and 1,600 s • mm2. The slice thickness was 2 mm, and the field of view was 7.5 × 7.5 mm2 with an
in-plane resolution of 256 × 128 pixels. Figure 27.5A shows a transaxial proton image of a nerve obtained with a b value of 0.

FIGURE 27.5. A: Transaxial proton image of the sciatic nerve of a mouse. B: Graph illustrating the variation of the magnetic
resonance signal as a function of the orientation of the diffusion gradient. The circles (open and closed) correspond to
measurements with the diffusion gradients applied in the plane perpendicular to the nerve axis. The triangles correspond to
diffusion measurements obtained by using the gradient parallel to the nerve axis. The anisotropy of the restriction to the
diffusion of water molecules in the cellular compartments is clearly shown.

The effect of restriction to molecular diffusion within the nerve is shown in Fig. 27.5B . Diffusion gradients applied along directions
gx = (1,0,0) and gz = (0,0,1), which were perpendicular to the nerve axis gy = (0,1,0) in these experiments, show that the presence of
more barriers (i.e., cell membranes and cytoplasmic fibers) hinder molecular diffusion across rather than along the nerve.

Macroscopic Studies
In a sense, the cerebral white matter can be considered as a finite set of discrete and topographically organized fiber pathways or
connections that convey connectivity within the brain, and the physical connections in their entirety would make up anatomic
connectivity. On the other hand, the physiologic outcome of activities of neuronal assemblies, which is coherent in nature and is
expressed in the correlated time structure of the firing pattern of its member neurons, would be the basis of functional connectivity
(i.e., the temporal correlation between remote neurophysiologic events) (36 ). In the context of a functional experiment, functional
connectivity would allow us an array of possible interpretations. The simplest of these solutions that can replicate the observed
functional connectivity describes the interactions and connections that are sufficiently active to be detectable at the time of
observation. This simplest solution is the effective connectivity, which accounts for the interaction that one neural system exerts on
another (37 ).

In vivo DTI-based fiber tract analysis is relevant for the study of structural–functional and anatomic–clinical relationships. The
details of systems neuroanatomy are critical for studies of lesion analysis and for the analysis and interpretation of metabolic and
functional neuroimaging data. Damage to specific fiber pathways correlates with the decreased cerebral metabolism pattern
observed in stroke patients. Functional activation studies in which PET and fMRI are used contribute to the knowledge of the spatial
distribution of cortical and subcortical processing elements. In the creation of effective connectivity models, actual knowledge of
brain anatomy is an integrative part because it is utilized for the design of the a priori model used in structural equation modeling
(38 ,39 ,40 and 41 ). Use of actual individual information regarding in vivo white matter fiber pathway topography and volumetry
may optimize and increase the predictive power of these models. These anatomic and neuroimaging studies of the constituents of
the fiber tracts and the connections of the human cerebral cortex will be important in acquiring an understanding of the distributed
neural circuits that subserve normal brain function. They will also pave the way for future morphometric studies of the white matter
fiber systems in normal populations, and for lesion–deficit correlations in patients with focal brain lesions.
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The significance of this method is underscored in the study of normal white matter neuroanatomy and also of several diseases that
affect the cerebral white matter, such as stroke, head trauma, spinal cord injury, and neurodegenerative diseases. Applications of
the DTI technique in white matter pathway analysis in normal persons and in cases of amyotrophic lateral sclerosis are illustrated.
Three normal tracts—the corticospinal projection, the corpus callosum, and the cingulum fiber system—have been selected for the
illustration because of their different orientations and also because they represent distinctive classes of connection (i.e.,
projectional, commissural, and associational, respectively). We have used “zorro” in these examples to show the various ways in
which tractography can be performed. Specifically, the corpus callosum and the corticospinal tracts were handpicked, whereas the
cingulum fiber system was reconstructed automatically in three dimensions. The results are shown in Fig. 27.6 and Fig. 27.7 .

FIGURE 27.6. Three-dimensional reconstruction of stems of individual pathways of a living normal young adult human subject.
Note that scaling of tracts is unequal. Red indicates medial–lateral, blue indicates superior–inferior, and green indicates
anterior–posterior orientation, respectively, as shown in the color-coded sphere in the center of the figure. For this caption,
three representative fiber tracts were selected based on class of pathway provenience and orientation. The corpus callosum, a
commissural tract with general mediolateral orientation, is shown in superior and lateral views. Specifically, its genu, body,
and splenium are colored in red because they run mediolaterally, whereas its anterior (forceps minor) and posterior (forceps
major) forcipes are colored in green because they are oriented anteroposteriorly. The corticospinal tract, a projectional
pathway with superior–inferior orientation, is depicted in blue in a frontal view. Finally, the cingulum bundle, a long,
associational corticocortical fiber tract with anterior–posterior orientation, is colored green above the body of the corpus
callosum for its major trajectory, whereas it is colored blue along its vertically oriented portions in front of the genu and
behind the splenium of the corpus callosum. See color version of figure.

FIGURE 27.7. Three-dimensional (3D) reconstruction of fiber tracts within the three-dimensionally rendered brain of a normal
young adult. A: A combined 3D representation of the cingulum bundle (green) and the corpus callosum (red). B: A composite
3D rendition of the corticospinal projection (blue) and the corpus callosum (red). Red indicates medial-lateral, blue superior-
inferior, and green anterior-posterior orientation, respectively. See color version of figure.

Amyotrophic Lateral Sclerosis

In amyotrophic lateral sclerosis (ALS), as degeneration of the upper motor neuron occurs, a progressive damage to the corticospinal
tract has been documented in the caudorostral direction (42 ,43 ,44 and 45 ). We used DTI in two patients (cases 1 and 2) to
document pronounced bilateral reduction in the size of the corticospinal tract at the level of the medulla oblongata. Case 1
illustrates an integrated MRI examination of structure, function, and metabolism. A change in morphology of the corticospinal tract
can be appreciated in conventional MR images or in the T2-EP images at its ventral surface shown in Fig. 27.8 A,D. However, these
MRI techniques do not allow an accurate definition of the borders of the tract. With the aid of tractographic DTI, the corticospinal
tract is readily visualized as it courses rostrocaudally. In addition, bordering structures can be visualized, such as the inferior olivary
nucleus; thus, a more precise definition of the caudal borders of the corticospinal tract allows quantification of the fiber pathway at
this anatomic level. Fractional and lattice anisotropy indices were measured in a 13-voxel region (voxel size = 2 × 2 × 2 mm3) for the
corticospinal tract in the medulla, as shown in Fig. 27.8C . The average fractional anisotropy index was .49 ± .02, and the lattice
anisotropy index was .30 ± .01. In this case, in addition to DTI, we collected single-voxel MRS measurements in medulla, and the
data indicated metabolic changes—loss of NAA (N = acetylaspartate) and possibly elevated glutamate/glutamine (glx), shown in Fig.
27.8H . Similarly, we acquired MRS measurements in the right and left primary motor cortex, shown in Fig. 27.8H . Of note is that
the observed metabolic pattern showed a slightly decreased NAA/Cr (creatine) ratio in the left motor cortex in comparison with the
contralateral counterpart. Finally, fMRI was acquired while flexion was performed (frequency of 1.5 Hz) with either the left or right
hand. An expanded area of activation was observed in the left hemisphere that corresponded with this patient's motor
symptomatology (i.e., right-hand weakness much greater than left-hand weakness) (Fig. 27.8I ).
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FIGURE 27.8. Comparison of diffusion tensor imaging in the brainstem (medulla oblongata) of a normal control (D,F) and two
patients with amyotrophic lateral sclerosis, ALS case 1 (A–C) and ALS case 2 (E,G). A T2 echo-planar image (T2-EPI) and tensor
orientation map (TOM) are included at each level. In this image, green voxels indicate anteroposteriorly, red indicate
mediolaterally, and blue indicate superoinferiorly oriented diffusion directions. H: Four magnetic resonance spectra from an
ALS patient (case 1). The spectra in the top row come from a point resolved spectroscopy sequence (PRESS) with TR (repetition
time)/TE (echo time) 2,000/144 ms in either left motor cortex (hand area), right motor cortex (hand area), or occipital cortex.
The occipital cortex serves as a control region that is essentially unaffected by ALS. A slight asymmetry in the NAA/Cr and
glx/Cr values between left and right motor cortex perhaps reflects the large motor asymmetry in this patient. Also shown on
the bottom are PRESS spectra from medulla in the same patient and from a normal control (TR/TE 2,000/30 ms). Note the
lower NAA/Cr values in the patient and the higher intensity in the glx region. I: This figure shows activation in the hand motor
cortex after performance of hand flexion (frequency of 1.5 Hz) with either the left or right hand. Note the expanded area of
activation in the left brain, which correlates with the asymmetry of symptoms. The left hand is essentially normal, whereas
the right hand is much more severely affected. No difference was noted in frequency of performance of the task with the left
or right hand. Also shown is a time course from a region of interest in left motor cortex showing the response of the BOLD
(blood oxygenation-dependent) signal to the off and on periods. AG, angular gyrus; CALC, intracalcarine cortex; CGa, cingulate
gyrus, anterior; CGp, cingulate gyrus, posterior; CN, cuneiform cortex; CO, central operculum; F1, superior frontal gyrus; F2,
middle frontal gyrus; F3o, inferior frontal gyrus, pars opercularis; F3t, inferior frontal gyrus, pars triangularis; FMC, frontal
medial cortex; FO, frontal operculum; FOC, frontal orbital cortex; FP, frontal pole; H1, Heschl gyrus; INS, insula; JPL,
juxtaparacentral cortex; LG, lingual gyrus; OP, occipital pole; OF, occipital fusiform gyrus; OLi, lateral occipital cortex,
inferior; OLs, lateral occipital cortex, superior; PAC, paracingulate cortex; PCN, precuneus; PHa, parahippocampal gyrus,
anterior; PHp, parahippocampal gyrus, posterior; PO, parietal operculum; POG, postcentral gyrus; PP, planum polare; PRG,
precentral gyrus; PT, planum temporale; SC, subcallosal cortex; SCLC, supracalcarine cortex; SGa, supramarginal gyrus,
anterior; SGp, supramarginal gyrus, posterior; SPL, superior parietal lobule; T1a, superior temporal gyrus, anterior; T1p,
superior temporal gyrus, posterior; T2a, middle temporal gyrus, anterior; T2p, middle temporal gyrus, posterior; T3a, inferior
temporal gyrus, anterior; T3p, inferior temporal gyrus, posterior; TFa, temporal fusiform, anterior; TFp, temporal fusiform,
posterior; TO2, middle temporal gyrus, temporooccipital; TO3, inferior temporal gyrus, temporooccipital; TOF,
temporooccipital fusiform gyrus; TP, temporal pole (46). See color version of figure.

Integrated Magnetic Resonance Neurologic Examination

Diffusion tensor imaging can be an important part of a comprehensive neurofunctional MRI examination. Quantitative assessment of
anatomic volume as measured by morphometric MRI (46 ), of fiber pathway anisotropy and size by DTI, of metabolic state by MRS,
and of functional state by fMRI provides a highly multidimensional data space for the elucidation of structural, functional,
behavioral, and clinical correlates. For instance, the relative contributions of each of these imaging modalities can be assessed for
any developmental, aging, or degenerative process with the use of principal component analysis. This leads to an optimized,
temporally efficient MRI neurologic examination that captures the salient features of the structural, metabolic, and functional
states as they change over time. Monitoring these manifestations enables us to elucidate the neurobiological underpinnings of
normal brain development and aging in addition to the endpoints of etiology, natural history, and therapeutic intervention in disease
states (47 ). The ALS case 1 is an example of this application. We have proposed using this technique to assess the changes in both
motor cortical “remapping” and recruitment of additional cortical areas that may occur progressively in ALS. We successfully
performed fMRI in this same patient (Fig. 27.8A ), in addition to MRS and DTI. The data indicated larger areas of activation in the
left than in the right hemisphere and reflected the motor asymmetry of this patient. The pattern of activation in the right
hemisphere was essentially normal, whereas that in the left hemisphere showed recruitment of posterior parietal and association
areas. This pattern of “expansion” of cortical activation has also been observed in other neurodegenerative conditions, such as
Parkinson disease (48 ,49 ) and stroke (50 ,51 and 52 ).

CONCLUSION
Part of "27 - Diffusion Tensor Imaging "
An understanding of the human cerebral white matter, specifically its fiber pathways, is needed. Historically, this objective was
achieved to a certain extent in postmortem material, and the findings were therefore of limited practical value. The advancement
of neuroimaging technology with such techniques as DTI has made it possible to study human white matter fiber pathways in vivo,
and therefore in clinical conditions. The DTI method opens up a new approach, tractography, for studying the various white matter
fiber pathways that are particularly involved in normal cognitive
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processing and in certain disease states, such as language, developmental, psychiatric, and demyelinating disorders. In addition, this
imaging technique may allow a better understanding of the state of white matter pathways during development, aging, and recovery
following brain damage. Finally, the integration of DTI in a more comprehensive MR neurologic examination would allow us to
monitor the evolution of a pathologic condition and thus elucidate the endpoints of etiology, natural history, and therapeutic
intervention in disease states.

ACKNOWLEDGMENTS
Part of "27 - Diffusion Tensor Imaging "
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We thank the following persons for assistance in these projects: D. N. Pandya, E. Kaplan, J. P. Vonsattel, T. G. Reese, E. Kraft, D. N.
Caplan, and R. H. Brown, Jr. This work was supported in part by grants from the National Institutes of Health (NS27950, DA09467,
and NS34189 as part of the Human Brain Project), and from the National Alliance for Research on Schizophrenia and Depression
(NARSAD), the Fairway Trust, the Giovanna Armenise-Harvard Foundation for Advanced Scientific Research, and the Amyotrophic
Lateral Sclerosis Association (ALSA).

Dr. Sorensen receives research support from, is a consultant for, or has spoken on behalf of the following companies within the last
year: Siemens Medical Systems, General Electric Medical Systems, Neurocrine Biosciences Inc., Matrix Pharmaceuticals Inc.,
Millennium Pharmaceuticals Inc., Vertex Pharmaceuticals Inc., and Berlex Laboratories.
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In addition, Dr. Sorensen has an equity position in and holds the position of Medical Director at EPIX Medical Inc., a specialty pharmaceutical company based in Cambridge,
Massachusetts, engaged in developing targeted contrast agents for cardiovascular MRI.

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28
Activation Paradigms in Affective and Cognitive Neuroscience:
Probing the Neuronal Circuitry Underlying Mood and Anxiety
Disorders

Richard J. Davidson

Richard J. Davidson: Laboratory for Affective Neuroscience, University of Wisconsin, Madison, Wisconsin.

Virtually all forms of psychopathology are associated with disturbances in various aspects of affect and cognition. Although most
clinical research has relied on relatively coarse phenomenologic descriptions of symptoms, recent work in neuroimaging with
behavioral activation paradigms offers a new and more penetrating look at specific cognitive and affective processes in
psychopathology. This new trend is predicated on the view that we must go beyond phenomenology to understand the brain circuitry
that is associated with complex mood and anxiety disorders. Advances in our understanding of these conditions will emerge from
research that is designed to examine more specific cognitive and affective processing abnormalities. This work holds promise in
revealing additional targets for therapeutic intervention, both behavioral and pharmacologic. It also will be important in helping to
expose the heterogeneity of these disorders and in offering more meaningful ways in which to parse various subtypes. Finally, by
examining the impact of particular therapeutic interventions on functional brain activity elicited in the context of activation
paradigms, a better understanding of the impact of these interventions on specific subcomponents of the brain circuitry underlying
affect and cognition is likely to emerge.

In this chapter, some key elements of the circuitry that is most relevant to understanding mood and anxiety disorders are first
reviewed. The role of individual differences in the functional activity of this circuitry is then considered. The next section reviews
key approaches and findings of activation paradigms that have been used in this area. The chapter concludes with a summary and a
discussion of future trends in this rapidly developing area.

 CIRCUITRY OF AFFECT AND COGNITION IN MOOD AND ANXIETY DISORDERS

 PROBING THE NEURAL CIRCUITRY OF AFFECT AND COGNITION IN PATIENTS WITH MOOD AND ANXIETY DISORDERS: CONCEPTUAL
AND METHODOLOGIC CONUNDRA
 ACTIVATION STUDIES IN PATIENTS WITH MOOD AND ANXIETY DISORDERS
 SUMMARY AND CONCLUSIONS

CIRCUITRY OF AFFECT AND COGNITION IN MOOD AND ANXIETY

DISORDERS
Part of "28 - Activation Paradigms in Affective and Cognitive Neuroscience: Probing the Neuronal Circuitry Underlying Mood and
Anxiety Disorders "

The review presented in this section of the key components of the circuitry underlying aspects of emotion and cognition that are
most relevant to mood and anxiety disorders is gleaned mostly from studies of lesions experimentally produced in animals, the
human lesion literature, and neuroimaging studies in normal humans. The review focuses on various territories of the prefrontal
cortex, amygdala, hippocampus, and anterior cingulate cortex. Collectively, these studies provide important clues regarding the
types of activation paradigms that are most promising for use in patients with mood and anxiety disorders to probe the underlying
circuitry of affect and cognition. Research in which activation paradigms with neuroimaging are applied in patients with mood and
anxiety disorders is reviewed in a subsequent section.

Prefrontal Cortex
A large corpus of data at both the animal and human levels implicates various sectors of the prefrontal cortex (PFC) in both
cognition and emotion. The PFC is not a homogeneous zone of tissue; rather, it has been differentiated on the basis of both
cytoarchitectonic and functional considerations. The three subdivisions of the primate PFC that have been consistently distinguished
are the dorsolateral, ventromedial, and orbitofrontal sectors of the PFC. In addition, it appears that important functional
differences exist between the left and right sides within some of these sectors.

The role of the PFC in cognitive control has recently been reviewed (1 ), so it is not extensively considered here other than to
underscore that a major function of the PFC in general is “to extract information about the regularities
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across experiences and so impart rules that can be used to guide thought and action” (1 ). One of the principal roles of the PFC is to
represent goal-relevant information, a key component of both complex thought and emotion. As many studies at the nonhuman
primate level have now documented, reward-related information plays a key role in modulating the activity of PFC neurons. Activity
in both lateral and ventromedial zones of the PFC is associated with the identity and size of expected rewards (2 ). This component
of PFC activity is likely governed by a dopaminergic input from the ventral tegmental area of the midbrain (see ref. 1 for review).
Our notion of the role of the PFC in pre-goal attainment positive affect is based on this corpus of research, which is discussed below
(3 ,4 ).

The case for the differential importance of left and right PFC sectors in emotional processing was first made systematically in a
series of studies of patients with unilateral cortical damage (5 ,6 and 7 ). Each of these studies compared the mood of patients with
unilateral left or right-sided brain damage and found a greater incidence of depressive symptoms following left-sided damage. In
most cases, the damage was fairly gross and likely included more than one sector of the PFC and often other brain regions as well.
The general interpretation that has been placed on these studies is that depressive symptoms are increased following left-sided
anterior PFC damage because this brain territory participates in certain forms of positive affect, particularly pre-goal attainment
positive affect; damage leads to deficits in the capacity to generate this form of positive affect, a hallmark feature of depression
(8 ). It should be noted that not all studies support this conclusion. In a recent metaanalysis of lesion studies, Carson et al. (9 )
failed to find support for this hypothesis. Davidson (10 ) has previously reviewed many of these studies and has addressed a number
of critical methodologic and conceptual concerns in this literature. The most important of these issues is that according to the
diathesis stress model of anterior activation asymmetry proposed by Davidson and colleagues (11 ,12 and 13 ), individual differences
in anterior activation asymmetry, whether lesion-induced or functional, represent a diathesis. As such, they alter the probability
that specific forms of emotional reactions will occur in response to the requisite environmental challenge. In the absence of such a
challenge, the pattern of asymmetric activation will simply reflect a propensity but will not necessarily culminate in differences in
mood or symptoms. In a recent study of mood sequelae in patients with unilateral lesions with the largest sample size to date (n =
193), Morris et al. (14 ) found that among stroke patients, it was only in those with small lesions that the relation between left PFC
damage and depressive symptoms was observed. It is likely that larger lesions intrude on other brain territories and mask the
relation between left PFC damage and depression.

A growing corpus of evidence in normal intact humans is consistent with the findings derived from the lesion studies. Davidson and
colleagues have reported that induced positive and negative affective states shift the asymmetry in prefrontal brain electrical
activity in lawful ways. For example, film-induced negative affect increases relative right-sided prefrontal and anterior temporal
activation (15 ), whereas induced positive affect elicits an opposite pattern of asymmetric activation. This general pattern has been
replicated by others using similar measures (16 ,17 ). In positron emission tomography (PET) and functional magnetic resonance
imaging (fMRI) studies, with considerably better spatial resolution, similar PFC activations have been reported, although many
important methodologic details must be considered in interpreting the findings (see ref. 4 for review). The most important of these
is considered in a later section. In addition, a body of evidence supports the conclusion that individual differences in baseline levels
of asymmetric activation in these brain regions are lawfully related to variations in dispositional affective style (18 ).

The ventromedial PFC has been implicated in the anticipation of future positive and negative affective consequences. Bechara and
colleagues (19 ) have reported that patients with bilateral lesions of the ventromedial PFC have difficulty anticipating future
positive or negative consequences, although immediately available rewards and punishments do influence their behavior. Such
patients show decreased levels of electrodermal activity in anticipation of a risky choice in comparison with controls, whereas
controls exhibit such autonomic change before they explicitly know that a choice is risky (20 ,21 and 22 ).

The findings from the lesion method when effects of small unilateral lesions are examined and from neuroimaging studies in normal
subjects and patients with anxiety disorders converge on the conclusion that increases in right-sided activation in various sectors of
the PFC are associated with increased negative affect. Less evidence is available for the domain of positive affect, in part because
positive affect is much harder to elicit in the laboratory and because of the negativity bias (23 ,24 ). This latter phenomenon refers
to the general tendency of organisms to react more strongly to negative than to positive stimuli, perhaps as a consequence of
evolutionary pressures to avoid harm. The findings of Bechara et al. (19 ) on the effects of ventromedial PFC lesions on the
anticipation of future positive and negative affective consequences are based on studies of patients with bilateral lesions. It will be
of interest in the future to examine patients with unilateral ventromedial lesions to ascertain whether valence-dependent
asymmetric effects are also present, although most lesions in this PFC territory are bilateral.

Systematic studies designed to disentangle the specific role played by various sectors of the PFC in emotion are lacking, although a
growing corpus of work illustrates the functional differentiation among different sectors of the PFC in different aspects of cognitive
control (25 ). Many theoretical accounts of emotion assign it an important role
 P.375

in guiding action and organizing behavior toward the acquisition of motivationally significant goals (26 ,27 ). This process requires
that the organism have some means of representing affect in the absence of immediately present rewards and punishments and
other affective incentives. Such a process may be conceptualized as a form of affective working memory. It is likely that the PFC
plays a key role in this process (28 ). Damage to certain sectors of the PFC impairs an individual's capacity to anticipate future
affective outcomes and consequently results in an inability to behave in an adaptive fashion. Such damage is not likely to disrupt an
individual's response to immediate cues for reward and punishment, only the anticipation before and maintenance after an affective
cue has been presented. This proposal can be tested with current neuroimaging methods (e.g., fMRI) but has not yet been rigorously
evaluated. With regard to the different functional roles of the dorsolateral, orbitofrontal, and ventromedial sectors of the PFC,
Davidson and Irwin (4 ) suggested on the basis of both human and animal studies that the ventromedial sector is most likely involved
in the representation of elementary positive and negative affective states in the absence of immediately present incentives. The
orbitofrontal sector has most firmly been linked to rapid learning and unlearning of stimulus-incentive associations and has been
particularly implicated in reversal learning (29 ). As such, the orbitofrontal sector is likely key to understanding aspects of emotion
regulation (30 ). One critical component of emotion regulation is the relearning of stimulus-incentive associations that may have
been previously maladaptive, a process likely requiring the orbitofrontal cortex. The dorsolateral sector is most directly involved in
the representation of goal states toward which more elementary positive and negative states are directed.

Amygdala
A large corpus of research at the animal (mostly rodent) level has established the importance of the amygdala in emotional
processes (31 ,32 and 33 ). Because many reviews of the animal literature have appeared recently, a detailed description of these
studies is not presented here. LeDoux and colleagues have marshaled a large corpus of compelling evidence to suggest that the
amygdala is necessary to establish conditioned fear. Whether the amygdala is necessary to express that fear following learning and
whether the amygdala is the actual locus where learned information is stored is still a matter of some controversy (34 ,35 ). The
classic view of amygdala damage in nonhuman primates (resulting in major affective disturbances as expressed in the Kluver–Bucy
syndrome, in which the animal exhibits an abnormal approach, hyperorality and hypersexuality, and little fear) is now thought to be
a function of damage elsewhere in the medial temporal lobe. When very selective excitotoxic lesions of the amygdala are made that
preserve fibers of passage, nothing resembling the Kluver–Bucy syndrome is observed (36 ). This diverse array of findings suggests a
more limited role for the amygdala in certain forms of emotional learning, although the human data imply a more heterogeneous
contribution.

Although the number of patients with discrete lesions of the amygdala is small, they have provided unique information about the
role of this structure in emotional processing. A number of studies have now reported specific impairments in the recognition of
facial expressions of fear in patients with restricted amygdala damage (37 ,38 ,39 and 40 ). Recognition of facial signs of other
emotions have been found to be intact. In a study that required subjects to make judgments about the trustworthiness and
approachability of unfamiliar adults based on facial photographs, patients with bilateral amygdala damage judged the unfamiliar
persons to be more approachable and trustworthy than did control subjects (41 ). Recognition of vocalic signs of fear and anger was
found to be impaired in a patient with bilateral amygdala damage (42 ), which suggests that this deficit is not restricted to facial
expressions. Other researchers demonstrated an impairment of aversive autonomic conditioning in a patient with amygdala damage
despite the fact that the patient demonstrated normal declarative knowledge of the conditioning contingencies (43 ). Collectively,
these findings from patients with selective bilateral destruction of the amygdala suggest specific impairments on tasks that tap
aspects of negative emotion processing. Most of the studies have focused on perception; the data clearly show the amygdala to be
important in recognizing cues of threat or danger. The conditioning data also indicate that the amygdala may be necessary for
acquiring new implicit autonomic learning of stimulus–punishment contingencies. In one of the few studies to examine the role of
the amygdala in the expression of already learned emotional responses, Angrilli and colleagues (44 ) described a patient with a
benign tumor of the right amygdala who underwent an emotion-modulated startle study. Among control subjects, they observed the
well-known effect of startle potentiation during the presentation of aversive stimuli. In the patient with right amygdala damage, no
startle potentiation was observed in response to aversive versus neutral stimuli. These findings suggest that the amygdala may be
necessary for the expression of an already learned negative affect.

Hippocampus and Anterior Cingulate Cortex

In this section, the contributions of the hippocampus and anterior cingulate cortex (ACC) to emotion and cognition are briefly
mentioned. A more extensive discussion of the contributions of this circuit to emotional and cognitive processing can be found in
several recent reviews (4 ,45 ,46 and 47 ).

The hippocampus has been implicated in various aspects of memory (47 ), particularly declarative memory of the sort we experience
when we consciously recall an earlier episode. The contribution of the hippocampus to emotion and affective
 P.376

style has only recently begun to be gleaned from the available corpus of animal studies on its role in context-dependent memory
(48 ). This literature has generally supported a role for the hippocampus in the learning of context. For example, when an animal is
exposed to a procedure in which a discrete cue is paired with an aversive outcome, in addition to learning the specific cue–
punishment contingency, the animal learns to associate the context in which the learning occurs with the aversive outcome. Lesions
to the hippocampus abolish this context-dependent form of memory but have no effect on learning of the cue–punishment
contingency. The fact that the hippocampus has a very high density of glucocorticoid receptors and participates in the regulation of
the hypothalamic–pituitary–adrenal axis is particularly germane to the importance of this structure in regulating emotion. Basic
research at the animal level has demonstrated the powerful impact of glucocorticoids on hippocampal neurons (32 ,49 ). Data
indicate that the exogenous administration of hydrocortisone to humans impairs explicit memory that is presumably hippocampus-
dependent (50 ), although other data that suggest that in more moderate amounts, cortisol may facilitate memory (Abercrombie,
unpublished doctoral dissertation, Department of Psychology, University of Wisconsin at Madison, 2000). A number of investigators
using MRI-based measures have reported that hippocampal volume is significantly decreased in patients with several stress-related
disorders, including posttraumatic stress disorder (PTSD) (51 ) and depression (52 ,53 ), although several failures to replicate these
findings have also been reported (54 ). In the studies in which hippocampal atrophy has been found, the implication is that
excessively high levels of cortisol associated with the stress-related disorder cause hippocampal cell death and result in the
hippocampal atrophy seen on MRI. Although virtually all these studies have focused on the effects of hippocampal changes on
cognitive function, particularly declarative memory, we have proposed that the hippocampus also plays a key role in the context
modulation of emotional behavior (55 ). Moreover, we have suggested that it is in the affective realm that the impact of
hippocampal involvement in psychopathology may be most apparent, and that in persons with compromised hippocampal function,
the normal context-regulatory role of this brain region is impaired, so that they consequently display emotional behavior in
inappropriate contexts. This argument holds that what may be particularly abnormal in disorders such as PTSD and depression is not
the display of “abnormal emotion” but rather the display of perfectly normal emotion in inappropriate contexts. For example, in the
case of PTSD, extreme fear and anxiety were likely very adaptive in the original traumatic context. This extreme emotional response
probably plays an important role in facilitating an organism's withdrawal from a threatening situation. However, in PTSD, this
response is elicited in inappropriate situations. The patient with PTSD behaves like the animal with a hippocampal lesion in failing to
modulate emotional responses in a context-appropriate manner. These suggestions are only inferential at the present time.
Neuroimaging studies are needed to document the role of the hippocampus in this process in normal and disordered populations. In
addition, further study is needed to understand how and why the hippocampus may preferentially extract and process information
about context. Finally, some research (56 ) indicates that other structures with direct connections to the hippocampus (e.g., the
bed nucleus of the stria terminalis) play a role similar to that of the hippocampus. More work is needed to understand the
differential contributions of the various components of this circuitry.

Many studies that have used neuroimaging methods to probe patterns of brain activation during the arousal of emotion have
reported that the ACC activates in response to emotion. Several investigators (45 ,57 ) have recently distinguished between
cognitive and affective subdivisions of the ACC based on where activations lie in response to tasks that are purely cognitive versus
those that include aspects of emotion. The various tasks used to make these inferences are described in a subsequent section. Based
on the model of Carter et al. (58 ) of the role of the ACC in conflict monitoring in the cognitive domain, we have proposed that the
affective subdivision of the ACC may play a similar role in emotion (4 ). When emotion is elicited in the laboratory, something of a
conflict arises because social norms dictate certain rules for participant behavior that do not usually include the display of strong
emotion. Thus, the very process of activating emotion in the unfamiliar context of a laboratory environment might activate the ACC.
Carter et al. (58 ) have suggested that ACC activation results in a call for further processing by other brain circuits to address the
conflict that has been detected. In most individuals, automatic mechanisms of emotion regulation are likely invoked to dampen
strong emotion that may be activated in the laboratory. The initial call for the processes of emotional regulation may result from
ACC activation.

PROBING THE NEURAL CIRCUITRY OF AFFECT AND COGNITION IN

PATIENTS WITH MOOD AND ANXIETY DISORDERS: CONCEPTUAL AND
METHODOLOGIC CONUNDRA

Part of "28 - Activation Paradigms in Affective and Cognitive Neuroscience: Probing the Neuronal Circuitry Underlying Mood and
Anxiety Disorders "

In this section, some of the key conceptual and methodologic issues in the use of activation paradigms to probe dysfunctions in the
underlying neural circuitry of cognition and affect in patients with mood and anxiety disorders are considered. Issues specific to the
study of dysfunctions in the circuitry of emotion in children are considered in a recent review by Davidson and Slagter (59 ). A key
issue that is often neglected in the design of activation studies is the specification of how deficits in the process that is being
 P.377

studied may account for the symptoms of the disorder. For example, many of the early PET studies in patients with various types of
psychopathology used easy continuous performance tasks in which behavioral differences between groups were not expected to
occur, or they used unilateral somatosensory stimulation (see ref. 60 for review of early studies). Just what the hypothesized
relation was between abnormalities in activation patterns in response to such tasks and symptoms of the disorder being studied was
most often not specified in these earlier studies. The better the conceptual link between task performance and symptomatology,
the more useful an activation paradigm will be for revealing the underlying deficits in the disorder in question. Several examples of
strong conceptual connections between specific task-related deficits and symptomatology in both the cognitive and affective
domains are available and can be consulted by the interested reader (see ref. 61 for an example in the cognitive domain and ref. 30
for an example in the affective domain).

The use of tasks that require active performance on the part of subjects poses a host of methodologic issues that are crucial for
studies of psychopathology. One of the most important of these is matching the difficulty of an experimental task with that of a
control task. This is an issue with a long history in experimental research in psychopathology (62 ), although the neuroimaging field
has yet to appreciate its significance fully. When performance on two tasks is compared between groups, it is imperative that the
difficulty of the two tasks be matched. If one task is more difficult than the other task in normal subjects, than a differential deficit
on one versus the other task may be a consequence of differences in task difficulty and not specific to the processes that are
putatively required for performance of the task. Chapman and Chapman (62 ) have provided many examples of such artifactual
group differences that are products of variation in task performance rather than reflections of differential deficit. It is therefore
essential in neuroimaging studies for activation tasks to be matched in this way. If the tasks that are being compared in imaging
studies are not matched, then any difference found in activation between tasks may arise as a consequence of differences in the
difficulty level of the tasks. Unfortunately, the neuroimaging literature is replete with task comparisons for tasks that do indeed
differ in the level of difficulty and thus are particularly problematic for comparisons between groups. The challenge is to design
control conditions that are matched to the experimental conditions in regard to basic stimulus and response components, in addition
to task difficulty. In one of the few studies to have addressed this potential source of confound, Barch et al. (63 ), using fMRI, found
that the sustained PFC increases in working memory tasks were a function of specific task requirements when they compared such
tasks to control tasks that were matched in level of difficulty but did not require working memory.

In studies with patients, investigators frequently wish to examine changes over time with treatment. In this way, effects that may
be specifically associated with the symptoms of the disorder can be disentangled from those associated with vulnerability to the
disorder. The latter class of effects may also arise as a consequence of scarring—effects produced by having once had the disorder.
In experimental designs that require subjects to be scanned and administered tasks on two or more occasions, it is imperative to
have data on the test–retest stability of the effects in question. If the effects do not show stability over time, it becomes difficult to
interpret group differences in change over time in task activations. We have strongly advocated the psychometric assessment of
both psychophysiologic (64 ) and neuroimaging (65 ) measures. Such assessments can turn up important surprises. Resting regional
glucose metabolism measured with PET is frequently used to assess baseline differences in regional brain activation in various forms
of psychopathology. Using MRI coregistration and regions of interest, we recently examined the test–retest stability across a 6-month
period of such baseline measures of glucose metabolism in subcortical regions implicated in affective processing. We found that all
the regions we examined showed good test–retest stability, including the left and right hippocampus, left and right anterior caudate
region, left and right thalamus, and the left amygdala, but not the right amygdala (65 ). The right amygdala apparently varied over
time, in part because metabolic rate in this region was more affected by the stress of the first scan in comparison with activation
elsewhere.

Emotional pictures are frequently used to provoke changes in affect in imaging studies (66 ). When these pictures are used to
compare patients and controls over time, it is again important to establish that the effects produced are stable over time in normal
subjects. We used startle to probe the test–retest stability of the potentiation produced by negative pictures and the attenuation
produced by positive pictures, and we found poor stability when the same pictures were used on both occasions. It was only when
different pictures were used, matched on valence and arousal characteristics to the original set, that we found better stability (64 ).
These data underscore the importance of not assuming that effects will be stable over time and the utility of actually measuring the
test–retest stability of both task performance and activation changes in normal subjects before conducting a longitudinal study of
changes in patients.

The final issue I wish to raise here pertains to studies in which emotion is provoked by specific task manipulations, such as pleasant
and unpleasant pictures, guided imagery, monetary rewards and punishments, and symptom provocation with the use of actual
feared objects, pictures of objects, or imagined objects. When such paradigms are used, it is imperative for the investigator to
verify independently the presence of the intended affective state. Ideally, such verification should include more than self-report
measures. For example, peripheral biological indices (e.g., emotion-modulated
 P.378

startle, electrodermal activity) can often be effective when utilized in imaging studies to provide an independent index of the
effects of the intended emotion. Moreover, when such measures are used, correlations between activations produced by the task in
question and changes in the peripheral biological index can be computed and are often revealing. For example, Furmark et al. (67 )
found that subjects showing larger conditioned electrodermal changes in a classic conditioning task showed greater increases in
blood flow in the right amygdala during conditioning.

ACTIVATION STUDIES IN PATIENTS WITH MOOD AND ANXIETY

DISORDERS
Part of "28 - Activation Paradigms in Affective and Cognitive Neuroscience: Probing the Neuronal Circuitry Underlying Mood and
Anxiety Disorders "

Most of the extant imaging studies of patients with mood disorders have been performed with PET while the subjects are in a
baseline state. These findings have been recently reviewed elsewhere (68 ). Recent studies using these methods have reported
associations between the severity of particular symptom clusters and patterns of regional blood flow or metabolism (69 ,70 and 71 ).
These studies have underscored the importance of differentiating among various symptoms of depression and illustrate the lawful
relations that can be gleaned by examining associations between specific symptoms and patterns of regional brain activity. The few
studies using activation paradigms that have been conducted in patients with mood disorders have utilized complex cognitive tasks
designed to activate the PFC and ACC. Several studies from Dolan's group (72 ,73 and 74 ) assessed the relationship of regional
blood flow to performance on complex planning tasks during depressed mood in normal subjects and unipolar depressives. Depressed
subjects failed to show normal task-related increases in blood flow in regions of the PFC, ACC, basal ganglia, and thalamus.

Several reports have been published of deficits in task performance in depressed patients in which tasks were used that have been
extensively studied in previous neuroimaging or neuropsychological research. For example, Merriam et al. (75 ) studied Wisconsin
Card Sorting performance in a large group of patients with major depression who had been without medication for at least 28 days.
They found significant deficits on various indices of the Wisconsin Card Sorting task in these patients in comparison with controls.
Moreover, patients with more severe depression, reflected in the Hamilton Depression Scale, performed more poorly. Merriam et al.
(75 ) interpreted their data as consistent with suggestions of a dysfunction in prefrontal function in depression.

Other investigators have suggested that in addition to prefrontal deficits, right-sided parietal dysfunction can also contribute to
specific symptoms of depression (76 ). Henriques and Davidson (77 ), using extremely carefully psychometrically matched verbal and
spatial tasks chosen to reflect left- and right-sided posterior cortical function, found a selective deficit on the spatial cognitive task
(dot localization) in depressed subjects in comparison with controls. Moreover, in this study, measures of brain electric activity
paralleled the performance data and revealed deficits in activation in the right posterior scalp.

We have begun using positive and negative emotional pictures to probe affective processing in depressed patients and controls and
to examine changes over time with treatment (see ref. 78 for early preliminary findings). In more recent work with this same
paradigm, we have found that patients show a reduction in MR signal intensity in the amygdala in response to negative versus
neutral pictures with treatment, whereas controls tested at the same points in time do not. Moreover, the magnitude of MR signal
change in the amygdala predicts treatment response (55 ).

A unique strategy used in research on mood disorders is the short-term depletion of tryptophan among remitted depressed patients
maintained on selective serotonin reuptake inhibitors. The depletion of tryptophan, which reduces the presynaptic availability of
serotonin, often results in depressive relapse. Thus, this method can be powerfully harnessed to examine activation patterns during
the production of depressive relapse in mood-disordered patients. Bremner at al. (79 ) examined regional metabolic rate with PET
during tryptophan depletion and placebo. When they compared subjects who showed a depletion-induced relapse in symptoms with
those without relapse, they found that tryptophan depletion resulted in decreases in regional metabolism in the dorsolateral PFC,
thalamus, and orbitofrontal cortex in patients who relapsed, but not in patients without relapse. Furthermore, patients who
relapsed had a higher baseline (i.e., placebo) metabolism in several areas, including the dorsolateral PFC, orbitofrontal cortex,
hippocampus, and amygdala, than those who did not relapse, which possibly suggests that increased basal activity in these
structures increases vulnerability to depressive relapse.

We are currently using a task designed to elicit anticipatory positive affect, a form of positive affect that, as noted earlier in this
article, is probably implemented at least in part in the dorsolateral PFC. We have hypothesized that this form of positive affect is
abnormally decreased in patients with depression (80 ,81 ). The task we designed is a computerized “lottery” task in which subjects
are required to choose digits that may or may not match the digits displayed by a computer after a 10-second delay during which
the digits spin like a slot machine. We have found reliable attenuation of startle magnitude at selected points in time during this
task (82 ), and we are now studying a variant of this task in the scanner with fMRI in both normal persons and patients with
depression.

Many more studies have been performed in patients with various anxiety disorders (see ref. 83 for recent review). In general, most
studies that have used either symptom provocation or other procedures designed to activate the amygdala have found greater
activation in this region in response to
 P.379

such stimuli in anxious patients than in controls. For example, in two studies using script-driven imagery and PET to assess regional
blood flow, increased activation was found in the amygdala of patients with PTSD (84 ,85 ). In a more recent study comparing
patients with PTSD and controls, Rauch et al. (86 ) reported an increased activation of the amygdala in the PTSD patients in
response to masked facial expressions of fear versus masked expressions of happiness.

Right-sided activation in various territories of the PFC has been found as a general characteristic of anxiety when symptoms are
provoked in patients with several different anxiety disorders (e.g., obsessive-compulsive disorder, simple phobia, and PTSD) (87 ). In
a series of studies that used PET to measure regional cerebral blood flow, Fredrikson and colleagues (88; see ref. 89 for review)
reported increases in secondary visual associative regions in patients with snake phobia in response to the presentation of phobia-
relevant visual stimuli (e.g., pictures of snakes) versus control visual stimuli. Interestingly, in a separate group of patients with
arachnophobia, this pattern did not change after the administrative of diazepam when the subjects were rescanned (90 ). Using fMRI,
Birbaumer et al. (91 ) explored activation of the amygdala of patients with social phobia relative to that in healthy controls as they
were exposed to slides of neutral faces and aversive odor stimuli. The subjects in this study were all male; seven had been given a
DSM-IV diagnosis of social phobia and five were healthy controls matched for age, sex, and education. Neutral faces, which do not
lead to amygdala activation in nonpsychopathologic humans (92 ), and aversive odors, which are significantly associated with
amygdala activation in comparison with a no-odorant control condition (93 ), were presented to all the subjects. Birbaumer et al.
(91 ) compared activation in the thalamus and amygdala in the two groups. In both groups, odors elicited greater bilateral activation
in the amygdala than in the thalamus. In contrast, the social phobics responded to the faces with significantly greater bilateral
amygdala activation than did the controls. However, no difference in regional activation of the thalamus was found between the
two groups in response to the neutral faces. Interestingly, although significant amygdala activation was noted in the social phobics,
their subjective ratings of the faces did not differ from those of the controls.

SUMMARY AND CONCLUSIONS

Part of "28 - Activation Paradigms in Affective and Cognitive Neuroscience: Probing the Neuronal Circuitry Underlying Mood and
Anxiety Disorders "

This chapter began with discussion of some key components of the circuitry underlying affect and cognition that are most relevant
to an understanding of affective and cognitive dysfunction in patients with mood and anxiety disorders. Emphasis was placed on the
PFC, amygdala, hippocampus, and ACC. Next, some important conceptual and methodologic problems that plague research in this
area were considered. The relevance of the task chosen in activation studies to the underlying symptoms of the disorder should be
made explicit in this type of research. Several psychometric problems were then considered, including the issues of matching
experimental and control tasks according to level of difficulty and of establishing the reliability of tasks before using them in
longitudinal studies of patients in whom changes produced by treatment are being examined. Finally, in studies of emotion, the
importance of independent verification of elicitation of the intended emotion was emphasized.

Recent activation studies in patients with mood and anxiety disorders were reviewed. It should be apparent from this review that
studies using this strategy are currently lacking despite its obvious importance in revealing the abnormalities in circuitry that
underlie basic cognitive and affective processes. It is imperative that the next generation of clinical investigators be trained in the
methods and techniques of affective and cognitive neuroscience, the area where such activation paradigms are typically first
developed.

It is also imperative that the results of burgeoning research on cognitive and affective information-processing deficits in mood and
anxiety disorders (see ref. 94 for review) be used to develop new tasks that can be applied with neuroimaging to probe the circuitry
associated with specific types of processing anomalies. For example, an extensive corpus of literature has now documented biases in
forms of explicit memory in depression and biases in attention in various types of anxiety disorders. This information can be used to
design activation paradigms that are more closely linked to the various hypothesized underlying information-processing deficits.
Such research should help to uncover abnormalities in the circuitry underlying the processing of emotion and cognition in patients
with mood and anxiety disorders, and should also provide new targets for novel therapeutic approaches.

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29
Interactions Among Neuronal Systems Assessed with Functional
Neuroimaging

Christian Büchel

Karl Friston

Christian Büchel: Cognitive Neuroscience Laboratory, Department of Neurology, Hamburg University, Hamburg, Germany.

Karl Friston: Wellcome Department of Cognitive Neurology, London, United Kingdom.

In the late nineteenth century, the early investigations of brain function were dominated by the concept of functional segregation.
This approach was driven largely by the data available to scientists of that era. Patients with circumscribed lesions were found who
were impaired in one particular ability while their other abilities remained largely intact. Indeed, descriptions of patients with
different kinds of aphasia (an impairment of the ability to use or comprehend words), made at this time, have left a permanent
legacy in the contrast between Broca's and Wernicke's aphasia. These syndromes were thought to result from damage to anterior or
posterior regions of the left hemisphere, respectively. In the first part of the twentieth century, the idea of functional segregation
fell into disrepute and the doctrine of “mass action” held sway, according to which higher abilities depended on the function of the
brain “as a whole” (1 ). This doctrine was always going to be unsatisfactory. However, with the resources available at the time, it
was simply not possible to make any progress studying the function of the “brain as a whole.” By the end of the twentieth century,
the concept of functional segregation had returned to domination.

The doctrine is now particularly associated with cognitive neuropsychology and is enshrined in the concept of double dissociation
(2 ). A double dissociation is demonstrated when neurologic patients can be found with “mirror” abnormalities. For example, many
patients have been described who have severe impairments of long-term memory but whose short-term memory is intact. In 1969,
Warrington and Shallice (3 ) described the first of a series of patients who had severe impairments of phonologic short-term memory
but no impairments of long-term memory. This is a particularly striking example of double dissociation. It demonstrates that
different brain regions are involved in short- and long-term memory. Furthermore, it shows that these regions can function in a
largely independent fashion. This observation caused major problems for theories of memory, extant at the time, according to
which inputs to long-term memory emanated from short-term memory systems (4 ).

Functional brain imaging avoids many of the problems of lesion studies, but, here too, the field has been dominated by the doctrine
of functional segregation. Nevertheless, it is implicit in the subtraction method that brain regions communicate with each other. If
we want to distinguish between brain regions associated with certain central processes, for example, then we design an experiment
in which the sensory input and motor output are the same across all conditions. In this way, activity associated with sensory input
and motor output will cancel out. The early studies of reading by Petersen et al. (5 ) and Posner et al. (6 ) are still among the best
examples of this approach. The design of these studies was based on the assumption that reading goes through a single series of
discrete and independent stages; visual shapes are analyzed to form letters, letters are put together to form words, the visual word
form is translated into sound, the sound form is translated into articulation, and so on. By a comparison of suitable tasks (e.g.,
letters vs. false font, words vs. letters), each stage can be isolated and the associated brain region identified. Although subsequent
studies have shown that this characterisation of the brain activity associated with reading is a considerable oversimplification, the
original report still captures the essence of most functional imaging studies; a number of discrete cognitive stages are mapped onto
discrete brain areas. Nothing is revealed about how the cognitive processes interact or how the brain regions communicate with
each other. If word recognition really did depend on the passage of information through a single series of discrete stages, we
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would at least like to know the temporal order in which the associated brain regions are engaged. Some evidence comes from
encephaloelectrographic and myoelectrographic studies. In fact, we know that word recognition depends on at least two parallel
routes—one of meaning and one of phonology (7 ). Given this model, we would like to be able to specify the brain regions associated
with each route and have some measure of the strengths of the connections between these different regions.

In this chapter, we show how new methods for measuring effective connectivity allow us to characterize the interactions between
brain regions that underlie the complex interactions among different processing stages of functional architectures.

 DEFINITIONS
 EFFECTIVE CONNECTIVITY
 CONCLUSIONS

DEFINITIONS
Part of "29 - Interactions Among Neuronal Systems Assessed with Functional Neuroimaging "

In the analysis of neuroimaging time series (i.e., signal changes in a set of voxels, expressed as a function of time), functional
connectivity is defined as the temporal correlations between spatially remote neurophysiological events (8 ). This definition
provides a simple characterization of functional interactions. The alternative is effective connectivity, the influence one neuronal
system exerts over another (9 ). These concepts originated in the analysis of separable spike trains obtained from multiunit
electrode recordings (10 ,11 ). Functional connectivity is simply a statement about the observed correlations; it does not comment
on how these correlations are mediated. For example, at the level of multiunit microelectrode recordings, correlations can result
from stimulus-locked transients, evoked by a common afferent input* , or reflect stimulus-induced oscillations, phasic coupling of
neural assemblies mediated by synaptic connections (12 ). Effective connectivity is closer to the notion of a connection, either at a
synaptic (cf synaptic efficacy) or cortical level. Although functional and effective connectivity can be invoked at a conceptual level
in both neuroimaging and electrophysiology, they differ fundamentally at a practical level. This is because the time scales and
nature of neurophysiologic measurements are very different (seconds vs. milliseconds and hemodynamic vs. spike trains). In
electrophysiology, it is often necessary to remove the confounding effects of stimulus-locked transients (that introduce correlations
not causally mediated by direct neural interactions) to reveal an underlying connectivity. The confounding effect of stimulus-evoked
transients is less problematic in neuroimaging because propagation of signals from primary sensory areas onward is mediated by
neuronal connections (usually reciprocal and interconnecting). However, it should be remembered that functional connectivity is not
necessarily a consequence of effective connectivity (e.g., common neuromodulatory input from ascending aminergic
neurotransmitter systems or thalamocortical afferents), and when it is, effective influences may be indirect (e.g., polysynaptic
relays through multiple areas). In this chapter, we focus only on effective connectivity. More details about functional connectivity
can be found in Friston et al. (8 ).

EFFECTIVE CONNECTIVITY
Part of "29 - Interactions Among Neuronal Systems Assessed with Functional Neuroimaging "

A Simple Model
Effective connectivity depends on two models: a mathematical model, describing “how” areas are connected, and a neuroanatomic
model, describing “which” areas are connected. We shall consider linear and nonlinear models. Perhaps the simplest model of
effective connectivity expresses the hemodynamic change at one voxel as a weighted sum of changes elsewhere. This can be
regarded as a multiple linear regression, in which the effective connectivity reflects the amount of rCBF (regional cerebral blood
flow) variability, at the target region, attributable to rCBF changes at a source region. As an example, consider the influence of
other areas M on area V1. This can be framed in a simple equation:

where V1 is an n × 1 column vector with n scans, M is an n × m matrix with m regions and n observations (scans), c is an m × 1
column vector with a parameter estimate for each region, and e is a vector of error terms.

Implicit in this interpretation is a mediation of the influence among brain regions by neuronal connections with an effective strength
equal to the (regression) coefficient c. This highlights the fact that the linear model assumes that the connectivity is constant over
the whole range of activation and does not depend on input from other sources.

Experience suggests that the linear model can give fairly robust results. One explanation is that the dimensionality (the number of
things that are going on) of the physiologic changes can be small by experimental design. In other words, the brain responds to
simple and well-organized experiments in a simple and well-organized way. Generally, however, neurophysiologic interactions are
nonlinear, and the adequacy of linear models must be questioned (or at least qualified). Consequently, we focus on a nonlinear
model of effective connectivity (13 ).

Structural Equation Modeling

The simple model above is sufficient to analyze effective connectivity to one region at a time (e.g., V1 or V2). We will now
introduce structural equation modeling as a tool allowing for more complicated models comprising many
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regions of interest and demonstrate how nonlinear interactions are dealt with in this context. The basic idea behind structural
equation modeling differs from the usual statistical approach of modeling individual observations. In multiple regression or ANCOVA
(analysis of covariance) models, the regression coefficients derive from the minimization of the sum of squared differences of the
predicted and observed dependent variables (i.e., activity in the target region). Structural equation modeling approaches the data
from a different perspective; instead of variables being considered individually, the emphasis lies on the variance–covariance
structure.* Thus, models are solved in structural equation modeling by minimizing the difference between the observed variance–
covariance structure and the one implied by a structural or path model. In the past few years, structural equation modeling has
been applied to functional brain imaging. For example, McIntosh et al. (14 ) demonstrated the dissociation between ventral and
dorsal visual pathways for object and spatial vision by using structural equation modeling of positron emission tomographic (PET)
data in the human. In this section, we focus on the theoretic background of structural equation modeling and demonstrate this
technique with the use of functional magnetic resonance imaging (fMRI).

In terms of neuronal systems, a measure of covariance represents the degree to which the activities of two or more regions are
related (i.e., functional connectivity). The study of variance–covariance structures here is much simpler than in many other fields;
the interconnection of the dependent variables (regional activity of brain areas) is anatomically determined, and the activation of
each region can be directly measured with functional brain imaging. This represents a major difference from “classic” structural
equation modeling in the behavioral sciences, in which models are often hypothetical and include latent variables denoting rather
abstract concepts, such as intelligence.

As mentioned above, structural equation modeling minimizes the difference between the observed or measured covariance matrix
and the one that is implied by the structure of the model. The free parameters (path coefficients or connection strengths; c above)
are adjusted to minimize the difference between the measured and modeled covariance matrix.† (See ref. 15 for details.)

An important issue in structural equation modeling is the determination of the participating regions and the underlying anatomic
model. Several approaches to this issue can be adopted. These include categoric comparisons between different conditions,
statistical images highlighting structures of functional connectivity, and nonhuman electrophysiologic and anatomic studies (16 ).

With respect to anatomic connectivity in humans, the advent of new MR techniques promises a better characterization of neuronal
connectivity in humans. Diffusion tensor imaging measures the anisotropy of diffusion in the brain. The main anisotropy exists in the
white matter because the orientation of neuronal fibres (axons) allows molecules to diffuse more easily along the fiber than in other
directions. Therefore, the main direction of the diffusion tensor reflects the underlying orientation of white matter tracts. Through
tracing algorithms, it is now possible to infer the connectivity of individual regions (e.g., activations derived from an fMRI study) in
an individual brain (17 ) (Fig. 29.1 ).

FIGURE 29.1. Axial diffusion tensor image, obtained by using a TurboSTEAM diffusion sensitized pulse sequence on a Siemens
Vision 1.5T MR scanner. Voxel size 3 × 3 × 3 mm. Average of 20 replications. Needles in each voxel show the largest
eigenvector of the tensor (i.e., the main orientation of diffusion within this voxel). In white matter, the major axis of diffusion
is constrained by the orientation of white matter tracts and therefore provides a good estimate of the direction of fiber
bundles (17). As expected, the corpus callosum in the center of the image shows predominantly horizontal fibers connecting
both hemispheres. In the occipital cortex, parts of the optic radiation with a predominantly anterior–posterior fiber orientation
can be seen. The precision of the method is highlighted by the demonstration of corticocortical U fibers, magnified in the small
image. (From Nolte U, Finsterbusch J, Frahm J. Rapid whole brain diffusion mapping without susceptibility artifacts using
diffusion-weighted single-shot STEAM MRI. Proceedings of the eighth annual meeting of the International Society of Magnetic
Resonance in Medicine, Denver, 2000:807.)

A model is always a simplification of reality; exhaustively correct models either do not exist or are too complicated to understand.
In the context of effective connectivity, one has to find a compromise between complexity, anatomic accuracy, and interpretability.
Mathematical constraints on the model also exist; if the number of free parameters exceeds the number of observed covariances,
the system is underdetermined and no single solution exists.

Each estimated model can be analyzed to give an overall

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goodness-of-fit measure for use when different models are compared with each other. A “nested model” approach can be used to
compare different models (e.g., data from different groups or conditions) in the context of structural equation modeling. A so-called
null model is constructed in which the estimates of the free parameters are constrained to be the same for both groups. The
alternative model allows free parameters to differ between groups. The significance of the differences between the models is
expressed by the difference of the goodness-of-fit statistic. Consider the following hypothetical example. Subjects are scanned
under two different conditions (e.g., attention and no attention). The hypothesis might be that within a system of regions A, B, C,
and D, the connectivity between A and B is different under the two attentional conditions. To determine whether the difference in
connectivity is statistically significant, we estimate the goodness-of-fit measure for two models. Model 1 allows the connectivity
between A and B to take different values for both conditions. Model 2 constrains the path coefficient between A and B to be equal
for attention and no attention. If the change of connectivity between attention and no attention for the connection of A and B is
negligible, the constrained model (model 2) should fit the data as well as the free model (model 1). We can now infer whether the
difference of the two goodness-of-fit measures is significant. Nonlinear models can also be accommodated in the framework of
structural equation modeling by introducing additional variables containing a nonlinear function (e.g., f(x) = x2) of the original
variables (18 ). Interactions of variables can be incorporated in a similar fashion, wherein a new variable, containing the product of
the two interacting variables, is introduced as an additional influence. We will now demonstrate these ideas with an example. More
details of structural equation modeling, including the operational equations, can be found in ref. 15 .

Example: Learning
In the first example, we were interested in changes in effective connectivity over time as expected during paired-associates learning
(19 ). In the case of object-location memory, several functional studies have demonstrated activation of ventral occipital and
temporal regions during the retrieval of object identity and, conversely, increased responses in dorsal parietal areas during the
retrieval of spatial location (20 ). These results suggest domain-specific representations in posterior neocortical structures that are
closely related to those involved in perception, a finding that accords with the segregation of ventral and dorsal pathways in
processing categoric or spatial stimulus features, respectively. Another phenomenon observed in some learning studies is a decrease
of neural responses (i.e., adaptation) to repeated stimulus presentations. This repetition suppression has been replicated
consistently in primate electrophysiologic and human functional imaging studies (21 ). For object-location learning, it is intuitively
likely that two specialized systems need to interact to establish an association. Domain-specific representations or repetition
suppression is not sufficient to account for this associative component. In other words, functional segregation and localized response
properties cannot account for associative learning alone.
In our fMRI experiment, decreases in activation during learning, indicative of repetition suppression, were observed in several
cortical regions in the ventral and dorsal visual pathway. Within the framework of repetition suppression, it has been hypothesized
that decreases in neural responses are a secondary result of enhanced response selectivity (22 ). By analogy to the development and
plasticity of cortical architectures, this refined selectivity is likely to be a consequence of changes in effective connectivity within
the system at a synaptic level. We explicitly addressed this notion by characterising time-dependent changes in effective
connectivity during learning.
The experiment was performed on a 2-tesla (T) MRI system equipped with a head volume coil. fMRI images were obtained every 4.1
seconds with echo-planar imaging (48 slices in each volume). Six subjects had to learn and recall the association between 10 simple
line drawings of real-world objects and 10 locations on a screen during fMRI. Each learning trial consisted of four conditions:
encoding, control, retrieval, and control (Fig. 29.2A ). The behavioral data acquired during retrieval demonstrated that all six
subjects were able to learn the association between object identity and spatial location, for all 10 objects, within eight learning
blocks, as indicated by the ensuing asymptotic learning curves (Fig. 29.2B ).

FIGURE 29.2. Changes in effective connectivity over time in paired-associates learning. A: Design of the study. Blocks of
encoding and retrieval were alternated with control conditions. Subjects had to complete three individual learning sessions to
avoid the confounding effect of time. B: Behavioral performance data for each of the six subjects averaged across all three
learning sessions. C: Anatomic model. Processing of object identity is mainly a property of the ventral visual pathway, whereas
object location is a property of the dorsal stream. We focused on the interstream connections (mainly posterior parietal cortex
to posterior inferotemporal cortex) based on the hypothesis that learning the association of object identity and spatial location
leads to an increase in effective connectivity between the ventral and dorsal streams. (From Büchel C, Coull JT, Friston KJ.
The predictive value of changes in effective connectivity for human learning. Science 1999;283:1538–1541, with permission.)

The structural model used in the analysis embodies connections within and across ventral and dorsal visual pathways and was based
on anatomic studies in primates (Fig. 29.2C ). Primary visual cortex was modeled as the origin of both pathways. In addition to
“interstream” connections between dorsal extrastriate cortex and the fusiform region and between the posterior parietal cortex and
the posterior inferotemporal cortex, we included direct connections based on a hierarchic cortical organization. Given our
hypothesis relating to changes in effective connectivity between dorsal and ventral pathways, the path analysis focused on the
connection between posterior parietal cortex (PP, dorsal stream) and posterior inferotemporal cortex (ITp, ventral stream). We
divided each learning session into EARLY (first part) and LATE (second part) observations and estimated separate path coefficients
for each partition.
The path coefficient between PP and ITp increased significantly during learning in the group (p < .05) and was confirmed by an
analysis of individual subjects showing an increase in effective connectivity between PP and ITp of 0.27. In contrast to the
connections between streams, connections within the dorsal pathway decreased over time.
The estimated change in connectivity from PP to ITp
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clearly depended on the cutoff point between EARLY and LATE. To establish unequivocally a relationship between
neurophysiologically mediated changes in connectivity and behavioral learning, we examined the relationship between the temporal
pattern of effective connectivity changes and learning speed for all sessions and subjects. We estimated the differences in effective
connectivity for seven EARLY and LATE partitions by successively shifting the cutoff. The cutoff time at which the connectivity
change peaked was used as a temporal index of changes in effective connectivity (i.e., plasticity). The significant regression of k, a
measure of learning speed* , on this plasticity index indicated that for sessions showing fast learning (i.e., high value of k), the
maximum difference in path coefficients between PP and ITp was achieved earlier in the session (i.e., EARLY comprises fewer scans
relative to LATE) (Fig. 29.3 ). In other words, the temporal pattern of changes in effective connectivity strongly predicted learning
or acquisition.

FIGURE 29.3. Changes in effective connectivity predict learning. This graph shows the correlation between the temporal index
of changes in effective connectivity and learning. The temporal index is defined as the time of a maximum increase in
effective connectivity between posterior parietal cortex and posterior inferotemporal cortex. For example, a temporal index
of 3 indicates that the maximum increase in effective connectivity occurred between the third and fourth blocks. The numbers
denote the subject from which this temporal index of effective connectivity was obtained. Each subject was scanned during
three independent learning sessions; therefore, each number appears three times. A negative slope means that the maximum
increase in effective connectivity occurs earlier in fast learning. (From Büchel C, Coull JT, Friston KJ. The predictive value of
changes in effective connectivity for human learning. Science 1999;283:1538–1541, with permission.)

Example: Attention
Electrophysiologic and neuroimaging studies have shown that attention to visual motion can increase the responsiveness of the
motion-selective cortical area (V5) (23 ,24 ) and the PP (25 ). Increased or decreased activation in a cortical area is often attributed
to attentional modulation of the cortical projections to that area. This leads to the notion that attention is associated with changes
in connectivity.

Here we present fMRI data from an individual subject, scanned under identical visual motion stimulus conditions while only the
attentional component of the tasks employed was changed. First, we identify regions that show differential activations in relation to
attentional set. In the second stage,
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changes in effective connectivity to these areas are assessed with structural equation modeling. Finally, we show how these
attention-dependent changes in effective connectivity can be explained by the modulatory influence of parietal areas by using a
nonlinear extension of structural equation modeling. The specific hypothesis we addressed was that parietal cortex could modulate
the inputs from V1 to V5.

The experiment was performed on a 2-T MRI system equipped with a head volume coil. fMRI images were obtained every 3.2 seconds
with echo-planar imaging (32 slices in each volume). The subject was scanned during four different conditions: fixation, attention,
no attention, and stationary. Each condition lasted 32 seconds to give 10 volumes per condition. We acquired a total of 360 images.
During all conditions, the subjects looked at a fixation point in the middle of a screen. In this section, we are interested only in the
two conditions with visual motion (attention and no attention), in which 250 small white dots moved radially from the fixation point,
in random directions, toward the border of the screen at a constant speed of 4.7 degrees per second. The difference between
attention and no attention lay in the explicit command given to the subject shortly before the condition: just look indicated no
attention, and detect changes indicated the attention condition. Both visual motion conditions were interleaved with fixation. No
response was required.

Regions of interest were defined by categoric comparisons with use of an output statistical image (SPM{Z}) comparing attention with
no attention and comparing no attention with fixation. As predicted, given a stimulus consisting of radially moving dots, we found
activation of the lateral geniculate nucleus, primary visual cortex (V1), motion-sensitive area (V5), and posterior parietal complex.
For the subsequent analysis of effective connectivity, we defined regions of interest with a diameter of 8 mm centered around the
most significant voxel as revealed by the categoric comparison. A single time series, representative of this region, was defined by
the first eigenvector of all the voxels in the region of interest (15 ).

Our model of the dorsal visual stream included the lateral geniculate nucleus, V1, V5, and the PP. Although connections between
regions are generally reciprocal, for simplicity we modeled only unidirectional paths.

To assess effective connectivity in a condition-specific fashion, we used time series that comprised observations during the condition
in question. Path coefficients for both conditions (attention and no attention) were estimated by using a maximum likelihood
function. To test for the impact of changes in effective connectivity between attention and no attention, we defined a free model
(allowing different path coefficients between V1 and V5 for attention and no attention) and a constrained model (constraining the
V1 → V5 coefficients to be equal). Figure 29.4 shows the free-model and estimated path coefficients. The connectivity between V1
and V5 increases significantly during attention. Note also a significant difference in connectivity between V5 and the PP.

FIGURE 29.4. Structural equation model of the dorsal visual pathway, comparing attention and no attention. Connectivity
between right primary visual cortex (V1) and motion-sensitive area (V5) is increased during attention relative to no attention.
This is also shown for the connection between V5 and the posterior parietal cortex. (From Büchel C, Friston KJ. Effective
connectivity in functional brain imaging. Neural Networks 2000;13:871–882, with permission.)

The linear path model comparing attention and no attention

 P.389

revealed increased effective connectivity in the dorsal visual pathway in relation to attention. The question that arises is, which
part of the brain is capable of modulating this pathway? Based on lesion studies (26 ) and the system for directed attention
described in ref. 27 , the PP is hypothesized to play such a modulatory role.

We extended our model accordingly to allow for nonlinear interactions, testing the hypothesis that the PP acts as a moderator of the
connectivity between V1 and V5. Assuming a nonlinear modulation of this connection, we constructed a new variable, V1PP, in our
analysis. This variable, mediating the interaction, is simply the time series from region V1 multiplied (element by element) by the
time series of the right posterior parietal region.

The influence of this new variable on V5 corresponds to the influence of the PP cortex on the connection between V1 and V5 (i.e.,
the influence of V1 on V5 is greater when activity in the PP is high). The model is shown in Fig. 29.5 . Because our nonlinear model
could accommodate changes in connectivity between attention and no attention, the entire time series was analyzed (i.e.,
attention-specific changes are now explicitly modeled by the interaction term).

FIGURE 29.5. Structural equation model of the dorsal visual pathway

incorporating the interaction effect of right posterior parietal cortex
on the connection from right primary visual cortex (V1) to motion-
sensitive area (V5). (From Büchel C, Friston KJ. Effective connectivity
in functional brain imaging. Neural Networks 2000;13:871–882, with
permission.)

As in the linear model, we tested for the significance of the interaction effect by comparing a restricted and a free model. In the
restricted model, the interaction term (i.e., path from V1PP to V5) was set to zero. Omitting the interaction term led to a
significantly reduced model fit (p < .01), which indicated the predictive value of the interaction term.

The presence of an interaction effect of the PP on the connection between V1 and V5 can also be illustrated by a simple regression
analysis. If the PP shows a positive modulatory influence on the path between V1 and V5, the influence of V1 on V5 should depend
on the activity of the PP. This can be tested by splitting the observations into two sets, one containing observations in which the PP
activity is high and another one in which the PP activity is low. It is now possible to perform separate regressions of V5 on V1 by
using both sets. If the hypothesis of positive modulation is true, the slope of the regression of V5 on V1 should be steeper under high
values of PP.

Variable Parameter Regression

As demonstrated in the previous sections, the basic linear model can be seen as a linear regression. The regression coefficient is
then interpreted as a measure of the connectivity between areas. This interpretation of course implies that the influence is
mediated by neural connections with an effective strength equal to the regression coefficient. Using this approach, one immediately
makes the assumption that the effective connectivity does not change over observations because only a single regression coefficient
for the whole time series is estimated. This is unsuitable for the assessment of effective connectivity in functional imaging because
the goal in some experiments is to demonstrate changes in effective connectivity—for instance, as a function of different conditions
(e.g., attention and no attention) or simply time itself. In the framework of regression analysis, there are three ways around this
problem. Firstly, one can split the data in different groups according to the experimental condition (e.g., attention and no attention)
and then test for the difference of the regression coefficients. However, we may not know a priori the time course of the changes
that allow us to split the data in this way. A second, more general solution is to expand the explanatory variable in terms of a set of
basis functions to account for changes in connectivity. Here, we present another alternative, variable parameter regression, that
allows one to characterize the variation of the regression coefficient by using the framework of state–space models and the Kalman
filter (28 ,29 ).

Mathematical Background
Consider the classic regression model

where y is the measured data vector, x is a vector of explanatory variables, and β is the unknown parameter. Usually, β is estimated
as

However, β can also be estimated recursively with the advantage that inversion of a smaller matrix is necessary. This approach is
known as recursive least squares (30 ). This basic model is now extended to allow β to evolve over time.
 P.390
Variable parameter regression assumes T-ordered scalar observations (y1,… yT) generated by the following model:

where xt is an n-dimensional row vector of known regressors and βt is an n-dimensional column vector of unknown coefficients that corresponds to estimates of effective
connectivity. ut is drawn from a gaussian distribution. All observations are expressed as deviations from the mean.

A recursive algorithm known as the Kalman filter (29 ) can now be applied to estimate the state variable (β) at each point in time and also allows one to estimate the
log-likelihood function of the model. A numeric optimization algorithm is then employed to maximize the likelihood function with respect to P. As the Kalman filter is a
recursive procedure, the estimation of βt is based on all observations up to time t. Therefore, the filtered estimates will be more accurate toward the end of the sample.
This fact is corrected for with the Kalman smoothing algorithm, which is used post hoc and runs backward in time, taking account of the information made available
after time t. Details of the Kalman filter and smoothing recursions can be found in standard textbooks of time series analysis and econometrics (31 ,32 ).

Example: Attention to Visual Motion

To illustrate variable parameter regression, we use the single-subject data set from the study of attention to visual motion. We concentrate on the effect of attention on
the connection between the motion-sensitive area (V5) and the PP in the right hemisphere. Using structural equation modeling, we demonstrated that it is principally
this connection, in the dorsal visual stream, that is modulated by attention (15 ). In the current analysis, we are interested in whether variable parameter regression is
capable of reproducing these findings. We therefore have assessed the effective connectivity βt by regressing PP on V5. An alternate direction search, numeric
optimization, gave a χ2 statistic of 56.4. We therefore had to reject the null hypothesis of no variation at the 5% level. P was estimated to be 0.074 and σ2 was 0.23. The
ordinary regression coefficient β for the model y = xβ + u was estimated at 0.73. Figure 29.6 A,B shows the trajectories of the smoothed and filtered estimates (T)
together with the associated standard errors. It is clearly evident that is higher during the attention conditions than during the no attention conditions. Figure 29.6D
relates our technique to an ordinary regression. In this analysis, we constrained the variance term P to zero and reestimated . The trajectory of now converges
to β, the ordinary regression coefficient of the model y = xβ + u. As expected, the smoothed estimates are simply a constant (i.e., β = 0.73).

FIGURE 29.6. A,B: The trajectory of the smoothed and

filtered estimates (T) together with the associated
standard errors for the variable parameter estimation of
effective connectivity between motion-sensitive area
(V5) and posterior parietal cortex (PP). It is evident that
(the dynamic regression coefficient) is higher during
the attention conditions than during the no attention
conditions. C: Areas that significantly covaried with the
time-dependent measure of effective connectivity
between V5 and the PP [i.e., (T)]. The output
statistical image SPM{Z{ thresholded at p < .001
(uncorrected) overlaid on coronal and axial slices of the
subject's structural MRI. The maximum under the cross-
hairs was at 45, 21, 39 mm, Z = 4. D: The relationship
between our technique and an ordinary regression
analysis. In this analysis, the variance term P was set to
zero (i.e., fixed regression model). The trajectory of
now converges to β (= 0.73), the regression
coefficient of the model y = xβ + u. (From Büchel C,
Friston KJ. Dynamic changes in effective connectivity
characterized by variable parameter regression and
Kalman filtering. Hum Brain Mapping 1998;6:403–408,
with permission.)

We interpret as an index of effective connectivity between area V5 and the PP. In our example, the connection between V5 and the PP resembles the site of
attention modulation. This leads to an interesting extension, in which one might hypothesize that a third region is responsible for the observed variation in effective
connectivity indicated by the trajectory of (T). In other words, after specifying the site and nature of attentional modulation, we now want to know the location of
the source. We addressed this by using (T) as an explanatory variable in an ordinary regression analysis to identify voxels that covaried with this measure of
effective connectivity. Figure 29.6C shows the result of this analysis. Among areas with statistically significant (p < .001, uncorrected) positive covariation were the
dorsolateral prefrontal cortex and the anterior cingulate cortex. This result confirms the putative modulatory role of the dorsolateral prefrontal cortex in attention to
visual motion, as suggested by previous analyses (15 ).

Effective Connectivity versus Categorical Comparisons

One obvious advantage of the assessment of effective connectivity is that it allows one to test hypotheses about the integration of cortical areas. For example, in the
presence of modulation, the categoric comparison between attention and no attention might reveal prestriate, parietal, and frontal activations. However, the only
statement possible is that these areas show higher cortical activity during the attention condition as opposed to the no attention condition. The analysis of effective
connectivity revealed two additional results. Firstly, attention affects the pathway from V1 to V5 and from V5 to PP. Secondly, the introduction of nonlinear interaction
terms allowed us to test a hypothesis about how these modulations are mediated. The latter analysis suggested that the PP exerts a modulatory influence on area V5.

The measurements used in all examples in this chapter were hemodynamic in nature. This limits an interpretation at the level of neuronal interactions. However, the
analogy between the form of the nonlinear interactions described above and voltage-dependent (i.e., modulatory) connections is a strong one. It is possible that the
modulatory impact of PP on V5 is mediated by predominantly voltage-dependent connections. We know of no direct electrophysiologic evidence to suggest that extrinsic
backward PP to V5 connections are voltage-dependent; however, our results are consistent with this. An alternative explanation for modulatory effects, which does not
necessarily involve voltage-dependent connections, can be found in the work of Aertsen and Preissl (10 ). These authors show that effective connectivity varies strongly
with, or is modulated by, background neuronal activity. The mechanism relates to the efficacy of subthreshold excitatory postsynaptic potentials in establishing dynamic
interactions. This efficacy is a function of postsynaptic
 P.391

depolarization, which in turn depends on the tonic background of activity.

CONCLUSIONS
Part of "29 - Interactions Among Neuronal Systems Assessed with Functional Neuroimaging "

This chapter has reviewed the basic concepts of effective connectivity in neuroimaging. We have introduced several methods to
assess effective connectivity—multiple linear regression, covariance structural equation modeling, and variable parameter regression.
In the first example, structural equation modeling was introduced as a device that allows one to combine observed changes in
cortical activity and anatomic models. An application of this technique revealed changes in effective connectivity between the
dorsal and the ventral stream over time in a paired-associates learning paradigm. The temporal pattern of these changes was highly
correlated with individual learning performance, and therefore changes in effective connectivity predicted learning speed. The
second example of structural equation modeling focused on backward modulatory influences of high-order areas on connections
among lower-order areas. Both examples concentrated on changes in effective connectivity and allowed us to characterize the
interacting areas of the network at a functional level. Variable parameter regression was then introduced as a flexible regression
technique that allows the regression coefficient to vary smoothly over time. Again, we confirmed the backward modulatory effect of
higher cortical areas on those areas situated lower in the cortical hierarchy. Although this field is less than mature, the approach to
neuroimaging data and regional interactions discussed above is an exciting endeavor that is starting to attract more and more
attention.

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30
Measuring Brain Connectivity with Functional Imaging and
Transcranial Magnetic Stimulation

Mark S. George

Daryl E. Bohning

Mark S. George: Departments of Psychiatry, Radiology, and Neurology, Medical University of South Carolina, Charleston, South
Carolina.

Daryl E. Bohning: Department of Radiology, Medical University of South Carolina, Charleston, South Carolina.

 THE PROBLEM OF ATTRIBUTING CAUSALITY WITH OBSERVATIONAL FUNCTIONAL BRAIN IMAGING

 TRANSCRANIAL MAGNETIC STIMULATION
 BACKGROUND OF THE CONCEPT OF BRAIN CONNECTIVITY AND CIRCUITS
 INTEGRATING TRANSCRANIAL MAGNETIC STIMULATION WITH FUNCTIONAL IMAGING: PROBLEMS AND CHALLENGES
 REVIEW OF TRANSCRANIAL MAGNETIC STIMULATION FUNCTIONAL IMAGING STUDIES TO DATE
 CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH

THE PROBLEM OF ATTRIBUTING CAUSALITY WITH OBSERVATIONAL

FUNCTIONAL BRAIN IMAGING
Part of "30 - Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation "

Developments in functional imaging during the past two decades have allowed for significant advances in understanding how the
brain functions at a systems, circuit, or organ level. Positron emission tomography (PET), single-photon emission computed
tomography (SPECT), and blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) now allow researchers
to image brain activity (usually related to oxygen or glucose use) with crisp spatial and temporal resolution. For example, fMRI can
spatially resolve structures as small as 1 to 2 mm and view brain activity in time blocks as brief as 2 to 3 seconds. Although this time
resolution is crude relative to the speed of neuronal activity and information flow between brain regions (on the order of
milliseconds), these tools are nevertheless able to demonstrate the activity of clusters of brain cells through a sustained time
domain in association with a behavior or task. Unfortunately, these slow time frames cannot image the directional flow of
information through the brain, although exciting research in this area is under way. Thus, functional imaging tools alone have been
limited in their ability to demonstrate how brain regions work in a coordinated and connected fashion to modulate information and
regulate and produce behavior.

Therefore, a fundamental problem with conventional functional imaging to date has been the inability to probe and understand the
causal relationship between regional brain activity and behavior. For example, if a brain region uses more glucose
(fluorodeoxyglucose PET, or FDG PET) or oxygen (15O PET or BOLD fMRI) while a subject performs a behavioral act, one can safely say
that this regional activity correlates with the behavior. Most functional imaging researchers have correctly and appropriately used
the term correlate, rather than cause, knowing well that the exact causal relationship of the regional activity to the behavior
remains unclear after even the most fastidious study. For example, is the region producing the behavior? Or is the region trying to
inhibit or modulate the behavior? Or is the region only incidentally activated as part of the neural network?

A recent advance in this field involves combining functional imaging with transcranial magnetic stimulation (TMS), a new technology
that noninvasively stimulates the cortex. Used alone without brain imaging, TMS has been useful as a crude mapping tool for motor
functions (1 ). Recently, by combining TMS with functional imaging, researchers have begun to test directly theories about how
information flows within the brain (i.e., the functional connectivity of different brain regions). Thus, with this new combination of
imaging and noninvasive stimulation, the field can now move a step closer to making causal statements of brain function. In this
chapter, we introduce the technology of TMS and describe some of the important issues involved in integrating TMS with imaging to
address brain connectivity. We conclude by reviewing the most recent studies in this new field in which researchers have combined
noninvasive brain stimulation (TMS) with functional brain imaging.

TRANSCRANIAL MAGNETIC STIMULATION

Part of "30 - Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation "

Transcranial magnetic stimulation is a new method for noninvasively stimulating the brain (2 ,3 ). With TMS, a
 P.394

brief but powerful electric current is passed through a small coil of wires held against the scalp. This generates a powerful local
magnetic field, which passes unimpeded through the skull and induces a weaker and somewhat less focal electric current in the
brain (4 ,5 and 6 ). The highly localized TMS magnetic field typically has a strength of about 1 to 1.5 tesla (T) [about 30,000 times
the earth's magnetic field, or about the same intensity as the static magnetic field used in clinical magnetic resonance imaging (MRI)]
(7 ). Although different coil designs allow for more focal or more diffuse stimulation, current technology limits the depth of direct
stimulation to just below the skull in superficial cortex. The magnetic field declines exponentially with distance from the coil. MRI
techniques have enabled researchers actually to image the magnetic field of the TMS coil (8 ). Unfortunately, the actual important
physiologic effects are likely a consequence of the electric current density and the induced electric field in the area of cortex
(Appendix I ). Current theories hold that the induced electric fields cause neuronal depolarization or changes in neuronal activity,
which result in information flow and neurotransmitter release. Newer MRI sequences in development may someday soon allow us to
image the electric current density directly and, by applying this technology to high-resolution structural imaging, actually image the
induced electric field (D. LeBihan, personal communication; May, 1999).

Transcranial magnetic stimulation can be performed in outpatient laboratory settings in awake alert subjects (Fig. 30.1 ) and does
not intentionally cause a seizure, nor does it require anesthesia (9 ). Subjects usually notice no adverse effects except for occasional
mild headache and temporary discomfort at the site of the stimulation. Repeated rhythmic stimulation is called repetitive TMS
(rTMS). Recent technologic advances have led to the development of magnetic stimulators that can repeatedly stimulate faster than
once per second (1 Hz). By convention, stimulation faster than 1 Hz is called fast rTMS, and stimulation slower than 1 Hz is slow
rTMS. This distinction is important because some evidence from work in animals (10 ) and humans (11 ) suggests that stimulation at
different frequencies may have divergent and even antagonistic effects on neuronal activity (12 ,13 ). Importantly also, the risk for
seizures in healthy adults is virtually nil with slow rTMS, and so in the United States, research with slow rTMS does not require an
investigational device exemption from the Food and Drug Administration (14 ).

FIGURE 30.1. The chain of events by which transcranial magnetic stimulation produces changes in the brain and resulting
behavior. Transcranial Magnetic Stimulation (TMS): Time-varying electrical current in a coil produces ⇒ Focal 2 Tesla magnetic
field passes unimpeded through skull ⇒ Induces current in neurons ⇒ Behavioral change.

Over primary motor cortex, a TMS pulse of sufficient intensity causes movement in the opposite arm or leg (an intensity called the
motor threshold). Similarly, a single pulse of TMS over visual cortex can produce a subjective flash of light (or phosphene). Precisely
timed pulses can also interfere with, or augment, other complex tasks (see ref. 1 for review). Thus, TMS alone without imaging has
been used as a relatively spatially crude mapping technique, largely over the motor cortex. rTMS at frequencies of 4 Hz or higher
applied over Broca's area can cause temporary speech arrest (15 ). This ability to block function temporarily is frequency-dependent.
It is unclear which neurons are stimulated with TMS, and whether and how this varies as a function of intensity or frequency. It is
also not known how TMS causes speech arrest—whether through synaptic tetany or activation of local inhibitory interneurons.

In summary, the ability to stimulate the cortex noninvasively with TMS in an awake, alert human is an important new tool and
scientific advance. At present, knowledge is limited about the physiologic and pharmacologic actions of TMS, especially as they may
vary as a function of frequency, intensity, or length, in different brain regions, and in disease states versus health. Coupling TMS
with imaging will likely produce new knowledge in two different areas. First, it will probably advance understanding of how TMS
affects brain and thereby refine the clinical applications and therapeutic uses of TMS in neuropsychiatry. More importantly, from
the perspective of cognitive neuroscience, combining TMS with functional imaging will open up new avenues for the investigation of
brain circuits, connectivity, and the causal chain in brain–behavior relationships and is thus a powerful new research tool.

BACKGROUND OF THE CONCEPT OF BRAIN CONNECTIVITY AND

CIRCUITS
Part of "30 - Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation "
 P.395

Current Approaches to Functional Imaging Analysis and Connectivity

The first step in functional imaging is to find out which areas of the brain show activity (based on increased blood flow) when a
subject performs some mental or physical task, or mentally responds to a stimulus. Conceptually, this is simple—one compares
images of the brain acquired during periods when it is responding to some well-defined test stimulus with images acquired when it is
performing some well-defined control task. The assumption is that the differences in the two sets of images represent the
differences in brain activity during the test stimulus and during the control task. However, these are fairly subtle effects. A small
change in the signal can confound the data in unknown ways. The fMRI signal is inherently noisy and often changes because of
instability of the instrumentation and environmental influences on the subject. For this reason, the problem of determining the
areas of the brain that are being activated by the test stimulus and assigning a probability to that determination has been
extensively studied, and the field has developed commonly accepted methodologies for processing PET and fMRI data
(16 ,17 ,18 ,19 ,20 ,21 and 22 ).

Determination of Regional Activation

The concept of constructing an interpolated spatial map of a statistical parameter, significance probability mapping, was developed
in the analysis of multichannel electrophysiologic (EEG) data (23 ,24 ). In early functional imaging with PET, Fox and Mintun (16 )
introduced what they called change distribution analysis, which consisted of a subtraction of subject-averaged PET images. Present
fMRI-processing methodology draws on both these ideas. In its simplest form, a pixel-by-pixel t test is performed by comparing the
distribution of activation values for two different conditions during the course of an experiment. This gives a t map (i.e., an image
in which each pixel represents the Student's t statistic for the comparison of the test condition relative to the reference condition at
that location). By using the associated p values and the number of degrees of freedom, the t values can be converted to z values
(gaussian distribution: mean 0, variance 1) to obtain z maps. This is the basis for statistical parametric mapping (25 ), formally
described as the construction of spatially extended statistical processes, or maps, to test a hypothesis (usually about
neurophysiology) directly. Generally based on a linear and parametric model, statistical parametric maps (SPMs) are image processes
with voxel values that are, under the null hypothesis, distributed according to a known probability density function, usually gaussian.
In the same way that a t value is interpreted by reference to Student's t distribution, an SPM is interpreted by referring to the
probabilistic behavior of stationary gaussian fields (26 ) and can be used to make statistical inferences about regionally specific
findings (e.g., the probability of finding an activation focus by chance). In general, SPMs characterize experimentally elicited
changes in terms of (multiple) activation foci. Regions of the SPM with high or low values are interpreted as regional activations.
Thus, it is possible to locate areas of the brain that are “active” during the execution of some task (i.e., the signal in those areas
has a time-varying pattern that correlates with the pattern of the conditions in the experiment) (17 ).

Functional and Effective Brain Connectivity

Intuitively, it is common to think of brain functional connectivity as two or more separate anatomic areas of the brain that influence
each other in the performance of some mental or physical task (e.g., recalling a name or moving a finger), or to produce a mental
state (e.g., sadness). This action or state of the brain, in turn, affects the body through the somatosensory system, the sympathetic
and parasympathetic nervous system, and of course the brain's neuropharmacologic/neuroendocrine hypothalamic–pituitary–adrenal
system. Both in electrophysiology and functional neuroimaging, connectivity of different areas of the brain has been based on the
correlation between regions. In the case of electrophysiology, this means the EEG signal (24 ,27 ,28 ,29 ,30 ,31 and 32 ), and in
functional neuroimaging, this means the time course of regional blood flow or glucose use (18 ,21 ,33 ,34 )).

Friston et al. (19 ) emphasize the distinction between functional connectivity, the temporal correlations between remote
neurophysiologic events, and effective connectivity, the influence of one neural system on another (i.e., a functional as opposed to
a causal relationship). Viewed in this way, functional connectivity is simply the observed covariance among different brain systems.
It is an operational definition and says nothing about the causal relations of the observed correlations. To characterize distributed
brain systems, the functional connectivity (covariance) matrix, obtained from a time series of neurophysiologic measurements, is
subjected to principal component analysis (PCA) (20 ,35 ) (Appendix II ). The resulting eigenimages (principal components or spatial
modes) each identify a spatially distributed system, comprising regions of the brain that are jointly implicated by virtue of their
functional interactions (connectivity). This analysis of neuroimaging time series is predicated on established techniques in
electrophysiology (both EEG and multiunit recordings). For example, in the analysis of multichannel EEG data, the underlying spatial
modes that best characterize the observed spatiotemporal
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dynamics are identified with a Karhunen–Loeve expansion. Commonly, this expansion is in terms of the eigenvectors of the
covariance matrix associated with the time series. The spatial modes are then identical to the principal components identified with
a PCA.

Structural Equation Modeling

Principal component analysis and factor analysis approaches attempt to integrate the spatially distributed activations found in SPMs
into functional systems characterized by the eigenimages or spatial modes. However, one would like to go further and explore the
influence of one area on another (effective connectivity), not just the correlation (functional connectivity). Many anatomic
connections are reciprocal, and simple pairwise correlations cannot resolve asymmetric influences. Structural equation modeling is
an attempt to address this problem.

In describing their neural structural equation models, McIntosh and Gonzalez–Lima (21 ) use the terms anatomic model and
functional model (36 ). The anatomic model simply represents the discrete anatomic brain regions and the neuroanatomic
connections between them used in the structural equation models. These anatomic models have been derived from the observation
of patients with brain lesions and from animal studies, or inferred from neuroimaging studies and the analysis of SPMs. The
interregional correlations of activity are used to assign numeric weights to the connections in the anatomic model, which leads to
the functional model. A functional model, therefore, represents the influences of regions within the model on each other through
the anatomic connections, and both the magnitude and the sign of the path coefficients can be estimated. In some respects, the
functional model is close to the notion of effective connectivity (20 ,37 ) because it depicts the influence of one region on another.
The difference is that the influences in the functional model, unlike effective connections, are explicitly depicted as direct and
indirect effects through the anatomic model. Effective connectivity, as defined by Aertsen et al. (38 ), resembles most closely direct
effects in that an effective connection is the influence of one neural element on another irrespective of direct or indirect influences.
In structural equation modeling, effective connections, or total effects, are further decomposed into direct and indirect effects by
use of the anatomic model. A similar distinction can be made in covariance analysis, which is often characterized as exploratory
(objective) or confirmatory (theoretical) analysis. PCA and factor analysis are essentially exploratory techniques because no
constraints are placed on how the variance in the system is expressed. Structural equation modeling is typically thought of as a
confirmatory approach (confirmatory factor analysis) because a causal model is usually being confirmed or disconfirmed (39 ).

Transcranial Magnetic Stimulation as a Probe to Alter Connectivity Networks

Although the PCA and structural equation modeling techniques are well grounded statistically and quite powerful, they cannot
eliminate the possibility that apparent interactions between two regions may be a consequence of other factors. The activity of two
theoretically linked regions may be modulated, either directly or via changes in neurotransmitter release, by neurologic activity in a
third region that is outside of the field of view of the imaging experiment, or has not been included in the structural equation
anatomic model. The two regions may also be responding to different aspects of the test stimulus, either inherently because of the
nature of the task, or because of engagement of the subject in performing the task.

Since it was first developed, TMS has been used to test nerve connections, nerve excitability, and nerve conduction times. One
might think of this as two anatomic areas with a single connection. Paus et al. (34 ) and our laboratories at the National Institute of
Mental Health (40 ,41 ) and the Medical University of South Carolina (42 ,43 ) demonstrated that TMS might be combined with
neuroimaging to explore the connectivity of more complex three-dimensional networks in the brain to allow the direct assessment of
neural connectivity without requiring the subject to engage in any specific behavior.

Because TMS seems to have a disruptive effect in most areas of the brain, its most likely use will be to suppress the activity of a
region of the brain or disrupt communication between areas. This may be done by simply applying the TMS pulse at the moment the
task is performed or the stimulus is applied, and noting the changed response pattern. It may also turn out that it will be possible to
apply TMS after a precisely timed delay to modulate responses (44 ) and so investigate brain communications at time resolutions far
greater than that of the hemodynamic response, approaching that of EEG. Thus, TMS provides a noninvasive means of perturbing
brain circuits both spatially and at high temporal resolution. Because it is a noncognitive stimulus, its effects are less dependent on
subject engagement (“attention and performance”), and because it is a more direct and quantifiable stimulus, it more closely
relates to basic neurophysiologic parameters such as nerve excitability and conduction times.

INTEGRATING TRANSCRANIAL MAGNETIC STIMULATION WITH

FUNCTIONAL IMAGING: PROBLEMS AND CHALLENGES
Part of "30 - Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation "

Stimulating the brain with TMS while simultaneously imaging brain activity presents a host of unique technical problems, including (a)
physically placing the coil in the scanner
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and over the appropriate brain regions, (b) determining whether the TMS coil interferes with the functional image, and (c)
integrating the brief time domains of TMS with the slower temporal resolution of most modern imaging tools. We discuss several of
these issues and recent attempts at dealing with them.

Placement of the Coil—Structural or Functional Guidance

One of the most obvious problems in combining imaging and stimulation revolves around how to position the TMS coil over the skull.
Most researchers have used either a structural or functional guidance system.

Structural Guidance
The shape of the coil determines the magnetic field in the brain, and thus the pattern of induced electric current (7 ,45 ). For
circular coils, the magnetic field is most intense near the windings. When a circular coil is placed flat against the scalp, it induces a
toroidal ring of electric current in the underlying cortex that is of the same size as the coil itself but more diffuse. The electric
current distributions are assumed to be broad and the effects distributed. In contrast, with figure 8 coils, a focus at the intersection
of the two loops is roughly twice as intense as that obtained with a circular coil and the same current (Fig. 30.2 ). Although the
distribution of induced current is still fairly broad, stimulation over motor cortex demonstrates that it is sufficiently focal to cause
movement in one location; moving the coil less than a centimeter or even slightly changing the angle results in no movement, or
movement in different muscle groups.

FIGURE 30.2. Magnetic field of single-turn figure 8 coil in plane parallel to and one-fourth diameter from the coil: x, y, z
components and magnitude of field. (From Cohen LG, Roth BJ, Nilsson J, et al. Effects of coil design on delivery of focal
magnetic stimulation. Technical considerations. Electroencephalogr Clin Neurol 1990;75:350–357, with permission.)

The coil can be positioned in several ways, based on the underlying brain structure. Perhaps the best method is to acquire a
structural MRI scan of the head and then use image-guided systems to align the TMS coil precisely over a specific brain region.
Several groups are exploring this option by using structural MRI and then integrating the TMS with PET (46 ,47 ). Performing the
same mechanical alignment within an MRI scanner is more challenging because of the problems that arise when metal is used within
a powerful magnetic field. An intermediate approach, employed by the McGill group (34 ), is to position the coil according to a
probabilistic brain system keyed to landmarks on the subject being studied, rather than according to the subject's known anatomy as
determined by MRI. This method is much easier and is adequate if one is planning on using only group statements for the statistical
analysis (which requires spatially transforming the imaging data into a common brain atlas). Unfortunately, because the structural
morphology of the brain and the functional location of behaviors vary greatly between individuals, the probabilistic method suffers
from the problem that it is not certain within an individual whether the coil is positioned over the structure or region being studied.

The main difficulty with both image-based and structural localization is that unless one knows the exact relation of the induced
currents relative to the position of the TMS coil, one really does not know where to stimulate most efficiently. The induced electric
current is tissue-dependent, so it is not the same at different places on the scalp. Only if the currents had the same relation to the
position of the coil, and one could use MR guidance to place the coil over the same sulcus or gyrus in a subject's brain, could one
even be sure of stimulating the same structural area. However, brain conformation varies, so the induced currents might still
impinge on the cerebral cortex at a different angle.

Paus and colleagues (34 ) approached this problem by obtaining an image volume with MR from each subject and
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spatially transforming it into Talaraich space (a widely used common brain space) (48 ). After determining the Talairaich location
shown by neuroimaging studies to correspond to a function, they performed the inverse transform back into the MR image space for
each subject. Finally, using stereotactic guidance (49 ), they positioned the coil over this point, in effect ensuring that they were
probably stimulating the functional location of the behavior in all subjects. Krings et al. (50 ) used a frameless stereotactic system
to coregister TMS motor maps with fMRI data obtained during performance of a motor task. In two patients, they also performed
direct cortical stimulation, finding good correspondence among all three methods. This probabilistic technique is more or less
acceptable, depending on how consistently from one subject to the next a function is located within identifiable anatomic
structures, and on how the shapes of those structures vary. Thus, this technique still entails the problem that function does not
strictly map to the same location in different individuals.

Placement Based on Function

A different approach is to use TMS, electromyography, or functional imaging to determine the regions activated during a behavior
and then position the coil directly over the functioning region. For behaviors like movement or phosphene production over visual
cortex, one can bypass the imaging step, simply finding the scalp location with the desired behavioral effect and then keeping the
TMS coil at this spot throughout an imaging study (functional behavioral approach to placement). Unfortunately, outside primary
motor and vision areas, TMS does not produce easily viewed effects, so that this direct functional approach becomes impossible.
Despite its apparent simplicity, the functional behavioral approach of using elicited movement to guide coil placement to perform
TMS over the motor cortex is associated with certain problems. The movement elicited by the TMS is a reassuring, if somewhat
imprecise, way of being certain that one is in the correct area. However, even with comparable visible movement, one can be on
one side or the other of the target area, and it is difficult to know how much or how little additional stimulation is occurring.
Because this method reliably causes activation in large corticospinal circuits, we have used the functional behavioral approach for
initial studies of interleaved TMS/fMRI effects over motor cortex (51 ,52 ).
Whichever method of locating the site of stimulation is used, it is important that the TMS coil be positioned accurately and
repeatably, and then held securely in place so that its position relative to the brain is maintained throughout the stimulation. Each
group seems to have developed its own mounting systems. We have developed a system for accurately and repeatably positioning
the TMS coil within an MRI scanner (53 ). Both structural and functionally guided techniques have their place, and eventually
systems will likely be developed for relating the two.

Interaction of the Transcranial Magnetic Stimulation Coil and Image Acquisition

Yet a different technologic problem in this new area revolves around whether and to what extent the TMS coil interferes with the
functional image acquisition. Because of this concern, the early combinational studies used imaging techniques in which TMS was
delivered in a location other than that where the actual brain imaging was performed (54 ,55 ). Both FDG PET and perfusion SPECT
allow one to administer TMS and deliver the radiopharmaceutical agent away from the scanner. The tracer crosses the blood–brain
barrier and then settles into active regions. The subject can then be transported to the scanner for image acquisition.
Because of radiation dose limits and slow time resolution, neither of these techniques (FDG PET and perfusion SPECT) is suited for
thoroughly examining circuits and behavior with the combination of TMS and imaging. For this purpose, it is much better to have the
TMS coil directly within the scanner. However, one then has to understand to what extent, if any, the TMS coil interferes with the
acquisition of the functional images.
In PET, groups can perform an initial transmission scan before the functional image and then subtract the minor reduction in tracer
counts caused by the TMS coil. Obviously, solid core coils are not suited for this type of combinational imaging. With fMRI, the TMS
coil can produce both static and dynamic artifacts. Although it may be possible to correct for the static effects, as in PET, some
groups are so concerned about static artifacts that they have developed systems that quickly lift the TMS coil 2 to 3 cm from the
scalp during the actual MR image acquisition following a train of pulses (56 ). Both mechanical and pneumatic systems have been
developed to do this. Although it minimizes the potential impact of the artifact, mechanically moving the TMS coil produces
shimming and alignment issues on its own and does not allow for true interleaved imaging in real time. The dynamic artifacts
produced by TMS within an fMRI scanner are both more complicated and more difficult to account for (see Fig. 30.3 and ref. 53 for
a full discussion). Substantial progress has been made, so that this is not a major concern.

FIGURE 30.3. Researchers at the Medical University of

South Carolina have recently developed the technique of
performing transcranial magnetic stimulation (TMS) within
the bore of a conventional 1.5-T magnetic resonance
imaging scanner, the setup of which is depicted in (A). This
process produces dynamic TMS-induced eddy currents (B)
and static TMS-induced eddy currents (C). As seen in (B),
these dynamic eddy currents are approximately twice as
strong as the read gradient for about 20 milliseconds, and
then drop to approximately the same size as the read
gradient for another 20 milliseconds. Although the major
eddy currents have died out by 40 to 50 milliseconds after
the TMS pulse, some longer, low-level currents are still
present that cause significant image artifact (C).

Integration of Temporal Domains of the Scanner and Transcranial Magnetic

Stimulation
The final picture produced by each of the functional imaging tools represents summed brain activity over a measure
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of time. The averaged time domain ranges from 20 to 30 minutes for FDG PET, to 40 to 60 seconds for O PET and perfusion SPECT,
15

to 2 to 3 seconds for BOLD fMRI, to milliseconds for EEG and electromyography. The actual TMS pulse is very brief, on the order of
300 microseconds. Thus, it is important in all combined imaging studies to understand the relationship between the TMS activity and
the summed functional image. Moreover, because of the concerns of potentially causing a seizure with long trains of high-intensity,
high-frequency TMS, only certain TMS parameters can be used constantly over the time domains of some forms of imaging.
Unfortunately, some TMS effects, such as speech arrest, occur only with high-intensity and high-frequency stimulation.

Steady State
In this model, researchers perform TMS throughout an entire scan and then compare results with those of another scan in which all
conditions are the same except for the TMS. Even with this design, and stimulation frequencies of approximately 1/s (1 Hz), most of
the imaging is performed with the actual TMS machine off as a function of time.

Block Design
A different model is to scan in blocks in which periods of TMS are separated by periods of rest. An example of this is shown in Fig.
30.4 , which describes the interleaved TMS/fMRI
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setup. Figure 30.5 portrays results of a recent study in which TMS was administered over motor cortex. This study showed that TMS
at intensities slightly greater than motor threshold (110%) activates approximately the same number of pixels in the same region as
does a volition movement (Fig. 30.5C ). This study also revealed the relative magnitude of the TMS effect and the temporal
relationship to changes in blood flow.

FIGURE 30.4. To perform interleaved transcranial magnetic stimulation/magnetic resonance imaging (TMS/fMRI), one must
coordinate the TMS pulses with the MRI signal acquisition and interleave the two. A: An example of this process for a TMS rate
of 1 to 2 Hz. B: An example of serial blood flow changes underneath the TMS coil (over left motor cortex) and in a control
region. Note the increase from rest, r, in absolute blood oxygenation-dependent (BOLD) activity underneath the coil when it
discharges at 1 Hz (task, T), and how it decreases afterward (post, P).

FIGURE 30.5. One of the first studies in which this interleaved technique was used attempted to detect differences between
volitional and transcranial magnetic stimulation (TMS)-induced movement of the thumb. In (A), the TMS device was placed
over the left motor cortex of subjects, who alternately had TMS move their thumb (TMS) and then volitionally moved their
thumb in response to a tone (VOL). In (B) are averaged group time series of brain activity during TMS, volition, or a noise
control region (upper left). Note that for voxels that were activated in both tasks, the percentage rise in blood oxygenation-
dependent (BOLD) activity does not differ from baseline. Thus, TMS produced BOLD changes that are dynamically similar to
those of regular movement. In (C), the center of mass of the BOLD signal is virtually the same for both TMS and volition, within
the limit of resolution of the magnetic resonance imaging scanner (2 mm).

Single-Event fMRI
The method that is currently closest to the actual timing of TMS and brain events is single-event fMRI, or averaged-single-trials fMRI.
With this method of scanning, images are steadily acquired at a rapid rate while the performance of a single event is rapidly
interspersed. One can image the brain activity associated with a single TMS pulse by repeating the event many times and averaging
the images acquired at similar times after the events, much as electrophysiologists have done with evoked responses (57 ). Although
the BOLD response is relatively sluggish (on the order of 2 to 3 seconds), some groups are experimenting with the initial slope of the
response to attempt to increase time resolution (Fig. 30.6 ). Applying TMS pulses to different brain regions with different interpulse
interval times (milliseconds) may represent a unique way of improving the temporal resolution
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of BOLD fMRI and of studying information flow within circuits. With further refinement, the combination of single-pulse and paired-
pulse TMS and averaged-single-trials fMRI will probably be of considerable interest in in vivo neurophysiology.

FIGURE 30.6. One can also use an averaged-single-trials approach of transcranial magnetic stimulation (TMS) and functional
magnetic resonance imaging (fMRI)—that is, one can discharge a single TMS pulse and then measure the blood oxygenation-
dependent (BOLD) response (top). The time series above show the BOLD response in a control region, for the auditory cortex,
and in motor cortex. Note that a single pulse of TMS over motor cortex sufficient to cause the opposite thumb to move
produces more blood flow changes in auditory cortex (caused by noise) than it does in the motor cortex under the coil.

REVIEW OF TRANSCRANIAL MAGNETIC STIMULATION FUNCTIONAL

IMAGING STUDIES TO DATE
Part of "30 - Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation "

Transcranial Magnetic Stimulation Interleaved

Fluorodeoxyglucose PET
The first published combination of TMS and functional neuroimaging in real time was performed with FDG PET in a patient before
and after rTMS treatment for refractory depression (54 ). At a separate time, these investigators also injected the glucose while the
patient was intermittently stimulated at 20Hz over the left prefrontal cortex for 20 minutes. In comparison with her depressed scan
at baseline, her total brain metabolism rose following weeks of TMS treatment. Also, the scan that was taken during left prefrontal
TMS showed marked increases in activity, especially over the prefrontal cortex. Conclusions from this single case study are limited.

Complexity of the Issues as Demonstrated by Initial Simple Studies over Motor Cortex
A basic question for TMS and functional imaging is what happens to blood flow or activity in motor cortex while TMS is stimulating
the thumb. A straightforward hypothesis would be that TMS increases blood flow in a manner similar to that produced by volitional
movement. Confusion ensued when an early and still unpublished study of 1-Hz stimulation over the motor cortex for thumb showed
decreased glucose uptake at the putative site of stimulation and in the contralateral motor cortex (40 ). Stimulation was performed
at 1 Hz because FDG takes 20 minutes to settle into neurons and is thus a composite picture of brain activity over 20 minutes. This
paradoxical decrease in localized brain activity both under the coil and at the mirror or contralateral site during TMS was surprising,
but findings of decreased brain activity like this had been found in some electrophysiologic studies (12 ). The final image was a
summed picture of 20 minutes of brain activity. It is likely that TMS has multiple different effects during that time—increased
activity immediately with stimulation, decreases during the rest time between TMS pulses, and dynamic changes across the 20
minutes. Peter Fox and one of the chapter authors (MSG) (58 ) next sought to test this finding directly by using 15O PET rather than
FDG PET, with the TMS coil directly in the scanner. 15O PET has a shorter time frame (approximately 1 minute for tracer uptake) than
18
FDG PET (20 to 30 minutes). Therefore, imaging with 15O PET during stimulation requires that the TMS coil be placed inside the PET
gantry. Using the exact same design as in the FDG study, but scanning every 10 minutes for 1 minute, we found that slow (1-Hz)
rTMS over the motor cortex caused an increase in cerebral blood flow, although this was noted in only four subjects (58 ). Both of
these studies used a functional behavioral placement
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of the TMS coil over the optimal position for movement of the thumb and stimulated at or near the motor threshold with visual
confirmation that the TMS was producing activity in the motor circuit. Thus, the results of these two initial studies were confusing
and frankly contradictory.

To add even more confusion, Paus et al. (34 ) in the same year published a study combining 15O PET and TMS. In this study,
intermittent fast (10-Hz) rTMS over the frontal eye fields for 1 minute caused dose-dependent increases in blood flow at the
stimulation site and in visual cortex. In other words, when they increased the number of 10-Hz trains within the minute, blood flow
increased. Surprisingly, when the same investigators used the same rTMS parameters in the same subjects but shifted the coil to
motor cortex, they found a dose-dependent reduction in cerebral blood flow (59 ). Importantly, they positioned the coil based on a
probabilistic brain, and they also stimulated below motor threshold. No thumb movement occurred in these subjects.

Thus, the initial dream of using TMS and imaging to address connectivity problems in the brain has been hindered by a lack of
consensus about basic imaging and TMS questions. Using yet a different technology, BOLD fMRI, our group in several studies
consistently found that over much shorter time domains (7 to 30 seconds), TMS at motor threshold or above, positioned by a
functional behavioral approach, consistently produced increases in blood flow at the stimulation site and in connected regions, such
as the contralateral motor cortex and cerebellum (51 ,52 ). The issue now appears to be settled; the same National Institutes of
Health group that found decreases with FDG PET has recently completed a more fastidious 15O PET study. In this study, Speer and
colleagues found dose-dependent increases in blood flow in motor cortex with 1-Hz TMS, as was noted in the study of Fox et al. (58 )
and confirmed with the BOLD fMRI technique by our laboratory (A. Speer, personal communication; May, 2000).

There is now a small consensus in the existing literature that blood flow increases under the motor cortex in a dose-dependent
manner when the TMS coil is positioned by finding the appropriate spot for optimal thumb movement (functional behavioral
technique) and stimulation is above motor threshold (and activates large excitatory neurons). When the TMS coil is positioned in this
same region by a probabilistic approach, dose-dependent decreases have sometimes been found. Thus, some of the discrepancy in
the literature can be explained not only by differing time domains of the imaging technologies, but also by potential differential
effects caused by the method of coil placement. In this vein, using an identical study paradigm as their most recent TMS motor study,
the National Institutes of Health group (Speer and colleagues) stimulated the same subjects over the prefrontal cortex, defined
simply as a certain distance from the motor area (a very crude probabilistic approach, such as has been employed in many of the
TMS challenge and clinical studies). Paradoxically, this group found in these same subjects dose-dependent decreases in blood flow
over prefrontal cortex. In earlier work at 80% motor threshold, we found similar decreases in eight healthy adults when we used
perfusion SPECT, with a tracer uptake time of 30 to 40 seconds, to image cerebral blood flow during fast (20-Hz) left dorsolateral
prefrontal cortex rTMS (60 ). In comparison with a control scan with sham TMS, we found relative decreases under the coil site and
in the anterior cingulate and orbitofrontal cortex. In contrast, a recent BOLD fMRI study over prefrontal cortex by our group found
increases in blood flow at 120% motor threshold (61 ). With fMRI, one can examine individual differences, and a great deal of
heterogeneity of response was noted across subjects. We are currently performing repeatability studies within subjects over time to
address the inherent noise in this scanning system and the question of whether repeated TMS/fMRI studies yield consistent results.

The two most likely explanations for the opposite findings over motor and prefrontal cortex are that different brain regions react
differently, or that the method of TMS coil placement matters, and that the effects of clear stimulation of large corticospinal
neurons may be different from those of nonspecific stimulation of local inhibitory neurons with only probabilistic positioning.
Obviously, a series of studies is needed to settle this most important issue. For example, an important next study would be to test
directly the issue of blood flow as a function of functional behavioral versus probabilistic coil placement and see if functional
positioning produces increases in blood flow and probabilistic placement decreases, presumably secondary to differential stimulation
of excitatory versus inhibitory neurons.

BOLD fMRI
As mentioned above, the most promising, but also the most technically challenging, TMS imaging modality is a combination of TMS
and fMRI. Bohning et al. (51 ) first demonstrated the capability of interleaving TMS and BOLD fMRI with good spatial and temporal
resolution. This technique was initially thought impossible by many because of concerns about introducing a focal TMS magnetic
field (1 to 2 T) inside a clinical MRI scanner of 1.5 T. Our group has found that this technique, with the right precautions, is both
feasible and safe. Considerable progress has been made in devising a system for interleaving TMS with fMRI (53 ). Figure 30.4 shows
one subject's brain with areas of TMS-induced activation superimposed in color. The time–activity curve shows the changes in BOLD
signal over the course of the experiment as the TMS machine is alternately triggered at 1 Hz for 18 seconds and then turned off.

Work to date with this technique has shown that it is sensitive enough to detect subtle differences in brain blood flow response that
result from minor changes in TMS intensity (52 ).
 P.405

Additionally, direct comparisons of blood flow changes in motor cortex caused by TMS or volition show a surprising similarity between TMS-induced changes and those associated with
normal movement (71 ). For example, the location of the peak blood flow change is the same for TMS and normal movement (within 2 mm). Also, stimulating at around 1 Hz and just at
motor threshold activates roughly the same amount of brain tissue, and to the same degree. Thus, although many have the perception that TMS is causing supraphysiologic changes in
the brain, these data imply that TMS at these parameters is acting remarkably like normal physiology.

Using Interleaved TMS/fMRI to Address Issues of Connectivity: An Initial Study

Several electrophysiologic studies have suggested that 1-Hz TMS over time domains of 3 minutes or more is inhibitory (12 ). To test whether this inhibitory effect occurs at time
domains of several seconds, we performed TMS within an fMRI scanner and measured blood flow with the interleaved TMS/fMRI BOLD technique. Within a 1.5-T MRI scanner, five adults
were stimulated by applying a figure 8 TMS coil over the left motor cortex at the optimal spot for producing movement in the contralateral (right) thumb (abductor pollicis brevis). In
21-second epochs, subjects alternated between rest and a sequential finger opposition task in their left (nondominant) hand. In alternating-movement trials, TMS was applied either at
120% motor threshold or 10% of stimulator output (below the threshold for movement) (Fig. 30.7 ). Coronal echo-planar BOLD images were acquired continuously throughout,
interleaved so that TMS occurred 100 milliseconds after every fourth image acquisition.

FIGURE 30.7. Several transcranial magnetic stimulation (TMS) electrophysiology studies have demonstrated that low-frequency TMS over one motor cortex can inhibit the
opposite motor region. In this study, we applied TMS over the left motor cortex and had subjects perform a complex task with their nondominant (left) hand. TMS was applied
on alternate epochs. We hypothesized that TMS would inhibit the blood oxygenation-dependent (BOLD) response in the right motor cortex. We did not see this. Interestingly,
TMS produced an increase in BOLD response under the coil in an area of cortex that was already active. This simple study in which TMS was used to test connectivity highlights
many of the issues raised in this chapter concerning how and where to apply TMS, and whether baseline activity in the underlying brain matters in terms of response.

With this technique and at these short time intervals, TMS did not inhibit the local or remote BOLD response during movement. In fact, TMS of the left hemisphere caused a local 1.5%
increase in blood flow in addition to the 2.5% activation caused by the complex movement. We therefore concluded that the application of TMS over motor cortex for 21 seconds during
a motor task-enhanced motor cortex activation does not inhibit the BOLD response. Actually imaging the remote inhibitory or modulatory effect of TMS at a different site remains for
the future.

Averaged Single Trials

There is no doubt that a single TMS pulse applied to motor cortex is capable of causing a neuronal response because its consequences can be clearly seen in the form of an overt
movement of the contralateral extremity. However, to date, the only functional neuroimaging technique in which the response to single-pulse TMS has been observed is EEG. Our
laboratory recently sought to determine if interleaved TMS and fMRI could be used with an averaged-single-trials protocol to detect BOLD response to neuronal activation induced by a
single TMS pulse, and to measure its time course.

The technique is important because it allows a comparison of different TMS events by means of their associated BOLD responses. For example, with a single-event technique. it might
be possible to compare different TMS intensities, or coil orientations, or single versus paired stimulation (through one coil or possibly two different coils, one conditioning coil and one
test coil). Such studies could provide a bridge between electrophysiology (variation of motor evoked potential amplitudes) and fMRI (variation of BOLD response). Moreover, combining
TMS with precise timing relative to a behavior with the averaged-single-trials technique would likely make it possible to image the activity of brain circuits and their connections.

In an initial study in this area, five healthy volunteers were studied with interleaved TMS/fMRI and an averaged-single-trials protocol (57 ). The BOLD fMRI response to single TMS pulses
over the motor cortex was detectable in both ipsilateral motor cortex under the TMS coil and contralateral motor cortex, and also bilaterally in auditory cortex. The associated BOLD
signal increase showed the typical fMRI hemodynamic response time course. The response of the brain to a single TMS pulse over motor cortex at 120% of the level required to induce
thumb movement (1.0% to 1.5% signal increase) was comparable in both level and duration with the auditory cortex response to the sound accompanying the TMS pulse (1.5% to 2.0%
signal increase) (Fig. 30.5 ).

Thus, ultimately, TMS combined with fMRI may allow for more exact positioning of the TMS coil, with information obtained about the magnetic field produced and also about
alterations in physiology and biochemistry. Refinements of the averaged-single-trials technique and precise timing of TMS offer the potential of increasing the temporal resolution of
fMRI and promoting its evolution into a tool for assessing connectivity. Much background work is needed before this combined technique can achieve its potential.

Quantitative EEG
Ilmoniemi et al. (62 ) have combined high-resolution quantitative EEG (qEEG) with TMS and reported regional changes in spectral content that shifted over very brief episodes of time
and corresponded with known regional connections with primary motor cortex. High-resolution EEG clearly has the best temporal window of all the techniques (in the millisecond
range), although the spatial resolution is poor. Unfortunately, this group in Finland is the only one to date to be able to circumvent the technical problem of recording EEG
immediately after TMS and so avoid the artifact produced by the TMS pulse. This area has not advanced as rapidly as expected in the last 3 years, perhaps because of the complexity of
the technique.
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Transcranial Magnetic Stimulation and Multimodal Integration

Lastly, several groups have now used TMS in a complementary way to address systems neuroscience questions. The TMS aspect of
the study serves to confirm or validate a result from a purely functional imaging study. Two recent studies illustrate many of the
important aspects of this type of work.

Kosslyn and colleagues (63 ) used TMS to investigate secondary visual cortex (area 17) and visual imagery. As a first part of this study,
a traditional 15O PET study was performed in subjects while they visually imagined a stimulus. As predicted from previous imaging
studies in humans and animal studies, area 17 activated during this task. In a different cohort, with the use of probabilistic
positioning, TMS over putative area 17 interrupted this visual imagery task. This study suffered in several respects, most notably in
not knowing whether the TMS actually was applied over area 17. It nevertheless demonstrated the potential of using TMS as a
convergent method of testing brain–behavior theories.

In a more elegant and rigorous application of this approach, Desmurget and colleagues (64 ) used TMS and imaging to test the role of
the posterior parietal cortex in correcting the ongoing trajectory of movements (64 ). They scanned healthy subjects while they
pointed to visual targets that either remained stationary or moved during saccadic eye movements. Then, using a functional image-
based positioning system, they applied TMS over the left posterior parietal cortex during stimulus target presentation. The TMS
disrupted the normal path corrections that occur in moving objects. Thus, in this study, TMS indicated the necessary and critical role
of an area in the performance of a behavior and extended the traditional observational imaging approach.

CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH

Part of "30 - Measuring Brain Connectivity with Functional Imaging and Transcranial Magnetic Stimulation "

Transcranial magnetic stimulation combined with functional imaging offers the promise of a better understanding of brain circuits
and the causal relationship between behaviors and activity in distributed brain regions. Several studies with a variety of imaging
modalities have begun to use this approach. These studies have largely demonstrated that before the combination of TMS and
imaging can be used fully, much more work is needed to improve methods. Very basic questions remain largely unexplored. These
include how best to position the coil (functional behavioral versus probabilistic), how to adjust the intensity for nonmotor areas of
cortex, and whether to account for differences in depth into the brain (e.g., atrophy). Additionally, a true understanding of TMS
effects on the brain are still lacking, and this incomplete knowledge contributes to the lack of understanding of how best to use TMS
to address systems neuroscience questions. For example, do different frequencies of stimulation produce varying effects on local
metabolism, and if so, how? More complete knowledge of the local pharmacologic effects of TMS as a function of the many
parameters of use would greatly advance our ability to apply TMS/imaging in neuroscience research. It is obvious that a great deal
of systematic work is needed to understand this interesting tool.

However, although a better knowledge of TMS brain effects would expand and improve its use as a neuroscience tool, the ability to
combine noninvasive stimulation of the brain with real-time functional imaging is an important new technique that will no doubt add
to our ability to understand brain connectivity.

APPENDIX I
Determining The Appropriate Model For Calculating The Induced
Electric Current In The Brain
Although, typically, the spherical model has been used, this assumes that the brain is a sphere with uniform conductivity inside
spherical shells with different conductivities, corresponding to the skull and scalp. One group has gone so far as to use tissue
segmentations based on MR images and estimates of gray and white matter and cerebrospinal fluid from the literature and the
theoretic field of the TMS coil to perform finite element computations of the electric currents induced in individual brains (69 ).
Unfortunately, these computations of the electric currents were performed with special field computation software and a
supercomputer (69 ). Although the assumptions of most computational models that the brain is spherical and the cortex is an
isotropic, homogeneous volume conductor are simplistic, some important observations have been made. The charge accumulation on
the tissue surface tends to cancel the perpendicular component of the induced electric field, shielding the brain. This forces the
resultant electric field to lie predominantly parallel to the tissue surface and fall rapidly with depth (65 ,66 ,67 and 68 ). These
observations are also expected to be valid for models that more faithfully represent the actual shape and composition of the brain
by treating it as a summation of finite elements. To take into account the inhomogeneous conductivity of the brain, Cerri et al. (69 )
used MR images to segment the brain into white matter, gray matter, and cerebrospinal fluid, and a conductivity versus gray level
interpolation function derived from tissue conductivity data in the literature to obtain a three-dimensional conductivity map. They
then divided the brain into discrete resistive cells (quasistatic approximation) and used a supercomputer to determine the current
distribution that would be induced in the three-dimensional resistive network by
 P.408

an external magnetic field pulse However, such methods are not generally available and are still an approximation. A means of imaging the induced electric field is what is really
needed.

APPENDIX II
Principal Component Analysis And Singular Value Decomposition

Principal Component Analysis

Principal component analysis is a mathematical device that uses the intrinsic spatial and temporal properties of a set of fMRI data to reduce its dimensionality. PCA does not refer to a
specific statistical model, entails few assumptions, and makes no comment on the significance of the resulting spatial modes. By orthogonalizing the covariance matrix, PCA extracts
its important features in terms of principal components, or eigenvectors. These vectors are the linear combinations that account for independent or orthogonal amounts of variance in
the observed data. In terms of functional connectivity, a principal component represents a spatially distributed brain system, comprising a subset of brain region, within which many
temporal intercorrelations exist. Because any one principal component is orthogonal to the remaining principal components, these systems are functionally unconnected from each
other, even though any single area may be implicated in more than one system. To perform PCA, a mathematical technique called singular value decomposition is usually used (70 ).

Singular Value Decomposition

Given a set of M linear algebraic equations relating a set of N unknowns, xj, j = 1,2,…

or, in matrix form,

where the as and bs are known. If N = M, there are as many equations as unknowns, and there is a good chance of finding a unique solution set of xjs.

If M < N or M = N but the equations are not all linearly independent, then there are effectively fewer equations than unknowns. In this case, either there is no solution, or else there is
more than one solution vector x. In the latter event, the solution space consists of a particular solution xp added to any linear combination of (typically) N-M vectors (which are said to
be in the nullspace of the matrix A). The task of finding the solution space of A is called singular value decomposition of a matrix A.

Singular value decomposition explicitly constructs orthonormal bases for the nullspace (N-M dimensions) and the range (M dimensions) of the matrix, finding the least-squares best
compromise solution.

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31
In Vivo Molecular Imaging: Ligand Development and Research
Applications

Masahiro Fumita

Robert B. Innis

Masahiro Fumita: Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut.

Robert B. Innis: Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, Connecticut.

In positron emission tomography (PET) and single-photon emission computed tomography (SPECT), tracers labeled with radioactive
isotopes are used to measure protein molecules (e.g., receptors, transporters, and enzymes). A major advantage of these two
radiotracer techniques is extraordinarily high sensitivity (˜ 10-9 to 10-12 M), many orders of magnitude greater than the sensitivities
available with magnetic resonance imaging (MRI) (˜ 10-4 M) or magnetic resonance spectroscopy (MRS) (˜ 10-3 to 10-5 M). For example,
MRI detection of gadolinium occurs at concentrations of approximately 10-4 M (1 ), and MRS measures brain levels of γ-aminobutyric
acid (GABA) and glutamine at concentrations of approximately 10-3 M (2 ,3 ). In contrast, PET studies with [11C]NNC 756 in which a
conventional bismuth germanate-based scintillator is used can measure extrastriatal dopamine D1 receptors present at a
concentration of approximately 10-9 M (4 ). Because many molecules of relevance to neuropsychiatric disorders are present at
concentrations of less than 10-8 M, radiotracer imaging is the only currently available in vivo method capable of quantifying these
molecular targets.

PET and SPECT quantify the distribution of radioactivities in the brain, the direct in vivo correlates of in vitro autoradiographic film
techniques such as receptor autoradiography, Western blots, and Northern blots. Thus, the future possibilities of radiotracer imaging
are broad and exciting—and include targets of receptors, signal transduction, and gene expression. From this broader perspective,
PET and SPECT methodologies are described as “in vivo molecular imaging.”

Although in vivo molecular imaging is a promising technique, several barriers— physical, monetary, and chemical—to its successful
application in neuropsychiatric disorders must be addressed. Physical barriers include limited anatomic resolution and the need for
even higher sensitivity. However, recent developments with improved detector crystals (e.g., lutetium oxyorthosilicate) and three-
dimensional image acquisition have markedly enhanced both sensitivity and resolution. (5 ). Commercially available PET devices
provide resolution of 2 to 2.5 mm (6 ,7 ). Furthermore, the relatively high cost of imaging with SPECT, and especially PET, can be
partially subsidized by clinical use of the devices. Recent approval of U.S. government (i.e., Medicare) reimbursement of selected
PET studies for patients with tumors, epilepsy, and cardiac disease has significantly enhanced the sales of PET cameras and their
availability for partial use in research studies. Thus, the major barriers for the expanded use of PET are not physical or monetary,
but rather chemical in nature. Simply stated, the major barrier to radiotracer imaging of molecular targets may well be the
difficulties associated with developing the radiotracers themselves. Labeling the appropriate precursor typically is not the major
impediment. Almost all candidate ligands contain carbon and hydrogen, and the positron-emitting nuclides 11C and 18F can usually be
incorporated as an isotopic variant (11C for 12C) or an atomic substitute (18F for 1H). As described in the next section, the most
common obstacle to the development of in vivo tracers is the relatively small window of appropriate combinations of lipophilicity,
molecular weight, and affinity. For a molecule to pass the blood–brain barrier, relatively small molecular weights, less than 400 to
600 daltons (d), and moderate lipophilicity are required. However, in brain, relatively low lipophilicity and high affinity are required
to achieve high ratios of specific to nonspecific binding. In contrast, the requirements for in vitro ligands are much less stringent
because the blood–brain barrier can be removed by homogenization or tissue sectioning and most nonspecific binding washed away.
The special requirements of in vivo probes (low molecular weight, high affinity, and just the right amount of lipophilicity) are
discussed in the next section.

 REQUIRED PROPERTIES OF AN IN VIVO TRACER

 IN VIVO QUANTIFICATION OF TRACER UPTAKE
 ESTIMATION OF ENDOGENOUS NEUROTRANSMITTER LEVELS
 INTERACTION AMONG NEUROTRANSMISSION SYSTEMS
 USE OF RADIOTRACER IMAGING IN THERAPEUTIC DRUG DEVELOPMENT
 IMAGING POST-RECEPTOR SIGNAL TRANSDUCTION
 IMAGING REGULATION OF GENE EXPRESSION
 CONCLUSIONS

REQUIRED PROPERTIES OF AN IN VIVO TRACER

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "
 P.412

In addition to a high degree of affinity and selectivity, several other properties are required for useful in vivo tracers.

Combination of Small Molecular Weight, Appropriate Lipophilicity, and High

Affinity
Tissue uptake of a drug (whether radioactive or not) is often analyzed within the theoretic framework of a compartment, which is
defined as a space in which the concentration of a drug is uniform. Within brain tissue, the time-dependent concentration of drug is
described for at least three compartments—free [C2f(t)], nonspecifically bound [C2ns(t)], and specifically bound [C3(t)] radiotracer (Fig.
31.1 ). Free and nonspecifically bound ligand, C2f(t) and C2ns(t), cannot be washed away, the way they are in in vitro studies.
Therefore, a high ratio of specific to nondisplaceable uptake in brain (C3/C2) is required to obtain reliable data; to reduce the
number of unknown variables, C2f(t) and C2ns(t) are often combined in a single compartment and defined as a compartment, C2(t))
(Fig. 31.2 ).

FIGURE 31.1. Compartmental description of radioactively labeled

tracer

uptake in brain.

FIGURE 31.2. Three-compartment model (two tissues). To reduce

the number of variables, C2f(t) and C2ns(t) in Fig. 31.1 are combined
in a single compartment, C2(t).

Three major factors (i.e., lipophilicity, molecular weight, and affinity) determine the in vivo characteristics of a tracer. It is easy to
understand that small molecular weight and a high degree of lipophilicity are required to pass the blood–brain barrier because it is
composed of a lipid bilayer. However, lipophilicity has opposing effects on the brain uptake of a tracer. Increasing lipophilicity
enhances the permeability of the compound, but it also tends to increase plasma protein binding, thereby decreasing the
concentration of free drug available to cross the blood–brain barrier. From rat experiments in which 27 tracers of various chemical
classes were used, Levin (8 ) obtained the following simple equation to derive a capillary permeability coefficient, Pc, from an
octanol/water partition coefficient, P, and the molecular weight, Mr.

This equation was obtained for the tracers with molecular weights of less than 400 and relatively low lipophilicity (average log P, -
0.34; standard deviation, 2.3). Because a higher lipophilicity is required for brain imaging tracers to be taken up adequately in brain,
the equation only partially characterizes capillary permeability. High lipophilicity (higher value of log P) increases the binding to the
plasma components (protein and cell membranes) (9 ) and reduces the capillary permeability coefficient expressed relative to the
total plasma concentration of drug (10 ,11 ). Therefore, both low lipophilicity and high lipophilicity decrease the penetration of
imaging agents across the blood–brain barrier, so that a parabolic curve is created (Fig. 31.3 ).

FIGURE 31.3. Relationship between apparent lipophilicity (log KW)

at pH 7.5 and peak striatal uptake (percentage of administered
dose per gram of tissue in rat brain). Peak uptake occurred
between log KW of 2.4 and 2.8. Each point is the mean of four
animals ± standard deviation. (From Kessler RM, Ansari MS, de
Paulis T, et al. High-affinity dopamine D2 receptor radioligands. 1.
Regional rat brain distribution of iodinated benzamides. J Nucl
Med 1991;32:1593–1600, with permission.)

Although low lipophilicity decreases nonspecific binding in brain, it also decreases blood–brain barrier permeability (11 ). For any
particular chemical series, optimal parameters
 P.413

of affinity and lipophilicity generate a tracer with the best “signal-to-noise” ratio to measure the target molecule, as shown in Fig.
31.3 for a series of benzamide ligands for the dopamine D2 receptor. It should be noted that iodination for SPECT tracers usually
increases lipophilicity in addition to molecular weight. Therefore, depending on the position in the optimization curve (Fig. 31.3 ),
either an iodinated or a non-iodinated compound may show the most desirable in vivo properties.

Rapid Labeling of Precursor

The radionuclide must be incorporated quickly into appropriate precursor molecules because of the relatively short half-lives (t1/2) of
the isotopes (e.g., 11C, 20.4 minutes; 18F, 110 minutes; 123I, 13.2 hours). Therefore, precursors must be available that allow quick
labeling in one (but usually no more than two) synthetic steps just before the imaging study is performed.

Appropriate Clearance from Specific Binding Compartment

Following the bolus injection of radioactive tracer, the time–activity curve of an organ (e.g., brain) and its subcomponents (e.g.,
striatum) is characterized by uptake (rising) and washout (declining) phases. If the uptake phase is slow relative to the t1/2 of the
radionuclide, reasonably accurate data may be acquired only for the rising portion of the time–activity curve. Although parameters
related to receptor density and affinity can be derived for selected targets with only the uptake portion, most quantitative methods
calculate such parameters with both uptake and washout phases of the tissue time–activity curve. Thus, the tissue clearance of the
tracer must typically be matched with the t1/2 of the radionuclide. For example, 123I can be used to quantify tracers with much slower
tissue kinetics than 11C. The rate of tissue clearance is in part determined by the affinity of the tracer, with ligands of higher affinity
tending to “stick” longer to the target molecules. So, as with lipophilicity, the affinity of the candidate tracer should be high
enough that significant tissue uptake occurs, but it should not be so high that washout is delayed beyond the usable measurement
time of the radionuclide.

In summary, a low molecular weight is almost always mandatory, at least for tracers that cross the blood–brain barrier via passive
diffusion. Lipophilicity should be high enough to allow adequate permeability of blood–brain barrier, but not so high as to cause
unacceptable binding to plasma proteins or high levels of nonspecific binding in brain. Finally, the affinity of the tracer must
balance the opposing goals of tight binding and fast washout from the brain. That is, a high affinity ligand is needed to provide high
levels of tight binding of the ligand to the preceptor. However, if the binding is of such high affinity that the ligand shows negligible
washout from the brain during the course of a typical study, then the washout rate cannot be determined and critical kinetic data
are unavailable to calculate receptor levels in the brain. Such parameters are not required for therapeutic agents (in which fast
uptake may not be even helpful) or for in vitro tracers (in which most nonspecific binding can be washed away). For example, the
nondisplaceable uptake of [18F]haloperidol (12 ) and [11C]cocaine (13 ) is unacceptably high for optimal PET imaging of their
molecular targets, dopamine D2 receptor and dopamine transporter, respectively, although they can provide valuable data about
the disposition of the psychoactive drugs themselves. It should also be noted that desirable properties of radioactive tracers are
usually different from those of therapeutic agents. For, example, slow clearance of haloperidol from brain (12 ,14 ) may maintain
effective receptor occupancy for a long period of time. If the distribution in the nonspecific binding compartment [C2ns(t) in Fig.
31.1 ] is large, the compartment may act as a reservoir of the drug and provide stable levels in brain. The stringent requirements for
an optimal radioactive tracer easily explain why only a tiny percentage of in vitro tracers and therapeutic agents are useful as in
vivo imaging ligands.

Negligible Influence of Radioactively Labeled Lipophilic Metabolites

If the parent tracer generates lipophilic radioactive metabolites, they may enter the brain in significant concentration and confound
the imaging study. If they do not bind to the molecular target (inactive metabolites), they will increase nonspecific binding [C2ns(t) in
Fig. 31.1 ] and thereby decrease the signal-to-noise measurement of the target. On the other hand, if the radioactive metabolites
are active (i.e., bind to the target), quantification is highly confounded because the measured signal represents undetermined
proportions of parent tracer and metabolite, each of which may have a different affinity for the target. The problem of lipophilic
radioactive metabolites may sometimes be avoided by appropriate selection of the labeling position. For example, a tracer for the
serotonin 5-HT1A receptor WAY 100635 can be labeled with 11C at either an external (O-methyl) or internal (carbonyl) position. [O-
methyl-11C]WAY 100635 is rapidly metabolized in humans (15 ). The metabolic cleavage of WAY 100635 generates two moieties; the
11
C-containing methyl component is lipophilic and enters the brain, but the 11C-containing carbonyl component is polar and does not
enter the brain. The internal carbonyl labeling is more difficult than the external O-methyl labeling, but this clever radiochemistry
has markedly improved the signal-to-noise measurement of 5-HT1A receptors in brain (16 ).

Many tracers currently used for imaging studies produce at least somewhat lipophilic metabolites. However, the quantities produced
or their kinetics in passing blood–brain barrier are such that they do not commonly confound the measurements. For example, if the
uptake and washout of the parent tracer are fast relative to the production of radioactive
 P.414
metabolites, then their component of the total measured activity may be negligible during the imaging study.

IN VIVO QUANTIFICATION OF TRACER UPTAKE

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

In vivo quantification of molecular targets with radiotracer imaging is much more complicated than in vitro measurements for several reasons: (a) For in vivo experiments, tracers are
intravenously administered and not directly applied to the target tissue. Therefore, the delivery of a tracer to the target tissue is influenced by its peripheral clearance (i.e.,
metabolism and excretion). (b) Total tissue activity is measured, and the separate specifically bound, nonspecifically bound, and free components (Fig. 31.1 ) are usually estimated
with relatively complicated mathematical analyses. (c) The spatial resolution of cameras is limited, and the activity in a region of interest is influenced by tracer uptake in adjacent
areas. The details of in vivo quantification are beyond the scope of this chapter, and interested readers should refer to other sources (e.g., refs. 17 ,18 ). The following section
provides a simplified overview of the typical parameters and methods of quantification used in radiotracer imaging.

Binding Potential
In addition to having appropriate chemical and physical characteristics, a useful ligand must provide imaging results that are “amenable to quantification” because analogue/visual
images are likely to be of limited utility in neuropsychiatric research. The most commonly measured receptor parameter is the binding potential (BP), which equals the product of
receptor density (Bmax) and affinity (1/Kd, where Kd is the dissociation binding constant). Thus, increased uptake could reflect either an increased number of receptors or the same
number of receptors, each of which has a higher affinity for the ligand. To measure both Bmax and Kd separately, at least two experiments or two injections are necessary, in which
formulations of the tracer with both high and low specific activity are used (19 ,20 ). Because the injection of a tracer with low specific activity (i.e., high mass dose) causes significant
occupancy of the molecular target, the potential pharmacologic effects of the tracer must be both safe and acceptable within the experimental paradigm. If these studies are not
performed, Bmax and Kd cannot be measured separately, and only their ratio (BP = Bmax/Kd) is used as the outcome measure (21 ).

Methods to Measure Binding Potential

In vivo quantification has followed the well-established Law of Mass Action applied to ligand–receptor interactions under equilibrium conditions:

where B is the concentration of radiotracer bound to the receptor and F is the concentration of free radiotracer (i.e., not bound to proteins) in the vicinity of the receptor. Because
radiotracer imaging typically involves the injection of a miniscule mass dose of ligand, the concentration of free radiotracer is quite low. That is, F<<Kd, with the result that

Thus, BP can be simply estimated as the equilibrium ratio of bound (B) to free (F). With this fairly standard three-compartment model (i.e., two tissue compartments and one blood
compartment; Fig. 31.2 ), B is denoted as C3 and F as f1*C1. Thus, B/F = C3/(f1*C1). This equation makes the reasonable assumption for drugs that pass blood–brain barrier by passive
diffusion that the concentration of free tracer in plasma (f1*C1) equals that in brain under equilibrium conditions. Note that Eqs. 1 and 2 refer to equilibrium binding conditions.
Following the bolus injection of tracer, the ratio of receptor-bound tracer (B) and free tracer (F) changes dramatically and is not under equilibrium conditions. Figure 31.4 provides a
schematic representation of radiotracer concentrations in brain and plasma following a bolus injection with Fig. 4A showing early time points and Fig. 4B showing the entire data. With
use of the complete time–activity curves from brain (measured with a PET or a SPECT camera) and arterial plasma (directly sampled and measured in a gamma counter), the goal of
compartmental modeling is to estimate the ratio of B and F under equilibrium conditions. In other words, if the free level could be maintained at a constant level, how many times
higher than the free level (F) would the concentration of receptor-bound tracer (B) finally and stably be?

FIGURE 31.4. A,B: Simulated time–activity curves of parent tracer in plasma and compartments in brain, and (C) ratio of specific to nondisplaceable uptake. The plasma parent
curve was created by the following formula:

where A1, A2, and A3 were 60, 5, and 2 kBq/mL, respectively, and T1, T2, and T3 were 1, 20, and 100 minutes, respectively. A linear increase of the input curve was assumed
between 0 and 1 minute. The curves of brain compartments were created with the rate constants K1 = 0.2, k2 = 0.03, k3 = 0.04, and k4 = 0.02 min-1. The ratio of specific to
nondisplaceable uptake gradually increases and reaches the equilibrium value 2 (= k3/k4) (C). This time point is almost equal to the time when specific uptake reaches to the
maximum value (time of peak equilibrium) (B). After this time point, the ratio of specific to nondisplaceable uptake shows a further increase, which indicates that an
equilibrium value of 2 can be obtained at only one time point.

Several methods to estimate receptor parameters have been applied in radiotracer imaging and are briefly summarized below.

1. Compartmental modeling. Often viewed as the “gold standard,” compartmental modeling typically requires concurrent and lengthy measurements of parent compound (separated
from radioactively labeled metabolites) in plasma and of the brain time–activity curve. Kinetic parameters (K1, k2, k3, k4) are estimated from this so-called arterial input function (i.e.,
C1) and the “impulse response function” of the brain (i.e., sum of C2 and C3). The goal of compartmental modeling is to determine the values of the rate constants between these
compartments (Fig. 31.2 ), which when applied to the measured values of C1 generate a brain time–activity curve similar to that actually measured with the PET or SPECT camera. The
underlying concept is that the equilibrium ratio of B and F is equal to a ratio of kinetic rate constants.

The major disadvantage of kinetic analysis of compartmental

 P.415

modeling may often be logistic in nature because arterial sampling may be poorly tolerated, measurement of parent radiotracer in
plasma may be technically difficult, and prolonged periods of data acquisition may be needed for both plasma and brain activities.
The subsequent methods were developed in large part as “simpler” techniques to obtain receptor measurements that closely
correlate with or directly equal those obtained in a complete compartmental analysis.

2. Peak equilibrium method. Based on good theoretic grounds, the “peak equilibrium method” [commonly attributed to Farde et al.
(19 ) for radiotracer imaging] selects a unique equilibrium time as that when specific binding achieves its maximal level. Specific
binding is operationally defined as activity in a target region (e.g., striatum) minus that in a background tissue region (e.g.,
cerebellum). From a practical perspective, a subject is continuously imaged for an hour or two following a bolus injection of tracer.
Activities in target and background regions are plotted, and specific binding is calculated at each time point as the difference of the
two curves (Fig. 31.4B ). At the time of peak specific activity, a measure proportional to binding potential is calculated as
specific/nondisplaceable = (target - background)/background, as in (striatum-cerebellum)/cerebellum for a D2 tracer.

One major advantage of the peak equilibrium method is that plasma measurements are not required. Its major limitations include
the following:

(a) Background activity in brain is proportional but not equal to the free level of tracer (F). Thus, the outcome measure is not equal
to BP; rather this ratio of specific to nondisplaceable uptake is proportional to BP.

(b) Nonspecific binding in the background region may not equal that in the target region, a situation that can be caused, for
example, by different proportions of white and gray matter in the two regions. The assumption of equivalent nonspecific binding has
been evaluated (usually in animals) with displacement of radiotracer binding by high doses of a nonradioactive drug that also binds
to the site. The assumption would be supported by equivalent levels of residual activities in target and background regions with
complete receptor blockade.

(c) The time curves of free levels in target and background regions would be predicted not to overlap exactly.
 P.416

For example, during the uptake portion of the curve, the free level would be lower in the target region because both receptors and
nonspecific sites bind the tracer. Thus, at the time of peak specific uptake, the free level in the target region would not be the
same as that in the background region. Depending on the kinetics of specific and nonspecific binding, the resulting discrepancy
might be significant.

3. Constant infusion methods. Stable levels of drugs, including radiotracers, can be achieved with constant (sometimes called
continuous) intravenous infusion of the drug (Fig. 31.5A ). At some point, typically referred to as steady state, the amount of drug
entering the blood will equal that leaving the vascular compartment. The levels of both total and free drug in plasma will
subsequently be stable. At a somewhat later time point, the amount of drug binding to a receptor in an organ will equal that coming
off the receptor; the level of receptor-bound drug will subsequently be stable. In an analogy to in vitro receptor binding studies,
this stable condition is a state of equilibrium receptor binding.

FIGURE 31.5. Brain time–activity curves in a bolus plus constant infusion/equilibrium (A) and a bolus/kinetic study (B) of
[123I]epidepride. A: [123I]Epidepride was given as a bolus (145 MBq) followed by constant infusion with bolus/infusion ratio of 6.0
hours (the amount of the tracer given for 6 hours by constant infusion was equal to that of the bolus) in a 33-year-old man.
Note that equilibrium was achieved only in low-density regions (thalamus/hypothalamus and temporal cortex), not in a high-
density region (striatum), with this bolus/infusion ratio. To achieve equilibrium in all regions, including striatum, a higher
bolus/infusion ratio and longer infusion are required. B: [123I]Epidepride (371 MBq) was give as a bolus to a 24-year-old man.
(Part B reprinted from Fujita M, et al. Kinetic and equilibrium analyses of [123I]epidepride binding. Synapse 1999;34:290–304,
with permission.)

The constant infusion (or so-called equilibrium) method is computationally much simpler than compartmental modeling and does not
require extensive brain imaging or arterial plasma measurements. The concentration of receptor-bound tracer (B) can be estimated
as target minus background. The level of free tracer in plasma (F) can be measured in either venous or arterial plasma because the
body as a whole is in a condition of steady state. From a practical perspective, the subject is connected to an intravenous pump for
several hours, and then a single image is acquired (e.g., 30-minute duration) and a single venous blood sample is obtained. The
major disadvantage of this technique is that many hours of infusion may be required to achieve steady-state conditions in both
plasma and brain. In addition, data are acquired during a relatively short interval (e.g., 30 minutes) of a long infusion period (e.g., 7
hours). Thus, activity measurements before and after the relatively brief acquisition are not used, and the resulting radiation
exposure to the subject can be viewed as “wasteful.” In contrast, “all” activity is measured and used in the analysis of a
bolus/kinetic study (Fig. 31.5B ). From a practical perspective, the total amount of injected activity is often fairly equivalent for a
bolus/kinetic and a constant infusion/equilibrium study. However, the total activity in brain after many hours of decay may be quite
low and cause statistical counting errors.

In summary, three basic methods can be used to estimate receptor binding potential: (a) compartmental analysis of a bolus/kinetic
study, (b) peak equilibrium, and (c) equilibrium imaging following constant infusion of the tracer (with or without an initial bolus of
tracer). For all three methods, the target parameter is typically Bmax/Kd, which equals the equilibrium value of B/F under tracer
occupancy conditions (i.e., < 10% of the receptor occupied by tracer). The “true” binding potential (Bmax/Kd) can be calculated if the
free concentration of tracer in plasma is measured with the assumption
 P.417

that free concentration in plasma equals that in brain. In this case, the measurement of BP is the ratio of receptor-bound activity in
brain to free plasma activity (F). Because a blood sample is not used in the peak equilibrium method, the true BP cannot be
measured. An alternate outcome measure for each of these three methods uses the nondisplaceable activity in a background region
of brain as a value proportional to free tracer concentration. This so-called poor man's binding potential is a ratio of activities in
different regions of the brain (specific to nondisplaceable), and therefore plasma measurements are not required.

ESTIMATION OF ENDOGENOUS NEUROTRANSMITTER LEVELS

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

Molecular imaging probes can be used not only to measure a specific target but also, under appropriate conditions, to estimate
concentrations of endogenous compounds that compete with the tracer for binding to the target. For example, a D2-receptor probe
can be used not only to measure D2 receptors but also the extent of competition of this binding caused by endogenous dopamine. In
fact, the most extensively studied indirect measurements have been the interaction of dopamine with D2-receptor ligands. These
studies are briefly reviewed, and the special characteristic of a good ligand for such indirect measurements are discussed.

Tonic and Phasic Release of Dopamine

Dopamine transmission in striatum is thought to occur in two different modes, tonic and phasic (22 ,23 ). Tonic dopamine release
represents the steady-state level of dopamine in the extracellular space, which is estimated to be in the nanomolar range. On the
other hand, in phasic release, high extracellular concentrations of dopamine (millimolar range) are released within or near a
synapse during an action potential. Close relationships have been proposed between abnormalities in phasic and tonic dopamine
release and the symptoms of schizophrenia. Namely, excessive phasic release causes psychosis, and decreased tonic release causes
cognitive deficits and negative symptoms (24 ).

Phasic release has typically been initiated by intravenous administration of a stimulant such as amphetamine or methylphenidate.
These agents elevate synaptic dopamine concentrations either by releasing dopamine in a reverse manner via a dopamine
transporter (amphetamine) or by blocking dopamine transporter-mediated reuptake of dopamine (methylphenidate). In an imaging
study, the elevation of synaptic dopamine levels is estimated by the decrease in D2 radiotracer binding following stimulant
administration in comparison with control conditions. Just as careful quantification is required for direct radiotracer binding to a
molecular target, a similar if not even more rigorous approach is required for these indirect methods to ensure that measurements
of both the tracer and the competing displacer represent “equilibrium” values and not just transient pharmacokinetic “artifacts.”
To support the validity of these measurements, microdialysis studies have been performed in conjunction with D2-receptor imaging
and a stimulant challenge (25 ,26 ). Although D2-ligand displacement correlated with the increase in extracellular dopamine
measured with microdialysis, the relative increase in extracellular dopamine (1,000% to 4,000%) was much greater than the
percentage of displacement of the ligand binding (5% to 15%). Thus, D2-receptor displacement is a “low-gain” monitor (i.e.,
underestimation) of the increase in extracellular dopamine. The reasons that the changes in binding are so much lower (although
still, it is hoped, linear) relative to the increase in extracellular dopamine are unclear. This “low-gain” monitoring may reflect the
fact that the imaging measurements cannot temporally track and therefore lag behind the chemical changes they are designed to
measure. In other words, the displacement of radiotracer from the brain region occurs over a much slower time course than the
relatively rapid changes in extracellular dopamine. Nevertheless, these stimulant-induced displacement studies appear to provide
some reflection of changes in synaptic dopamine levels because they are relatively well correlated and because depletion of tissues
levels of dopamine can block the effects of amphetamine (27 ).

The tonic release of dopamine has been estimated by the increase in D2-receptor binding induced by the depletion of endogenous
dopamine. The removal of dopamine “unmasks” receptors, which then become available for radiotracer binding. The percentage of
unmasking reflects the percentage of D2 receptors occupied by dopamine under basal or tonic conditions. Dopamine depletion has
been induced in both animals and humans, with a resulting increase in D2 radiotracer binding (28 ,29 ). One limitation of these
studies, especially in humans, is the difficulty of knowing whether depletion is essentially complete, so that the full extent of
dopamine occupancy of the receptor has been measured. For example, if differences in unmasking are found in two subjects, does
that reflect different levels of endogenous dopamine—or just different levels of dopamine depletion? A second limitation of this
depletion paradigm is that increased receptor binding may not reflect “unmasking” but rather an up-regulation of D2-receptor levels,
similar to that often found in denervation supersensitivity. One mechanism to minimize this potential confound is to perform the
measurements as soon after dopamine depletion as possible. However, one clear advantage of the depletion paradigm in comparison
with the stimulant-induced increase is that the depleted levels can typically be stably maintained during the scan. Thus, the relative
slowness of the imaging measurements does not present a pharmacokinetic confound, as it does in studies with stimulant-induced
release of dopamine.

Both stimulant and depletion studies have been performed in patients with schizophrenia. In general agreement
 P.418

with the hypothesis of Grace, the “phasic” increase in synaptic dopamine (assessed following amphetamine administration) has been
found to be elevated in drug-free schizophrenic patients, and the decrease in D2 radiotracer binding correlates with a transient
increase in psychotic symptoms (30 ,31 and 32 ). In addition, stimulant depletion studies have found greater unmasking of striatal
D2 receptors in patients with schizophrenia, which suggests that basal/tonic synaptic dopamine levels are higher in this disorder
(33 ).

Affinity of Radiotracer
The relationship between affinity of the radiotracer and the sensitivity of its binding to endogenous dopamine is a source of
confusion. Under in vitroequilibrium conditions and at tracer levels of radioactively labeled ligand, both the Michaelis–Menten and
Cheng–Prusoff equations predict that the percentage of receptor occupancy by a competitive inhibitor (e.g., dopamine or other
neurotransmitters) depends on the affinity of the inhibitor for the receptor and is independent of the affinity of the radioactively
labeled ligand. However, such equilibrium binding conditions are achieved for neither the tracer nor the displacer if each is injected
as a bolus. Even under these conditions, the sensitivity of radioligand binding to endogenous dopamine levels is theoretically (at
least based on the in vitro theories) independent of the affinity of the radioactively labeled ligand when both the tracer and the
displacer have achieved equilibrium binding conditions. However, if either the radiotracer (as in the bolus injection paradigm) or
endogenous dopamine (as in stimulant-induced release) changes dynamically over time, the equilibrium condition is not achieved,
and the apparent sensitivity of the radioligand to endogenous dopamine levels is determined by the kinetic properties of the
radioligand (34 ,35 ). Equilibrium conditions can be achieved for both tracer and displacer in the dopamine depletion paradigm. For
example, equilibrium can be achieved with bolus plus constant infusion of the radiotracer, and stable dopamine depletion with
AMPT (α-methyl-p-tyrosine). The high-affinity D2 radioligand [123I]epidepride provides an instructive example of the differences seen
in kinetic and equilibrium studies. The kinetics of its uptake in brain are slow and do not show displacement by transiently increased
dopamine levels induced with amphetamine (36 ). However, stable low levels of dopamine induced with AMPT show unmasking of D2
receptors (37 ).

In Vivo Confounding Factors

Although the displacement of radioligand binding by neurotransmitter can be simply described with in vitro tissue homogenates,
several factors complicate the interpretation of in vivo experimental results. (a) The affinity states of some receptors for agonists,
but not antagonists, is regulated by the receptor to guanyl nucleotide-binding proteins (38 ). Therefore, in vivo measurements are
influenced by the affinity states and agonist or antagonist properties of the radiolabeled tracer, and results obtained with agonist
and antagonist tracers may be different. (b) Agonist binding typically causes receptor internalization of G protein-coupled receptors
(39 ,40 ). Thus, the endogenous agonist dopamine presumably facilitates the intracellular trafficking of D2 receptors (41 ), and
radiotracers may differ in their affinity for the internalized versus membrane-bound receptor. (c) A significant proportion of D2
receptors are located extrasynaptically (42 ,43 ), where the concentration of dopamine is lower than in the synapse. Thus,
neurotransmitters may occupy a smaller percentage of extrasynaptic receptors than of receptors within the synapse, and the in vivo
measurement may not truly reflect synaptic neurotransmitter levels.

INTERACTION AMONG NEUROTRANSMISSION SYSTEMS

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

Abnormalities in psychiatric disorders likely represent the complex interaction of several neurotransmitter systems in the brain. PET
imaging has recently been used to examine aspects of neurotransmitter interactions. For example, Dewy and colleagues (44 ,45 and
46 ) have pioneered studies on interactions among dopamine, GABA, and acetylcholine (ACh) systems in striatum. GABA neurons in
the striatum have inhibitory effects on nigral dopamine neurons, nigral dopamine neurons have inhibitory effects on striatal ACh
neurons, and striatal ACh neurons have facilitating effects on striatal GABA neurons. By estimating dopamine levels in striatum as
described above, Dewey and collaborators showed in human or anesthetized nonhuman primates that the blockade of cholinergic
transmission by benztropine (44 ) or scopolamine (45 ) decreased [11C]raclopride binding (increase in dopamine levels) and that
stimulation of GABAergic transmission by γ-vinyl-GABA (a suicide inhibitor of GABA transaminase) and lorazepam (a benzodiazepine
agonist) increased [11C]raclopride binding (decrease in dopamine levels) (46 ). In addition, they showed that a dopamine antagonist,
N-methylspiroperidol, induced a decrease in [N-11C-methyl]benztropine binding, indicating an increase in ACh levels (44 ).

Other interactions have also been studied with PET. In two human studies, an N-methyl-D-aspartate (NMDA) antagonist, ketamine,
decreased [11C]raclopride binding in striatum (47 ,48 ). In two human studies with similar techniques, the binding of [11C]raclopride
was decreased by stimulation of 5-hydroxytryptamine (5-HT) transmission with fenfluramine (a 5-HT releaser) (49 ) or psilocybin (a
mixed 5-HT2A and 5-HT1A agonist) (50 ). However, these results are discordant with those of previous studies in baboon, in which
citalopram (a 5-HT uptake inhibitor) increased [11C]raclopride binding (51 ). Key aspects of the interaction between dopamine and 5-
HT neurotransmitter
 P.419

systems may well be mediated by glutamate. One significant limitation of these studies is that no useful glutamatergic PET probes
have been developed to examine this important mediating neurotransmitter system. Furthermore, the linkage of pharmacologic
challenges can be difficult to interpret. For example, if a disorder is associated with an abnormal dopamine outcome measured with
PET in response to a 5-HT challenge, is the abnormal response caused by altered sensitivity of the dopamine or 5-HT system?

The results of these studies of interactions among neurotransmission systems have been interpreted under a simple assumption that
the binding of [11C]raclopride and other tracers is affected by synaptic neurotransmitter levels. This simple assumption has been
questioned by elaborate studies by Tsukada et al. (52 ). They measured dopamine synthesis, dopamine transporter, and dopamine
D2 receptor with L-[β-11C]methyldopa (L-[β-11C]DOPA), [11C]β-CIT, and [11C]raclopride, respectively, in combination with microdialysis
in conscious rhesus monkeys. Scopolamine did not change extracellular dopamine levels in the striatum but increased [11C]raclopride
binding by decreasing its affinity at the dopamine D2 receptor (52 ) Furthermore, ketamine decreased [11C]raclopride binding in the
striatum without increasing extracellular dopamine levels, and it increased both dopamine synthesis and dopamine availability (53 ).

Although interpretation may be difficult, and although the pharmacokinetics of either the tracer or displacer and changes in the
synthesis and reuptake of neurotransmitters and affinity of receptor binding may complicate the experiment, the authors feel that
challenges linked with radiotracer imaging are likely to provide useful information to allow a better understanding of the
pathophysiology of neuropsychiatric disorders.

USE OF RADIOTRACER IMAGING IN THERAPEUTIC DRUG DEVELOPMENT

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

Radiotracer imaging can provide useful information about molecules that are either the direct target of or indirect markers for the
effects of therapeutic drugs. For example, if both the tracer and therapeutic drug competitively bind to the same target, then
imaging can provide direct information on the extent and kinetics of receptor occupancy in the relevant tissue. In addition, the
molecular target measured by the tracer (e.g., amyloid) can be used as a “surrogate” biological marker to assess the efficacy of the
therapeutic agent (e.g., one designed to decrease amyloid deposition).

Measurement of Receptor Occupancy

Molecular imaging can provide useful guidance for two aspects of drug administration: dose and dosing interval. The dose is most
easily chosen with a known target occupancy and accepted range. For example, typical antipsychotic agents occupy 50% to 80% of
striatal D2 receptors (54 ). Thus, a new typical antipsychotic agent should show a reasonably acceptable side effect profile when
given at doses that occupy this range of D2 receptors. With regard to dosing interval, the drug may be retained in tissue much longer
than in plasma, and, therefore dosing intervals based on plasma pharmacokinetics may be too frequent. Such a situation has clearly
been shown for antipsychotic agents, with which a high rate of receptor occupancy extends well beyond low plasma levels (55 ,56 ).

The measurement of receptor occupancy and pharmacokinetics in brain combined with the evaluation of adverse reactions in a small
number of healthy subjects may provide valuable information for “go/no go” decisions at early stages of clinical drug development.
If targeted receptor occupancy is achieved without causing adverse reactions, studies in patients are justified. If not, further studies
may not be indicated. Even in the absence of a target level for receptor occupancy, it is reasonably safe to assume that doses
associated with greater than 95% occupancy are unnecessarily high, and that those with less than 10% occupancy are unlikely to be
efficacious.

The 5-HT2A antagonist M100907 may provide the best example of the use of PET receptor occupancy to provide useful information on
dose and dosing interval. By measuring receptor occupancy with [11C]N-methylspiperone in healthy human subjects, initially an
appropriate amount of a single dose (57 ) and then an appropriate dose and dosing interval were determined (56 ). Further, a similar
level of receptor occupancy was recently confirmed with [11C]M100907 in a small number of patients with schizophrenia (58 ).

Dopamine Transporter Imaging as a Biological Marker in Parkinson Disease

Imaging of a biological marker may provide information that is useful either for diagnosis or as a monitor of disease progression. In
Parkinson disease, the two most successful imaging targets used as biological markers are measures of dopamine synthesis with
[18F]FDOPA and of dopamine terminal innervation with ligands for dopamine transporter.

The symptoms of Parkinson disease are caused by the progressive loss of dopamine neurons in the nigrostriatal pathway. Therefore,
a reasonable method of diagnosis would be to “count” the number of dopamine neurons noninvasively. Such measurements are
possible with in vivo imaging tracers: [18F]FDOPA and various SPECT/PET tracers for dopamine transporter. However, these tracers
do not detect the same biological process. Whereas [18F]FDOPA detects metabolic activities at dopamine nerve terminals, the tracers
for dopamine transporter simply measure the density of the target protein. Because of this difference, the sensitivity to detect the
decrease in dopamine neurons
 P.420

may be different. In fact, both human and animal studies have indicated that the imaging of dopamine transporter is more sensitive
to dopamine neuronal loss (59 ). Dopamine transporter can be quantified with SPECT tracers, which can be used with lower cost
than can PET studies. Therefore, imaging of the dopamine transporter in movement disorders is widely performed in many
developed countries.

It is clinically important but sometimes difficult to differentiate essential tremor and Parkinson disease. Dopamine transporter
imaging clearly distinguishes these two groups, with only a small overlap (60 ,61 ). However, dopamine transporter imaging is not
able to distinguish idiopathic Parkinson disease from other parkinsonian syndromes, such as multiple system atrophy (62 ). Further,
these techniques have shown bilateral loss of dopamine transporter in hemi-Parkinson disease (i.e., the earliest stage of the
disorder), which suggests that even preclinical disease may be detected (63 ).

Restoring dopamine levels with L-DOPA is still the core medication treatment of Parkinson disease. However, long-term treatment
with L-DOPA frequently results in fading of the therapeutic effect and the development of serious motor and psychiatric side effects.
Although palliative treatment with L-DOPA is clearly of significant clinical benefit, current drug development is oriented toward
neuroprotective treatments designed to slow the loss of dopamine neurons and the consequent progression of symptoms. As a
biological marker of dopamine terminal innervation of the striatum, dopamine transporter imaging may well be useful to monitor
whether such new therapies actually slow the loss of dopamine neurons. In fact, SPECT imaging is sensitive to and can quantify
dopamine transporter loss in longitudinal studies of patients with Parkinson patients treated with conventional therapies (64 ,65
and 66 ). Therefore, with appropriate sample sizes, this imaging technique can quantify a relevant biological marker as a surrogate
measure of the efficacy of putative neuroprotective therapies.

IMAGING POST-RECEPTOR SIGNAL TRANSDUCTION

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

The majority of the imaging studies performed to date have focused on the synapse: transporters as presynaptic targets, receptors
as postsynaptic targets, and indirect measurements of the transmitter as a type of “intrasynaptic” target. However, measurements
of membrane receptors merely “scratch the surface” of the cell and ignore the multitude of important intracellular mechanisms
required for biological effects. Therefore, two promising areas for expansion in the near future are post-receptor signal transduction
and subsequent changes in gene expression. Abnormalities in second messenger systems have been postulated to play important
pathophysiologic roles in many psychiatric illnesses, including mood disorders (67 ) and drug addiction (68 ). However, only a small
number of PET and SPECT studies have been performed on these systems. Although tracers have been developed to image three
major biochemical cascades [i.e., cyclic adenosine monophosphate (cAMP), phosphoinositide (PI), and arachidonate pathways], all
existing ligands have moderate to significant technical limitations, and better ones are clearly needed. Some of these tracers are
lipids, and their nonspecific binding is too high. In addition, because most of these tracers do not bind to a single type of protein but
are metabolized by several enzymes, the interpretation of the results is not clear.

cAMP Cascade
Imaging of this signal transduction system was initially attempted by labeling its activator, forskolin, and a related analogue, 1-
acetyl-7-deacetylforskolin, with 11C (69 ,70 ). The binding of [11C]forskolin may be correlated with the activation of adenylate cyclase
(71 ). However, brain uptake of these tracers is very low (69 ).

Recently, imaging of this cascade was tried with an inhibitor of phosphodiesterase IV (PDE-IV), [11C]rolipram (72 ). PDE-IV is the
major subtype in brain of PDE, which hydrolyzes cAMP into 5′-AMP. PDE-IV is composed of four major enzyme subtypes, PDE-IV A, B,
C, and D, and all four subtypes are found in human brain (73 ). Rolipram binds to and inhibits all four PDE-IV subtypes with high
affinity. In one report of a rat ex vivo study, [11C]rolipram was a promising tracer exhibiting high specific brain uptake (72 ). Further
evaluation of the binding selectivity and kinetics must be performed in nonhuman primates and humans subjects.

Phosphoinositide System
Imahori and colleagues have studied the PI system in vivo using 11C-labeled (74 ) and 123I-labeled (75 ) 1,2-diacylglycerol. They
showed specific incorporation of the tracer in the chemical components of the rat PI system, such as phosphatidic acid,
phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bis-phosphate (74 ). Further, the tracer uptake
was increased by an agonist at the muscarinic ACh receptor, which is known to be coupled with the PI system (74 ). However,
absolute quantification of the tracer uptake is probably difficult because of the high lipophilicity. As explained above (“Required
Properties of an In Vivo Tracer”), high lipophilicity causes a high level of binding to plasma components (protein and cell
membranes), which reduces delivery of the tracer into brain. Under these circumstances, intersubject variability in the amount of
tracer delivered to brain may be primarily determined by its binding to plasma components and not by neuronal activity of the PI
system. If intersubject variability in f1 (the free fraction of tracer in plasma) is noted, tracer
 P.421

uptake cannot be compared among different subjects. Tracers with high lipophilicity and high binding to plasma components enter
the brain slowly, and [11C]diacylglycerol did indeed show such behavior (76 ). Further, whatever amount of a highly lipophilic tracer
actually crosses the blood–brain barrier tends to exhibit a high rate of nonspecific binding. In summary, because of the difficulty in
absolute quantification, the utility of this tracer as a quantitative measure may be significantly limited.

Arachidonate
The utility of [11C]arachidonate to detect in vivo activity of phospholipase A2 has been studied rigorously. After intravenous injection,
[11C]arachidonate is readily taken up (“pulse labeling”) and incorporated into cerebral phospholipids and other stable brain
compartments (77 ). By stimulating receptors that are linked to phospholipase A2, probably via the PI pathway, the labeled
phospholipids are catalyzed to generate arachidonate, and the regionally localized enhancement of phospholipid turnover increases
the uptake of [11C]arachidonate. The cholinomimetic arecoline has been shown to increase [14C]arachidonate levels, and this effect
can be blocked with a muscarinic ACh-receptor antagonist (78 ). For the quantification of the activity of this signal transduction
system, the measurement should not be affected by cerebral blood flow. Chang et al. (79 ) showed that the uptake of
[11C]arachidonate is relatively flow-independent in monkeys. As with radioactively labeled diacylglycerol, high lipophilicity may be a
significant limitation in absolute quantification. The potential dependence of brain uptake on the plasma free fraction may preclude
between-subject studies, although within-subject experimental designs may still be valid (e.g., before and after pharmacologic
challenge).

IMAGING REGULATION OF GENE EXPRESSION

Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

Signal transduction initiated with presynaptic firing does not terminate with the interaction of a transmitter with its receptors and
consequent second messenger generation. Instead, signal transduction may extend to the nucleus with alterations in gene expression.
A well-known example is the induction of the protooncogene c-fos by receptor–ligand interactions (80 ). In fact, oncogenes encoding
growth factors, membrane receptors, cytoplasmic and membrane-associated protein kinases, guanosine triphosphate-binding
proteins (GTP), and transcription factors play important roles in signal transduction and altered gene expression. These genes and
their cognate proteins will be important future targets for brain imaging. Much of what we know about these proteins in the central
nervous system is derived from studies of cancer biology. Thus, imaging oncogenes and their protein products is also an exciting
target for nuclear oncologists, in part because they are pharmacophores for drug development. Several modalities are applied in
cancer imaging, including (a) imaging oncogene products with radioactively labeled antibodies, (b) imaging messenger RNAs with
labeled antisense oligonucleotides (81 ), (c) imaging reporter gene products with labeled reporter probes (82 ,83 ), and (d) applying
conventional techniques with labeled small molecules that bind to particular oncogene products.

Because the blood–brain barrier presents a special obstacle in neuroimaging, most techniques successfully used in cancer imaging
are difficult to apply in brain imaging. For this reason, the first approach with antibodies is not possible in brain imaging unless the
integrity of the blood–brain barrier is disrupted, as in the case of brain tumors. The second approach, in which antisense
oligonucleotides are used, is also difficult because these multiply charged compounds do not cross the blood–brain barrier in any
appreciable amount. To make radioactively labeled oligonucleotide probes pass the blood–brain barrier, complicated techniques are
required, including the utilization of receptor-mediated transport (e.g., insulin and insulin-like growth factor) (84 ).

The third technique, the use of reporter probes, is being rigorously pursued, with possible applications in gene therapies. The best
imaging example is the use of labeled thymidine analogues (e.g., 5-iodo-2′-fluoro-2′-deoxy-1-β-D-arabino-furanosyl-uracil) in cells
expressing herpes simplex thymidine kinase. The probe is phosphorylated by the viral but not by mammalian thymidine kinases and
is thereby trapped within the cell, as in the brain uptake of 2-deoxyglucose analogues (85 ). The basic idea is that gene expression
can be monitored by radiolabeled substrate (“reporter probe”), which is metabolized by a transfected gene (“reporter gene”)
product and trapped in cells but not metabolized by endogenous enzymes of the host (82 ,83 ). A reporter gene can be different
from a therapeutic gene as long as parallel levels of expression are expected by sharing a common promoter.

At the moment, imaging of reporter probes is used to detect expression only of exogenously introduced genes. Endogenous gene
expression, which is interesting in psychiatric research, could be studied by using transgenes containing endogenous promoters fused
to a reporter gene (83 ). A limitation of these techniques in brain imaging is that the widely used reporter probes, the radiolabeled
substrates of herpes simplex type 1 thymidine kinase, do not show good permeability of the blood–brain barrier. This limitation can
be overcome by using dopamine D2 receptor as a reporter gene and D2 ligands as reporter probes (86 ,87 ). Because the expression
of functional D2 receptors may cause unwanted effects, further studies are being performed on the use of D2-receptor mutants,
which are not coupled with intracellular signaling but still maintain binding affinity for D2 ligands.
 P.422

Parkinson disease provides a useful example for gene therapy of a neuropsychiatric disorder (88 ). The concept of gene therapy for
Parkinson disease has grown directly from the promising results obtained by grafting fetal dopamine-producing neurons. However,
the limited availability and ethically controversial nature of the tissue source have restricted the utility of fetal grafts in this
disorder. As an alternative, a relatively unlimited supply of homogenous, well-characterized viral vectors could theoretically be
produced to deliver tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Attempts have also been made in animal
models to deliver neuroprotective or neurotrophic factors, such as superoxide dismutase and glial cell line-derived neurotrophic
factor, to prevent continued degeneration of dopamine neurons. Similar techniques of gene therapy have been investigated in motor
neuron degenerative diseases and Alzheimer disease. Reporter genes whose probes can cross the blood–brain barrier, such as D2
receptors, can monitor the expression of these transfected genes. On the other hand, dopamine release from the grafts could be
monitored by a conventional technique utilizing competition of radioligand binding to D2 receptors, as described in the section on
estimation of endogenous neurotransmitter levels. In fact, this technique of receptor displacement has been used to detect
dopamine release in patients with embryonic nigral transplants (89 ).

The fourth approach, the relatively conventional one of imaging with small probes for relevant gene or oncogene protein products,
is hampered by the development of useful and selective probes. As described in the section on the required properties of an in vivo
tracer, it is difficult to fulfill all the requirements for a successful brain-imaging agent. However, once good imaging agents are
developed, these targets can in general be imaged without the complicated techniques required in the other three modalities,
described above. Many new anticancer agents are being developed, and a significant number of these agents target signal
transduction systems, which may also play pathophysiologic roles in psychiatric disorders (90 ,91 ). For example, Ras
farnesyltransferase is a target for cancer chemotherapy and potentially also for brain imaging. After post-translational modifications,
including farnesylation, Ras binds to the cell membrane and transmits signals. Many inhibitors of Ras farnesyltransferase have been
developed as anticancer medications (92 ). Among them, a recently developed agent has a high affinity (93 ) and may be used as a
template from which brain imaging agents can be developed. In addition, a small molecule ligand for epidermal growth factor
receptor has been labeled with 11C and has shown brain uptake (94 ).

Rapid developments in molecular biology and the advent of gene-targeting techniques have enabled the study of individual genes in
mice by means of in vitro experimental techniques. Recently developed animal-dedicated PET devices (e.g., “rat PET” and
“microPET”) achieve high resolution of about 2 mm and can now image these animalsin vivo (6 ). These imaging studies may make it
possible to apply new findings in molecular biology to the study of patients with neuropsychiatric disorders in exciting ways.

CONCLUSIONS
Part of "31 - Molecular Imaging: Ligand Development and Research Applications "

Progress in molecular neurobiology has dramatically changed our understanding of psychiatric disorders. A significant proportion of
these findings have been obtained from animal experiments and postmortem human studies. A major challenge for neuroimaging in
future years will be to extended the application of these in vitro probes to in vivo imaging of patients. Radiotracer imaging with PET,
and to a lesser extent with SPECT, is ideally suited for such in vivo applications because of its extraordinarily high sensitivity and
improving anatomic resolution (now about 2 mm). This chapter has reviewed what is arguably the most difficult barrier to
accomplishing in vivo molecular imaging—the development of useful and quantifiable tracers. The blood–brain barrier is a challenge
to both the delivery of radiolabeled tracers and the quantification of brain uptake of the tracers. However, many successful tracers
have been developed to date. These probes have largely been synthesized as analogues of agents active at synaptic transmission and
have provided measures of presynaptic, postsynaptic, and even “intrasynaptic” targets. Relatively little progress has been made in
measuring intracellular signal transduction or gene expression. These two areas are clearly important targets for future ligand
development. By bridging new findings in molecular neuroscience and clinical studies, in vivo molecular imaging will likely
contribute to a greater understanding of psychiatric pathophysiology and, it is hoped, enhance the development of improved
pharmacotherapies.

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32
Event-Related Potentials and Magnetic Fields in the Human Brain

Steven A. Hillyard

Marta Kutas

Steven A. Hillyard: Department of Neurosciences, University of California, San Diego, La Jolla, California.

Marta Kutas: Department of Cognitive Science, University of California, San Diego, La Jolla, California.

To uncover the neural bases of a cognitive process it is important both to identify the participating brain regions and determine the
precise time course of information transmission within and among those regions. Although neuroimaging techniques based on
cerebral blood flow or metabolism (e.g., positron emission tomography [PET] and functional magnetic resonance imaging [fMRI]) are
providing increasingly detailed pictures of the anatomic regions activated during cognitive activity, these methods lack the temporal
resolution to reveal the rapid-fire patterning of neuronal communication. Noninvasive recordings of the electrical and magnetic
fields generated by active neuronal populations, however, can reveal the timing of brain activity related to cognition with a very
high, msec-level resolution. This chapter gives an overview of how these temporally precise recording techniques have been used to
analyze perceptual and cognitive mechanisms in the human brain.
The changes in field potentials that are time-locked to sensory, motor, or cognitive events are known as event-related potentials
(ERPs) and the corresponding magnetic field changes are termed event-related fields (ERFs). Both ERPs and ERFs consist of precisely
timed sequences of waves of varying field strength and polarity (Fig. 32.1 ). These observed peaks and troughs in the waveform are
often referred to as “components.” Some authors, however, prefer to use the term component to refer to portions of the waveform
that originate from particular neural structures, whereas others consider ERP/ERF components to be those waveform features that
are associated with a particular cognitive process or manipulation (2 ). Both ERPs and ERFs are generated primarily by the flow of
ionic currents in elongated nerve cells during synaptic activity. Whereas synaptic currents flowing across nerve cell membranes into
the extracellular fluid produce ERPs, the flow of synaptic current through neuronal processes produce ERFs, thereby giving rise to
concentric magnetic fields surrounding the cell. When a sufficient number of neurons having a similar anatomic configuration are
synchronously active, their summated fields may be strong enough to be detectable at the scalp.

FIGURE 32.1. The characteristic waveform of the auditory event-related potential following a brief stimulus such as a click or
tone. The individual components (peaks and troughs) are evoked with specific time delays (latencies) after stimulus onset.
Note the logarithmic time base, which makes it possible to visualize the earliest waves (I–VI) generated in the auditory
brainstem pathways. Longer latency negative (N) and positive (P) components are generated in different cortical areas. Dashed
line shows increased negativity elicited by attended sounds (negative difference) or by deviant sounds (mismatch negativity),
and dotted line shows N2 and P3 components to task-relevant target stimuli. Adapted from Münte TF, Schiltz K, Kutas M. When
temporal terms belie conceptual order. Nature 1998;395:71–73.

When ERPs or ERFs are recorded from the surface of the head, the locations of the active neural generators can only be estimated
rather than visualized directly. The calculation of generator locations from surface field distributions is known as the inverse
problem, which typically has no unique solution. However, the validity of inverse source estimations can be considerably improved
by using algorithms and models that take into account the geometry of the cortical surface, biophysical properties of the intervening
tissues, constraints from neuroimaging data, and statistical likelihood of alternative source locations (3 ,4 ). In general, the
localization of active neural populations is more straightforward with surface recordings of ERFs than with ERPs, because magnetic
fields, unlike electrical fields, are minimally distorted by the physical properties of the intervening tissues.
ERP and ERF recordings have been used extensively to investigate the spatiotemporal patterns of brain activity associated with a
variety of perceptual, cognitive, and linguistic processes. The general research strategy has been to discover the mapping between
the components of the waveform and specific cognitive processes that are engaged by a particular task. When an ERP/ERF
component can be shown to be a valid index of the neural activity underlying a cognitive operation, that component can yield
valuable information about the presence or absence of that operation and its timing with respect to other cognitive events. In many
cases, such data have been related to psychological models of the underlying processing operations and used to test alternative
theoretical positions. In addition, by localizing the neural generators of such components, inferences can be made
 P.428

about the participating anatomic circuits that can be interfaced with neuroimaging and neuropsychological data. This chapter
describes recent advances made using this approach for analyzing the neural and cognitive mechanisms of preattentive sensory
processing, selective attention, mental chronometry, memory storage and retrieval, and language comprehension and production.
The use of ERP/ERF recordings to evaluate cognitive disorders associated with neurobehavioral and psychopathologic syndromes also
is reviewed.

 PREATTENTIVE SENSORY PROCESSING

 SELECTIVE ATTENTION
 MENTAL CHRONOMETRY
 MEMORY
 LANGUAGE
 CONCLUSION
 ACKNOWLEDGMENTS

PREATTENTIVE SENSORY PROCESSING

Part of "32 - Event-Related Potentials and Magnetic Fields in the Human Brain "

Much of the early ERP waveform, and some later components as well, represent sensory-evoked neural activity in modality-specific
cortical areas. These evoked components vary with the physical parameters of the stimuli and in many cases are associated with the
preattentive encoding of stimulus features. In the visual modality, for example, the early C1 component (onset latency 50 to 60
msec) originates in retinotopically organized visual cortex (5 ) and varies in amplitude according to the spatial frequency of the
stimulus (6 ). Similarly, the early auditory cortical components P50 and N100 (and their magnetic counterparts, M50 and M100) arise
in part from generators in tonotopically organized supratemporal auditory cortex and reflect the encoding of perceived pitch (7 ).

In general, ERP amplitudes decrease when the time between successive stimulus presentations is made shorter than the refractory
or recovery period of the component under study. Although the neural processes underlying ERP refractory effects are not well
established, some candidate mechanisms include synaptic fatigue, active inhibition, and the persistence of sensory memory for the
preceding stimulus. In line with this latter idea, the refractory period of the auditory M100 has been found to have a similar time
course to that of sensory or “echoic” memory for stimulus loudness (8 ).

The P50 and Sensory Gating

The refractory properties of the auditory P50 (P1) component have been studied extensively over the past 20 years as a possible
marker of abnormal sensory input control in schizophrenia (9 ,10 ,11 and 12 ). In the standard paradigm, pairs of auditory stimuli
are presented with an ISI of 0.5 sec, and the amplitude ratio of the P50 evoked by the second stimulus (S2) relative to the first
stimulus (S1) is calculated. In general, schizophrenic subjects do not show as large a reduction in the P50 amplitude to S2 relative to
S1 as do normal controls. This refractory reduction of P50 amplitude to S2 has been interpreted as a sign of preattentive sensory
gating, which occurs because the initial S1 automatically activates an inhibitory system that suppresses responsiveness to S2 (9 ,10 ).
This inhibitory system presumably prevents irrelevant information from ascending to higher levels of cortical processing. The
abnormally large S2/S1 amplitude ratio for P50 seen in schizophrenics was thus considered evidence for impaired sensory gating,
which was suggested to be the principal sensory deficit of the disease process.

This pattern of more rapid P50 recovery in schizophrenia has been widely reported, but there have been some notable exceptions
that raise questions about the exact conditions needed to produce the effect (13 ,14 and 15 ). A more serious question, however, is
whether existing studies have, in fact, demonstrated a reliably abnormal S2/S1 ratio of the auditory P50 in schizophrenics. This
concern stems from the way the
 P.429

P50 has typically been measured—as the maximal positive amplitude within a time window (e.g., 40 to 80 msec) that encompasses
the P50 peak. Such peak measures may be artificially inflated by increased levels of background noise in the EEG recordings,
originating from either intracranial or extracranial sources. Thus, if a patient group has higher EEG noise levels, then the measured
P50 amplitudes tend to be greater because the noise peaks are at times mistaken for the actual peak of the P50. This type of error
is more pronounced when measuring the P50 to S2, because its amplitude is diminished relative to the noise owing to refractory
effects. Reports of increased variability and lower reliability of P50 measures in schizophrenics (12 ,16 ) suggest that background
noise levels are indeed higher in the patient groups. Several studies, however, have reported that the P50 evoked by S1 is smaller in
schizophrenics (11 ,12 ,16 ), which suggests that overall response amplitudes may be lower and/or response latencies more variable
in these patients. Further studies are needed to determine whether the actual S2/S1 amplitude ratio is reliably higher in
schizophrenics, or whether this reported effect is a product of noise-sensitive measurement procedures.

Even if the S2/S1 ratio for P50 were determined to be greater in schizophrenia, there would still be some question about its
functional interpretation. There is little evidence that refractory reductions of ERP amplitudes to stimuli repeated at 0.5 sec ISIs are
associated with any reduction in the perceptual information reaching higher centers. Nor does it appear that the amplitude of P50
to S2 is reduced only when S2 is irrelevant (17 ), calling into question the hypothesis that the P50 refractory effect reflects the
selective gating of irrelevant versus relevant sensory inputs. In addition, it has been reported that schizophrenic patients showing
the most severe perceptual anomalies did not differ from normals in their P50 refractory effects (15 ). Thus, there seems to be
scant evidence that reduced P50 refractoriness in schizophrenia, if such exists, is related to the selective gating or filtering of
irrelevant sensory information in the auditory cortical pathways.

Auditory Feature Encoding

The preattentive coding of auditory features is indexed with considerable precision by the mismatch negativity (MMN) component
and its magnetic counterpart, the MMNm. The MMN is a scalp-negative component with a latency of 120 to 250 msec that is
specifically elicited by a deviant sound in a repetitive auditory sequence (18 ,19 and 20 ). The MMN can be triggered by any
discernible change in the ongoing sounds, such as deviations in frequency, intensity, duration, rise-time, timing, and spatial location.
MMNs also have been recorded to changes in more complex sound properties such as the phonetic structure of speech sounds and the
patterning of tone sequences (20 ,21 ). Näätänen (22 ) has proposed that the MMN is generated by an automatic comparator process
that contrasts current auditory input against a multidimensional trace of the previous repeating sound's features held in
preperceptual sensory memory. This mismatch detection process may represent an early stage in the alerting and orienting of the
organism toward novel and potentially important changes in the acoustic environment.

The MMN provides a window on auditory sensory or “echoic memory” because it is initiated by a mismatch with the memory traces
of the preceding sounds (23 ). Indeed, the maximal interstimulus interval (ISI) at which the MMN can be maintained is of the order of
10 sec, corresponding well with behavioral estimates of the duration of echoic memory (19 ,20 ). The MMN also can be used to study
more permanent auditory memory traces, such as those for the phonemic characteristics of one's native language (19 ) as they
emerge during the first year of life (21 ).

It has been proposed that a supratemporal component of the MMN originating in auditory cortex reflects the preattentive sensory
store and automatic change detection process, whereas a frontal component indexes the involuntary orienting of attention to the
deviant event (24 ,25 ). For speech sounds, however, the MMN/MMNm appears to arise from sources in auditory cortex of the left
hemisphere, in accordance with proposals that the left posterior temporal cortex is the locus of language-specific auditory traces
(19 ).

Given that the MMN may be elicited with minimal cooperation from the subject, it has found wide applicability for the diagnosis and
evaluation of a variety of neurobehavioral and psychiatric disorders (24 ,26 ). Schizophrenic patients have reduced or prolonged
MMNs to pitch or duration deviants, with the degree of abnormality depending on the specific parameters of the stimulus deviance
(24 ,27 ,28 ). These findings provide clear evidence for a deficit in preattentive auditory processing in schizophrenia, although there
is some debate about whether the impairment is primarily in temporal processing (28 ) or auditory encoding and trace formation
(27 ). A different pattern of abnormality has been observed in Alzheimer's disease, with MMN amplitude reductions becoming more
prominent at longer ISIs (29 ), suggesting a more rapid decay of auditory sensory memory traces. MMN abnormalities indicative of
auditory processing deficits have also been reported in cases of learning disorders, language and speech impairments, depression,
autism, parkinsonism, and HIV infection. (See refs. 19 , 21 , 24 , and 29 for reviews.) Drugs that interfere with NMDA-receptor
mediated neurotransmission also disrupt the MMN, which is consistent with models of schizophrenia that posit a disturbance in
glutaminergic/NMDA functioning (27 ).

SELECTIVE ATTENTION
Part of "32 - Event-Related Potentials and Magnetic Fields in the Human Brain "

The brain's attentional systems include a central control network with projections to the sensory pathways of the different
modalities that enable the selective modulation of
 P.430

incoming information. A good deal of research on attention over the past few decades has been aimed at determining whether
incoming sensory information is selected at “early” or “late” levels of processing—that is, before or after stimulus properties are
fully analyzed (30 ). Current evidence from both behavioral and physiologic studies indicates that attention can select stimuli at
different levels of the sensory pathways, depending on the features being attended and the task requirements.

Auditory Attention
In the auditory modality, ERPs have demonstrated that attentional selection occurs at early levels of cortical processing, but not in
the brainstem pathways (31 ). In dichotic listening tasks with rapidly presented tones to the left and right ears, the earliest ERP
component that is reliably influenced by paying attention selectively to one ear is a small positive wave with a latency of 20 to 50
msec (termed the P20–50), which has been localized using magnetoencephalography (MEG) to sources in or near primary auditory
cortex. This short-latency modulation provides evidence for an attentional mechanism of sensory gain control at the earliest levels
of cortical processing. A much stronger attentional modulation of auditory input takes place at 50 to 70 msec after stimulus onset in
the form of a negative difference (Nd) potential that augments the amplitude of the evoked N1 wave to attended-channel sounds
(Fig. 32.1 ). This N1/Nd attention effect also has been localized to auditory cortex by MEG recordings and is considered to be an
index of further processing of attended sound information, or alternatively, of the closeness of match between incoming stimuli and
the acoustic features that define the attended channel (30 ). These negative ERP modulations indicate the precise timing with which
different auditory features are attended or rejected (32 ) and provide strong evidence for early selection theories of attention.
Schizophrenic subjects reportedly show abnormally reduced Nd amplitudes when attending to multiple sound features, suggesting a
deficit in their control functions for allocating attentional resources during selective listening (33 ).

In recent studies, auditory ERPs have been used to study how attention is allocated in a noisy environment with multiple, competing
sound sources (34 ). When subjects listened selectively to sounds coming from one loudspeaker in a free-field array, the spatial
focusing of auditory attention took place in two distinct stages: an early, broadly tuned input selection occurring over the first 80 to
180 msec (indexed by N1/Nd) was followed by a more sharply focused selection of target sounds that began at around 250 msec
(indexed by the late positive P3 component). These findings indicate that auditory spatial attention is deployed as a sharply tuned
gradient around an attended sound source in a noisy environment. Under these conditions congenitally blind persons were found to
have sound localization capabilities superior to those of sighted control subjects (35 ). ERP data indicated that this enhanced
capability of blind persons is mediated at least in part by an attentional tuning mechanism that operates within the first 100 msec
after sound onset.

Visual Attention
Covertly directing attention to a specific location in the visual fields typically results in faster and more accurate detections or
discriminations of stimuli at that location. Recordings of brain activity in both humans and animals have identified a number of sites
along the visual pathways where afferent information is modulated under the influence of visual-spatial attention. Neurophysiologic
studies in monkeys demonstrated strong influences of spatial attention on neural activity in extrastriate cortical regions, including
retinotopic areas V2, V3A, and V4 and higher areas of both the ventral (inferior temporal lobe) and dorsal (area MT, posterior
parietal lobe) processing streams (36 ). These findings are congruent with human ERP studies showing that stimuli at attended
locations elicit enlarged P1 (70 to 130 msec) and N1 (150 to 190 msec) components (Fig. 32.2 ), which have been localized to
generators in extrastriate visual cortex (37 ). This amplitude enhancement of the P1 and N1 waves occurs with little or no change of
the component latencies, suggesting that spatial attention exerts a gain control or selective amplification of attended inputs within
the visual-cortical pathways in the interval between 70 and 200 msec after stimulus onset (38 ).

FIGURE 32.2. A: Visual event-related potential

waveforms from several scalp sites in response
to stimuli in left visual field in study by
Martínez and colleagues (41). Subjects were
required to attend to one field at a time while
randomized stimulus sequences were presented
concurrently to left and right fields. Note
increased amplitude of P1 and N1 components
when stimuli were attended and lack of change
in C1 component. B: Voltage topographies of C1
component and of the P1 attention effect
(increased positivity with attention) in two
different time ranges. C: Locations of dipolar
sources calculated for the C1 component (in
primary cortex of calcarine fissure, left) and
the early and late P1 attention effects (dorsal
and ventral extrastriate cortex, respectively,
right).

In experiments that combined PET neuroimaging and ERP recordings, the calculated dipolar sources of the P1 attention effect were
found to correspond closely with regions of increased blood flow in retinotopically organized extrastriate areas, including areas
V3/V4 and the posterior fusiform gyrus (37 ). Significantly, however, the earlier C1 component (onset at 50 msec), which appears to
originate from generators in primary visual cortex (area V1), was found to remain invariant with changes in the direction of spatial
attention. These findings suggest that spatial attention modulates the flow of visual information at a higher level than the primary
cortex.

Recent studies in monkeys, however, reported that stimulus-evoked activity in area V1 may be affected by spatial attention when
competing stimuli are present in the visual field (39 ). The participation of V1 in spatial attention has also been inferred from recent
fMRI studies in humans (40 ). In a study combining fMRI with ERP recordings, Martínez and colleagues (41 ) observed focal increases
in blood flow (BOLD signal) in area V1 as well as areas V2, V3/VP, and V4 at sites corresponding to the retinotopically mapped
position of the attended stimulus; however, the amplitude of the C1 component again remained invariant (Fig. 32.2 ). The earliest
influence of attention was on the P1 component (75 to 130 msec), which was localized through dipole modeling to dorsal and ventral
extrastriate sources. It was suggested
 P.431

that the attentional modulation of V1 activity revealed by fMRI may take place at a latency longer than the initial geniculo-striate
response represented by the C1 and is consequent on delayed feedback of enhanced visual signals back to V1 from higher
extrastriate areas. Such long-latency modulations in V1 have been observed in animals and may enhance figure/ground contrast in
attended regions of the visual field (39 ).

The spatial allocation of attention has also been studied with the steady-state visual evoked potential (SSVEP), which is the
oscillatory response of the visual cortex evoked by regularly repeating stimuli such as a light that flickers at a rate of 8 Hz or more
(42 ). The amplitude of the SSVEP elicited by such a flickering stimulus is substantially increased in amplitude when attention is
directed to its location, thereby indexing amplification of visual inputs within the spotlight of attention. The continuous nature of
the SSVEP as a measure of cortical facilitation makes it suitable for measuring the time course of attention shifts among stimuli in
the visual fields.

In contrast with spatial attention, when stimuli are selected on the basis of nonspatial features such as color, shape, or spatial
frequency, a different pattern of ERP components is observed. Stimuli having the attended feature elicit a prominent “selection
negativity” (SN) over the posterior scalp that begins at 120 to 220 msec and may extend for several hundreds of msec (37 ). The
onset of the SN provides a precise measure of the time point at which a particular feature is discriminated and selectively processed,
and localization of its neural generators points to the brain regions involved in the selection. When stimuli are selected on the basis
of two or more features concurrently, recordings of the SN can indicate whether the features are selected independently or in an
interactive, contingent manner (43 ).

MENTAL CHRONOMETRY
Part of "32 - Event-Related Potentials and Magnetic Fields in the Human Brain "

Motor preparation, execution, and evaluation are indexed by a series of electric (and magnetic) components both preceding and
following movement onset. Prime among the ERPs indexing preparation is the readiness potential (RP), which is a slowly ramping
negativity that starts about 1 s before the onset of a voluntary or self-paced movement and peaks around movement onset (44 ).
The initial, bilaterally symmetric portion of the RP is generated in the supplementary
 P.432

motor area (SMA). Approximately 200 to 500 msec before movement onset the RP becomes asymmetric, being maximal over central
scalp contralateral to the active musculature. This lateralized portion of the RP has a somatotopic distribution over the motor
cortex and has been localized to the primary motor cortex (45 ). This asymmetric portion of the RP can be seen in stimulus-locked
averages (46 ) and serves as the basis for an index of differential motor preparation termed the lateralized readiness potential (LRP).
Subtracting for each hand separately the activity over the ipsilateral from that over the contralateral central site, and then
averaging the activity for the two hands together derive the LRP.

The LRP has proven especially useful in studies of mental chronometry aimed at answering questions about the dynamics of
information processing. For example, LRP data have shown that whether a person responds quickly and accurately is in large part a
function of whether he or she is prepared to do so before the stimulus appears. Appropriate preparation leads to fast and correct
responses, whereas inappropriate preparation leads to fast but wrong responses, and no preparation at all leads to slow but
accurate responses. More important, LRP data revealed that people develop biases that influence how they prepare to respond. (See
refs. 47 and 48 for review.)

In a number of sensory-motor discrimination tasks, LRP recordings have provided strong support for “continuous flow” models that
specify that transmission of perceptual information to the response system occurs continuously rather than in discrete, all-or-none
stages. Such studies have revealed partial transmission of perceptual information (e.g., about letter identity) to the response system
and have provided a means of tracking the time course of the extraction of various types of information. The LRP also has been used
to determine the timing of “the point of no return;” that is, the time in the course of response preparation beyond which response
execution cannot be stopped (47 ).

Readiness potentials have been examined in a number of patient populations. They are abnormal in a large majority of individuals
with Parkinson's disease (PD), presumably because of abnormal activation of the SMA by the basal ganglia (49 ,50 ). Because the
early part of the RP is sensitive to attention, it has been suggested that motor performance in PD patients might be improved by
having them attend to movements that they might otherwise try to execute automatically (51 ). Individuals with tardive dyskinesia
(TD) also show abnormal RPs that are larger in amplitude than those of normal controls and schizophrenic patients without TD (52 ).
Unlike voluntary leg movements, involuntary myoclonic leg movements in patients with restless leg syndrome do not elicit an RP,
suggesting that these involuntary movements have a subcortical or spinal origin (53 ).

Purposeful movements are generally monitored and evaluated so that remedial action may be taken if an error in committed.
Performance monitoring of this sort is indexed by an ERP known as the error-related negativity (ERN), which peaks about 100 msec
after the onset of the electromyographic activity associated with an erroneous response. ERN amplitude covaries with the perceived
inaccuracy of a response (54 ) and is influenced by how similar the given response is to the correct one. An ERN is also elicited by a
feedback stimulus that lets the subject know an error was made. ERN generators have been localized to the anterior cingulate gyrus
and are modulated by lateral prefrontal cortex (55 ). Patients with lateral prefrontal cortical damage are impaired in correcting
their behaviors and produce equal-sized ERNs for correct and incorrect responses. ERN amplitude is sensitive to mood and
personality variables (56 ), especially when correct responses are rewarded and/or incorrect responses are punished (57 ).

MEMORY
Part of "32 - Event-Related Potentials and Magnetic Fields in the Human Brain "

Working Memory
Unexpected events typically require us to revise or update our current working mental model of the environment. Donchin and
colleagues (58 ) proposed that this updating of the working memory (temporary, limited capacity) system is indexed by the P3 (also
known as the P300 or P3b) component. (See refs. 59 and 60 for alternative views.) The P3b is a positive, broadly distributed
component with a centro-parietal maximum and peak latency between 300 and 800 ms. It is elicited by infrequent target events in a
sequence of higher probability nontarget events that are being attended, although irrelevant stimuli that draw attention may also
trigger a P3. In general P3 amplitude grows with the relevance, salience, and utility of the target or “oddball” stimulus. The P3 can
be elicited by many different simple and complex events, including the occasional absence of a predicted stimulus. (See ref. 61 for
review.) The differences in the distribution and timing of P3s in various modalities are consistent with the proposal that there are
multiple working memory stores.

P3 amplitude is inversely related to the overall probability of the target events, and varies with the fine structure of event
sequences. The more difficult the categorization of the target events, the longer the P3 latency. P3 latency is not correlated with
the variance in reaction time that is caused by response execution, thereby making it a rather pure measure of stimulus
evaluation/categorization time. The combined sensitivity of the P3 to attention and stimulus evaluation makes it a good index of the
availability and allocation of capacity-limited perceptual resources. Measurements of P3 latency and RT together can be used to
pinpoint the processing locus of individual differences in performance, as was done, for example, to analyze cognitive slowing with
normal aging (62 ). Similarly, P3 data have demonstrated that the prolongation of response time for the second of two decisions
made in rapid succession (“psychological
 P.433

refractory period”) is owing to interference at a stage that follows perceptual categorization, presumably that of response selection
(63 ).

Updating working memory has consequences for an individual's subsequent performance. For example, the relative amplitude of the
P3 on a trial when a subject commits an error is predictive of the performance (accuracy and response speed) on the next trial;
moreover, the larger the P3 to an item, the greater the likelihood that it will be remembered subsequently. (See ref. 64 for review.)
Intracranial recordings in individuals with epilepsy have revealed P3-like potentials in the hippocampal region of the medial
temporal lobe (65 ). The scalp-recorded P3, however, primarily reflects activity in a number of neocortical (frontal, central, parietal,
temporal-parietal junction) and perhaps subcortical generators and is mediated by several neurotransmitter systems (66 ). Not
surprisingly, therefore, many patient populations show abnormally small or delayed P3bs including schizophrenic patients,
individuals at risk for alcoholism, patients with probable Alzheimer's dementia, and individuals with attention deficit and
hyperactivity disorder, among others (61 ,66 ).

The storage of information in working memory may be modulated by attention and appears to be strongly suppressed during the
“attentional blink” that follows detection of the first of two target stimuli presented in a rapid sequence. Luck and colleagues
showed that the P3 was absent in response to targets that went undetected during the attentional blink, suggesting that no updating
of working memory occurred. (See ref. 67 for review.) However, undetected target words did elicit late negative ERPs, indicating
that they had been identified at the level of lexical/semantic processing. Thus, the ERP data provided strong evidence that the
attentional blink acts at the postperceptual stage of working memory storage.

A frontally distributed late positivity (P3a) is elicited by rare and unexpected stimuli for which there is no memory template readily
available. It appears to reflect an orienting response to stimulus novelty and is reduced in patients with prefrontal cortical injury
(68 ). Maintenance of information in working memory is also reflected in sustained ERPs lasting on the order of seconds. These
potentials vary in their scalp distribution as a function of the information being held in working memory, consistent with proposals
of independent short-term buffers for verbal and nonverbal information, among others (69 ).

Long(er)-Term Memory

Encoding
Encoding processes (transforming sensory input into a lasting representation) are associated with an increased positivity between
200 and 800 msec that spans several components. Items that call for preferential processing because they stand out, for example,
are better recalled and elicit larger P3 components (70 ). Likewise, the more deeply (semantically) an item is analyzed, the more
likely it is to be remembered, and this is reflected in greater late positivity (71 ). Even among items that are all deeply processed,
those that will in fact be remembered later elicit a larger positivity during encoding than those that will be forgotten (Fig. 32.3 ).
These late components produced during encoding that are predictive of subsequent memory performance are collectively termed
Dm effects (71 ).

FIGURE 32.3. Averaged event-related potentials (ERPs) from midline parietal site (filled in circle in map on the right) sorted
as a function of subsequent memory in a cued recall test. The responses to words subsequently recalled (solid line) are
overlapped with those subsequently not recalled (dashed line). Participants were presented the first three letters of a word
and asked to use this stem as a clue for verbally recalling the words they had just studied. The voltage map of this difference
related to memory (Dm) effect at 550 ms was computed by subtracting the ERPs to words subsequently not recalled to ERPs
from those subsequently recalled. A: Semantically anomalous word. B: Unexpected word. Adapted from Paller KA. Recall and
stem-completion priming have different electrophysiological correlates and are modified differentially by directed forgetting.
J Exp Psychol Learn Mem Cogn 1990;16:1021–1032.

Dm effects are larger in semantic than in nonsemantic tasks and are not seen for items that have no preexisting representation in
long-term memory. Van Petten and colleagues (73 ) suggested that this positivity indexes the richness of associative elaboration
engendered by the to-beremembered event. Consistent with this proposal, the Dm effect varies with the encoding task and
information retrieved from long-term memory and shows substantial variability in onset latency, duration, and scalp topography
(74 ).

Retrieval
Retrieval processes are indexed by several ERP effects that vary with whether or not the rememberer is in a retrieval mode,
whether memory is queried directly or indirectly, what aspect of the memory is being queried, and whether or not the retrieval
attempt is successful (75 ,76 ). Retrieval itself is indexed by slow potentials sustained over several seconds with an amplitude
determined by the difficulty of the retrieval and a scalp topography determined by the nature
 P.434

of the information retrieved (77 ). These results fit with the notion that the brain areas involved in explicit memory are the same as
those carrying out the initial encoding and perception and argue against the concept of a single, amodal memory store.

In a typical retrieval paradigm items are presented twice, and ERPs to the first and second (i.e., repetition) presentations are
compared. When subjects are asked to recognize and detect the repeated items, the task is considered to probe memory directly or
explicitly. By contrast, when the old or new distinction is irrelevant, as in tasks involving lexical decision, semantic judgment, or
identification of visually degraded words, the stimuli may only tap memory indirectly or implicitly and may not produce actual
recollection. In both implicit and explicit memory tasks, stimulus repetition produces large and reliable ERP effects. The first is a
reduction in the amplitude of negativity between 250 and 500 msec (N400) that is associated with semantic processing (76 ). The
N400 is reduced by repetition, whether or not the task explicitly calls for detection of repeated items, even in amnesic individuals
with damage to the medial temporal lobes (78 ). Some authors have linked a frontal subcomponent of the N400 to repetition
independent of recognition (79 ).

Another ERP consequence of word repetition is a change in the amplitude of a late positive component (LPC), which typically begins
around 400 to 500 msec, and and is somewhat larger over the left than right scalp. There is mounting evidence that this LPC reflects
conscious recollection. Factors that influence perceptual priming do not modulate LPC amplitude (80 ), whereas factors that
influence recognition memory do. There is an LPC repetition effect whether memory is tested implicitly or explicitly. When
participants are asked to indicate whether an item is old or new, correctly recognized old items elicit larger LPCs than do
unrecognized old items or correctly recognized new items, although its distribution across the scalp varies somewhat with the
materials (81 ). The LPC to correctly recognized old items is larger for confident than less confident decisions, and for items that
participants actually “remember” (82 ).

When a subject attempts to remember some aspect of the context in which an item was studied or some attribute of the item that
it shared with others in the study task, a large, late, frontally distributed (sometimes right lateralized) positivity is elicited (83 ).
This large positivity over prefrontal sites occurs in addition to the standard LPC effect. The prefrontal locus of this ERP source
retrieval effect fits with the known impairments that patients with frontal lobe damage have in retrieving source information about
items that they recognize (84 ).

LANGUAGE
Part of "32 - Event-Related Potentials and Magnetic Fields in the Human Brain "

Semantic Analysis
The semantic analysis of verbal and nonverbal stimuli is indexed by the N400 component (85 ). The N400 is a broadly distributed
component, with a negative-going peak over centroparietal sites often with a slightly right predominance; in young adults, it has an
onset around 200 msec and a peak around 400 msec. The largest N400s are elicited by unexpected, semantically anomalous words in
a sentence (Fig. 32.4 ). However, all potentially meaningful items (e.g., words and pseudowords, environmental sounds, pictures,
 P.435

faces) can elicit some N400 activity with an amplitude that is determined by a variety of factors. With little or no contextual
constraint, N400 amplitudes are inversely related to the frequency of the eliciting word in the language (86 ).

FIGURE 32.4. A: A prototypical N400 recorded at the vertex in

response to a semantically anomalous word (dashed line) at the
end of a sentence, compared with the ERP to the expected ending
(solid line), and an anomalous ending that is semantically related
to the expected ending (dotted line). Note reduced N400 for
nonsensical ending (“hive”) that is semantically related to
expected ending. B: The N400 recorded at a midline parietal site
elicited by a word that fits with the ongoing discourse context
(solid line) versus that to a word that is less expected and does
not fit as well with the ongoing discourse context (dashed line).
Data taken from van Berkum JJA, Hagoort P, Brown CM. Semantic
integration in sentences and discourse: evidence from the N400. J
Cog Neurosci 1999;11:657–671.

The N400 is typically considered an ERP index of semantic processing or contextual integration because its amplitude is modulated
by its relation and fit to the ongoing context, be it a single word, a sentence, or a multisentence discourse. (See refs. 87 and 88 for
review.) N400 amplitudes are enlarged to a word in unrelated word pair or in an incongruous or weak context relative to the
response to the same word in a related pair or strong congruous sentence. The N400 in these cases is almost identical in timing and
distribution over the head, indicating that by 400 msec at the latest, lexical, sentential, and discourse processes all converge to
influence language comprehension in a similar manner. Visual half-field studies of the N400 show that the left hemisphere, in
particular, uses the organization of semantic memory tapped by context words to aid in its online predictions, whereas the right
hemisphere waits and integrates (89 ).
As would be expected of an index of semantic processing and contextual integration, N400 amplitude is greatly attenuated and its
latency delayed in aphasic patients with moderate to severe comprehension deficits (90 ) N400 latency is also prolonged with normal
aging and various dementias. Although ERP evidence for a differential organization of semantic memory in schizophrenia is equivocal,
a delay in N400 latency has been reported. (See ref. 87 for review.) Intracranial recordings from patients with epilepsy show
potentials functionally similar to the scalp N400 in the anterior fusiform gyrus (91 ).

Syntactic Analysis
The processing of language at a syntactic level is indexed by a several ERP components, both negative and positive. Many, although
not all, syntactic violations elicit a late positivity variously called the P600 or the syntactic positive shift (SPS) (92 ,93 and 94 ). The
P600 is typically elicited when some aspect of sentence structure violates the rules of the language—for example, if the subject of
the sentence does not agree with its verb in number or if a word in a phrase is out of order. The P600 also may be elicited when
processing difficulties arise at a structural level (87 ). Some researchers have proposed that the P600 belongs to the family of P3
waves (95 ). In addition to the P600, many syntactic violations also elicit a left anterior negativity (LAN), which some researchers
have interpreted as an index of working memory usage (96 ,97 ).
The fact that an N400 or P600 is elicited shortly after a semantically anomalous or grammatically incorrect word, respectively,
regardless of its ordinal position in a sentence, is most consistent with those models of sentence processing that emphasize the
immediate and online nature of comprehension (98 ). That is, the language processing system seems to use all information as it
becomes available, often to predict what words or ideas are likely to come next (89 ,99 ). That processing at a semantic and
syntactic level yields different patterns of electrophysiologic activity suggests that the processes differ, if not the representations.
Further, the presence of different ERP patterns to various syntactic violations indicates that syntax is not a unitary phenomenon
mediated by a single neural generator. Many aspects of sentence processing at semantic, syntactic, referential, thematic, prosodic,
and discourse levels are indexed by transient ERP effects and/or slow potentials that encompass the entire sentence (100 ,101 ). In
short, the reported patterns of ERP effects are inconsistent with a view of language comprehension that gives syntactic analysis
precedence over semantic analysis or a system wherein syntactic processes are isolated from all other processes. Instead, ERP data
provide considerable evidence for parallel processing, interaction, and top-down effects during language processing. The brains of
readers and listeners work very much online using all information as it becomes available to anticipate upcoming items, concepts,
and schemas to achieve the aim of an efficient and error-free understanding of the incoming language (even if at times these
predictions may lead to misunderstanding).

Language Production
As in language comprehension, many of the controversies in language production revolve around the issue of the relative timing of
the different levels of processing that are engaged. Although there is a general consensus that producing a coherent utterance
involves information at the levels of meaning, syntax, and phonetics there is no agreement as to whether meaning comes first and
then phonologic form (i.e., a serial model), these processes overlap somewhat in time (i.e., a cascade model), or they unfold in
parallel (102 ). Two ERP measures—the LRP and N200—can be used to track the time course of information availability as people
prepare to speak, even if they never actually utter a word. In studies using a two choice go/no-go paradigm, subjects were shown a
picture of an item on each trial about which they were asked to make two decisions (Fig. 32.5 ). Across experiments, decisions were
based on semantic, syntactic, and phonologic aspects of the pictured item and its name. The timing of the N200 and LRP on no-go
trials indicated that semantic information becomes available before syntactic information (by about 80 msec), which is in turn
available before phonologic information (by about 40 msec) (103 ,104 ). Electrophysiologic data from the scalp thus support a serial
model of speech production, indicating that people first figure out what they want to say and then choose exactly how to say it.

FIGURE 32.5. Overlapped are the N200 difference waves (no-go

minus go event-related potentials) recorded at a midline
prefrontal site (as marked on head icon) when the decision to
respond or not (go/no-go) was contingent on a semantic (dashed
line) versus a syntactic (solid line) attribute of the pictured
object. Note that the N200 effect contingent on the semantic
analysis peaks around 380 ms, whereas that contingent on syntax
peaks around 500 ms. Data from Schmitt B, Münte TF, Kutas M.
Electrophysiological estimates of the time course of semantic and
phonological encoding during implicit picture naming.
Psychophysiology 2000 Jul;37(4):473–484.

CONCLUSION
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 P.436

Specific components of ERPs and ERFs recorded from the surface of the head are sensitive to a wide range of sensory, perceptual,
motor, mnemonic, and linguistic processes. It appears that many cognitive acts engender synchronous neural activity patterns that
produce electrical and magnetic fields precisely time-locked to informational transactions in the brain. Recordings of ERPs/ERFs
thus provide critical information about the timing and neural substrates of the processing stages that underlie cognitive activity.
These physiologic data are being used increasingly to test alternative functional models and to constrain psychological theories
(2 ,64 ,105 ).

Considerable progress has been made in demonstrating reliable associations between ERP/ERF components and a wide range of
psychopathologic syndromes. In no case, however, is a single ERP/ERF component absent or abnormal in such a way as to be
diagnostic. Rather, a given syndrome (e.g., schizophrenia) usually manifests abnormalities in one or more parameters of several
different ERP components, and a given component (e.g., the P3) appears abnormal across a range of neurobehavioral syndromes.
This is to be expected, except in the unlikely (and perhaps nonexistent) case in which the psychopathology would only affect a
single, isolated cognitive subprocess that had a unique ERP/ERF marker. Thus, instead of seeking a single ERP marker, it seems more
likely that various patient populations will be distinguished by different profiles of ERP effects across a number of different tasks
(much like the approach taken in neuropsychological testing). Many of the same interpretational issues that are of concern with
neuropsychological testing may become relevant for testing with an ERP battery, together with some that are specific to these
physiologic measures. For example, the considerable synaptic plasticity of the neocortex suggests that even normal individuals'
component amplitudes and latencies are likely to show considerable variability, depending on their life experiences. Such variability
within the normal population clearly exacerbates the difficulty of uniquely identifying ERP/ERF markers of specific clinical
syndromes. Further progress in achieving diagnostic specificity and sensitivity may require comparing ERPs/ERFs across multiple
tasks in each patient to reveal reliable abnormalities that are related to specific cognitive manipulations. Such ERP/ERF
abnormalities will become increasingly informative about the specific processing mechanisms that are dysfunctional in patient
groups as the cognitive specificity of the distinctive components is sharpened through studies in normals and as better methods are
developed for measuring and isolating those components. These developments should make it possible to incorporate ERP/ERF data
into multimeasure diagnostic batteries to aid in classifying and subtyping psychopathologic syndromes.

Recent technical advances have made it possible to obtain more accurate information about the neural bases of ERPs/ERFs and their
relationships with cognitive and behavioral variables. The neural generators of surface recorded ERP/ERF activity can be localized
with increased precision using algorithms that exploit more accurate bioelectric models of the head and constrain the generators to
lie within the cortical mantle as reconstructed from MRI scans. (See ref. 105 for review.) Source localizations can be further
improved by incorporating functional imaging data (e.g., from fMRI) into the inverse calculations, thereby providing a more veridical
picture of the spatiotemporal patterning of cognitive-related brain activity (3 ,4 ,106 ). New approaches also have been developed
for decomposing these complex patterns of brain activity arising from multiple, concurrently active generators into functionally
meaningful subcomponents. Among these, the technique of Independent Component Analysis (107 ) has shown considerable promise
for decomposing ERP data sets from multiple task conditions into temporally independent and spatially localizable components that
may be related to cognitive operations on the one hand and to fMRI activation patterns on the other. Newer spatiotemporal filtering
procedures (e.g., wavelet filtering) have improved our ability to extract the ERP/ERF signal from ongoing brain activity and other
background noise. (See ref. 105 for review.) These methods ultimately may allow reliable detection of event-related signals on a
single-trial basis without relying on the usual computer averaging procedure. Single-trial analyses are important not only for
achieving a closer correspondence between brain activity and behavioral performance but also for ascertaining the degree of trial-
to-trial variability that may characterize different clinical syndromes. All of these techniques will substantially increase the utility of
ERP/ERF recordings for analyzing the neural bases of both normal and disordered cognition.

ACKNOWLEDGMENTS
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 P.437

This work was supported by NIH grants MH-25594, HD-22614, and AG-08313. We thank Carole Montejano, Matt Marlow, and Tom Urbach for assistance with manuscript preparation.

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Section IV
Drug Discovery and Evaluation
Herbert Y. Meltzer

Drug Discovery and Evaluation - Introduction

It is fitting to include in Neuropsychopharmacology: The Fifth Generation of Progress a group of chapters concerned with many of the important
issues related to: (a) the development of new drugs (e.g., the role of preclinical models to develop and test targets for new drugs; (b) the role of
biological markers and imaging studies to provide indices of drug action in humans or predict clinical response; (c) advances in clinical trial design;
(d) the optimal utilization of existing drugs (e.g., pharmacokinetics); (e) pharmacogenomics in relation to drug metabolism, discovery, and
development; (f) the ethical issues concerning clinical trials and neuropsychiatric research in general; (g) governmental (at least United States)
regulation of the process of developing and utilizing new drugs; and (h) the evaluation of the impact of drug treatment on outcome of
neuropsychiatric disorders from the perspectives of economics, clinical endpoints, and humanistic considerations. Although the majority of these
issues are the same in many parts of the world where this book will be read, there are clear differences in ethical attitudes toward clinical research
and governmental regulation of drug development and utilization around the world (despite the Declaration of Helsinki). It is beyond the scope of
this volume to consider the worldwide variations in these issues. All the chapters that have preceded this section bear on the subject matter addressed
here because new drug development depends so heavily on our understanding of the function of the brain in general, neurotransmitter and modulator
receptors and elimination mechanisms, theories of the etiology of the major neuropsychiatric disorders, efficacy and side effects of existing
treatments, and an understanding of the mechanism of action of existing drugs.

The process of new drug development has changed greatly in the few years since the last volume in this series and is likely to change even more
rapidly in the immediate future. A great advance was the development of combinatorial chemistry and rapid, robotic characterization of the
pharmacologic profile of the vast libraries of compounds produced by this shotgun approach, which, when successful, leads to the elegant and
expensive custom syntheses of candidate compounds by sophisticated organic chemical procedures that are now often computer-derived or by
methods involving cell and molecular biology to produce peptides or other organic substances. Together with greatly improved methods for analysis
of structure-activity relationships, it has been possible to develop putative pharmaceuticals with the desired pharmacologic profile. The old cliché,
beware what you desire because you may get it, is relevant here, because it much easier now to come up with the desired pharmacologic profile than
it is to be certain that what is sought is what should be sought. There is not yet sufficient understanding of what is needed in the way of an optimal
antidepressant, anxiolytic, antipsychotic, mood stabilizer, antidementia, or other type of drug, especially when a truly novel compound is sought.

The chapter by Geyer and Markou on the role of preclinical models in the development of psychotropic drugs mainly focuses on animal models for
the major psychiatric disorders. These authors point out that this approach may seem somewhat old-fashioned compared to approaches such as high
throughput screening and utilization of molecular biological techniques to develop targets based on gene expression and identification methods;
however, they correctly state that preclinical models are required (emphasis added) to provide initial assessment of the functional effects of novel
compounds in the integrated organism. We are not yet to the point where new chemical entities go directly into patients or even normal volunteers
without some evidence that clinically relevant effects might be present. We
 P.442

can expect major advances in the development of preclinical models as our knowledge of disease processes and our ability to alter the genome in
laboratory animals increase. Knockout and knockin mouse models will increasingly guide drug discovery and testing. The importance of research
designed to identify new drug targets based on the Human Genome Project and the ensuing effort to characterize the genes involved in
neuropsychiatric disorders and the action of drugs used to treat neuropsychiatric illness is discussed in various chapters throughout this volume rather
than in a single chapter in this section.

The use of biomarkers (i.e., natural history markers), biological activity marker, and surrogate markers is thoroughly explored by Wong and
colleagues, who note that the importance of biomarkers as a means to reduce the cost of drug development, improve the ability to predict outcome,
and expedite the identification of desired endpoints (e.g., no more than 80% occupancy of striatal 2 receptors in order to minimize the development of
extrapyramidal side effects), is increasing all the time. The extraordinary development of a variety of brain imaging methods, including magnetic
resonance imaging, functional magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography,
appears to be particularly suited for this purpose. Given the cost associated with a failed clinical trial, someday it may be possible to bring brain
imaging into routine clinical practice to guide drug dosage and choice. However, more classical methods such as neuroendocrine testing or
examining the effect of treatments on peripheral processes such as changes in saliva, serum, and blood cells still can be valuable at various stages of
drug development.

Clinical trials abound in psychiatry. Good clinical trials are much more rare. Problems in trial design, identification of appropriate patients,
recruitment, retention, and ethical issues surrounding the use of placebos are very much with us and show signs of becoming more rather than less
intractable in the near future. The cost of clinical trials in Western countries has grown enormously, leading to fewer and smaller trials that are often
market-driven rather than designed to answer the most important research questions. Kane describes a number of efforts that have been made to
improve clinical trial design and to cope with the increasing limitations that current ethical viewpoints have placed on this process. It is a sign of
progress that broader outcome measures other than global psychopathology are increasingly the focus of clinical trials. For example, the recognition
that cognition may be a more important endpoint than the reduction of positive or negative symptoms in the evaluation of a new drug for
schizophrenia is an enormous advance because it refocuses the goal of new drug development and allows for distinguishing between new and
existing drugs on a much more meaningful basis.

Pharmacokinetics, pharmacodynamics, and drug disposition in relation to new drug development and their subsequent utilization have never been
better summarized than they are in the chapter by Greenblatt and colleagues. Advances in this area yield the information needed to use drugs wisely.
This area of research has matured to the point where the fundamental principles are well understood and can be readily incorporated into the
processes of drug development and utilization. Information about drug interactions that affect efficacy, elimination, and toxicity are ever more
essential in the current area of polypharmacy.

Özdemir and colleagues discuss pharmacogenetics, the field that explores individual differences in drug responses that depend on genetic factors and
genetic-environmental interactions. A key part of this refers to genetic variations in the liver enzymes that metabolize drugs. Greenblatt and
colleagues consider this as well as the genetic factors that determine pharmacodynamics, and thus directly impact on efficacy and side effects. It is
clear that this is critically important to psychopharmacology and will become even more so with the completion of the Human Genome Project. They
also consider how genomic research will play an increasingly important role in drug discovery and development, including the design of safer and
more efficient clinical trials. Their term "personalized therapeutics" is provocative. Have doctors not tried to do this since time immemorial? Genetic
information will aid the process to be more science than art (rather than the reverse).

Is medicine in need of new ethical compasses in clinical research? This would seem to be the view of the authors of the United States-based National
Bioethics Advisory Commission (NBAC) or the newest version of the Declaration of Helsinki. NBAC, in particular, has singled out research on the
mentally ill for more stringent regulation and unique standards. The chapter on ethical aspects of neuropsychiatric research by Pinals and Appelbaum
thoughtfully analyzes these recommendations as well as the fundamental principles that should guide policy in this area. Professional societies such
as the ACNP have developed guidelines for investigators in an effort to show that there is an awareness of the obligation to protect subjects who
agree to research to the greatest possible extent with minimal diminution of the information to be gained from the study. A "safe" study from which
we can learn little or nothing of use because of design flaws may be less ethical than others where the absolute risk may be greater, but still within
acceptable limits, whereas the potential gain in knowledge is far higher. Institutional review boards (IRB) are becoming increasingly restrictive
around clinical research with the mentally ill, based in part on a poor understanding of the intactness of their decisional capacities. It is imperative
that methods to assure competence to give consent that meets the legitimate concerns of IRBs are employed in all trials.

Paul Leber, formerly head of the Division of Psychopharmacology of the United States Food and Drug Administration (FDA), explicates the policies
of his former employer with the oratorical flourish he is renowned for now transmuted into the written word with equal elegance. This chapter
 P.443

explains what was (and probably still is) guiding the policies of the regulators of the FDA, which has worldwide influence directly and indirectly.
This chapter can serve as a primer on getting a new drug application approved by that agency.

Mahmoud and colleagues succinctly and clearly describe the process of evaluating treatment outcomes utilizing the Economic, Clinical, Humanistic
outcomes (ECHO) model. These three perspectives on outcome are intimately intertwined. Trouble arises when any one of the aspects is
overemphasized to the neglect of the others. There has been increasing awareness of the need for societal consensus on the importance of outcomes
as the cost of achieving the best outcomes now possible has risen greatly as a percentage of gross national product in both developing and developed
countries. Advances in medical research will surely suffer if there is insufficient attention to demonstrating that new therapies are more cost-effective,
not just more effective, than previous methods. The critical issue of distinguishing between efficacy and effectiveness research and the need for both
are thoroughly discussed.

In conclusion, this section on new drug development and clinical research issues should be of great importance to any reader who is interested in the

broader picture of alleviating neuropsychiatric disease burden through psychopharmacology.

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 P.445

33
The Role of Preclinical Models in the Development of Psychotropic
Drugs

Mark A. Geyer

Athina Markou

Mark A. Geyer: Department of Psychiatry, School of Medicine, University of California, San Diego, La Jolla, California.

Athina Markou: Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California.

This chapter critically discusses how preclinical models, primarily animal models, can be used in neurobiological research to
promote the development of psychotropic drugs as therapeutics for psychiatric disorders. The authors' previous chapter in
Neuropsychopharmacology: The Fourth Generation of Progress (1 ) extensively discussed the process of developing, validating, and
working with animal models relevant to psychiatric disorders. Various approaches to model development and validation criteria for
animal models were defined and evaluated. These basic principles of model development and validation were further elaborated in
the context of reviewing animal models of depression and schizophrenia. The present chapter is intended as a continuation and
addition to the previous chapter. Thus, assuming the fundamental principles of model development and validation established in the
previous chapter and briefly reviewed here, the present chapter focuses on additional aspects of model development, validation,
and use that need to be taken into consideration when using models as aids to the development of therapeutic approaches for
psychiatric disorders. These principles are clarified further by discussing a few exemplary issues relating to animal models used in
the study of depression, schizophrenia, and anxiety. Although the development of pharmacologic treatments for psychiatric
disorders is typically the major focus, the same basic principles of model use also can be applied in the development of
nonpharmacologic therapeutics for these disorders.

 DEFINITION OF A PRECLINICAL MODEL

 PURPOSES OF A PRECLINICAL MODEL
 NECESSARY AND SUFFICIENT CRITERIA FOR EVALUATING PRECLINICAL MODELS
 APPROACHES AND ISSUES RELATED TO MODEL DEVELOPMENT
 DRUG DISCOVERY AND DEVELOPMENT: PRECLINICAL MODELS AND CLINICAL TRIALS
 ACKNOWLEDGMENTS

DEFINITION OF A PRECLINICAL MODEL

Part of "33 - The Role of Preclinical Models in the Development of Psychotropic Drugs "

A model is defined as any experimental preparation developed for the purpose of studying a condition in the same or different
species. A model is comprised of both the independent variable (i.e., inducing manipulation) and the dependent variable (i.e., the
measure[s] used to assess the effects of the manipulation). The choice of the inducing manipulation is usually based on hypotheses
about the etiology of the disorder of interest or nontheoretic exploratory attempts to induce the abnormality (as reflected in the
dependent measures) that is considered relevant to the psychiatric disorder of interest. Pathologies having homology with those in
humans can be induced in animals more readily if the etiology of the disease is known. Unfortunately, the etiologies of psychiatric
diseases are largely unknown, making the choice of the independent variable particularly difficult. The choice of the dependent
measures is usually based on operational definitions of abnormalities believed to be pathognomonic, or at least symptomatic, of the
disorder of interest. As with the inducing manipulations, the selection of diagnostic criteria and determination of the core features
of a psychiatric disorder are also debatable. Thus, the selection of both the inducing manipulations and dependent measures that
comprise a model of a psychiatric disorder are based largely on theoretic arguments regarding both the etiology and core aspects of
the disorder. The choice of dependent variables is somewhat easier than the choice of the inducing manipulation because it can be
based on operational definitions of observable aspects of the disease, even if the chosen measure is not a core symptom of the
disorder.

Preclinical models could involve either human or nonhuman experimental preparations. Typically, models are nonhuman animal
preparations that attempt to mimic a human condition, including human psychopathology. Nevertheless, as implied in the definition
of a model provided above, preclinical models could also be human experimental preparations. Whether a human or a nonhuman
model should be used depends largely on the purpose of the model and the experimental question of interest (see the following).
The vast majority of preclinical models in use are nonhuman because such models provide two distinct advantages over
 P.446

human preclinical models. First, nonhuman models enable the investigation of the neurobiology of the phenomena of interest using
invasive techniques that cannot be used in humans. Second, if used properly, nonhuman animal models can significantly reduce the
cost of drug development by increasing (or decreasing) the degree of confidence in a particular pharmacologic approach before
undertaking expensive and time-consuming clinical trials in the psychiatric population of interest. Nevertheless, it should be
clarified that human preclinical models can also contribute importantly to this latter goal, if used properly (see the following).

PURPOSES OF A PRECLINICAL MODEL

Part of "33 - The Role of Preclinical Models in the Development of Psychotropic Drugs "

In developing and assessing an animal model, it is imperative to consider the explicit purpose intended for the model (2 ), because
the intended purpose determines the criteria that the model must satisfy to establish its validity and utility. For example, is the
purpose of the experimental system to model specific signs and symptoms or to model the entire diagnostic syndrome? Is the
purpose of the model to promote our basic understanding of the neurobiological, genetic, environmental, and other factors that
contribute to a mental disorder or the development of therapeutic agents for this disorder? Is the purpose of the model to rapidly
and efficiently screen compounds to identify drugs that may have similar therapeutic properties to an existing class of compounds,
or the identification of therapeutic targets that may lead to the development of compounds having novel mechanisms of action? The
preceding are just a few general examples of the various purposes that a model may be intended to fulfill. Such purposes and uses
explicitly guide the development and validation process for a particular model. Following, the necessary and sufficient criteria for
evaluating preclinical models are reviewed briefly. (See refs. 1 and 3 ,4 ,5 ,6 and 7 for more extensive discussions.) Then, some
general issues about preclinical models are discussed that also relate to the premise that the intended purpose of a model
determines the validation criteria that the model must satisfy.

NECESSARY AND SUFFICIENT CRITERIA FOR EVALUATING PRECLINICAL

MODELS
Part of "33 - The Role of Preclinical Models in the Development of Psychotropic Drugs "

The validity of a model refers to the extent to which a model is useful for a given purpose. Thus, depending on the desired purpose
of the test that one wishes to validate, different types of validity are relevant. Further, in considering the validity of a model, both
the independent and dependent variables need to be evaluated. The reliability and predictive validity of the model system are
relevant to both the independent variable and dependent measures and are the most important criteria to satisfy. (See refs. 1 and
8 ,9 and 10 for definitions of the various types of validity.) The additional criteria relevant to the independent variable (i.e.,
inducing manipulation) include etiologic, construct, and face validity, with etiologic validity being the most relevant. The criteria
relevant to the dependent variable include construct, convergent, discriminant, and face validity. Undoubtedly, the more types of
validity a model satisfies, the greater its value, utility, and relevance to the human condition. Nevertheless, it could be considered
circular logic if a model was required to satisfy all types of validity before being considered useful. Hence, it has been argued
previously that predictive validity and reliability are the only necessary and sufficient criteria for the initial evaluation of any
animal model (1 ).

Predictive validity of a model is broadly defined as the ability to make accurate predictions about the human phenomenon of
interest based on the performance of the model (1 ,9 ). In reference to animal models of human psychopathology, the term
predictive validity is often used in a narrow sense to refer to the model's ability to identify drugs with potential therapeutic value in
humans (i.e., pharmacologic isomorphism) (2 ). Although correct, this use of the term is limited because it ignores other important
ways in which a model can lead to successful predictions (7 ). For example, the identification of any variables that have similar
influences in both the experimental preparation and modeled phenomenon can demonstrate predictive value of the experimental
preparation and enhance one's understanding of the phenomenon.

Reliability refers to the consistency and stability with which the variables of interest are observed, and is relevant to both the
independent and dependent variables (1 ,5 ).

APPROACHES AND ISSUES RELATED TO MODEL DEVELOPMENT

Part of "33 - The Role of Preclinical Models in the Development of Psychotropic Drugs "

Modeling Specific Signs or Symptoms Versus Modeling Diagnostic Syndromes

As discussed previously (1 ), early attempts at model development focused on reproducing in animals a psychiatric syndrome in its
entirety. Such an approach, although useful in advancing the field at the time, has been largely abandoned because of increasing
awareness that such an approach is typically impractical, unrealistic, and fruitless for the following reasons. (a) The defining
symptoms of psychiatric disorders and even the diagnostic categories themselves are being revised and redefined continuously. (b)
One would not expect homology on all aspects of a disorder between two species (e.g., one would not expect a rodent to exhibit a
complete schizophrenia syndrome). (c) Modeling a syndrome in its entirety would require the validation of multiple endpoints. (d)
There is considerable heterogeneity within each of the major diagnostic categories of psychiatric disorders. (e) The validation
process for such a model needs to be extensive, thorough, and all-inclusive, and thus not different from the scientific process aimed
at elucidating
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the neurobiological and behavioral mechanisms mediating a psychiatric disorder.

Most recent approaches to the development of animal models rely on mimicking only specific signs or symptoms associated with
psychopathologic conditions, rather than mimicking an entire syndrome. These specific signs or symptoms may be: (a) observables
that have been identified in psychiatric populations that may or may not be pathognomonic for or even diagnostic symptoms of the
disorder, but can be defined objectively and measured reliably; or (b) more theoretically based measures of psychological constructs
that are believed to be relevant to the psychiatric disorder under investigation (2 ,7 ). The latter approach involves the definition of
a hypothetical construct and subsequent establishment of operational definitions suitable for the experimental testing of the
validity of the construct in both human and nonhuman animals. The narrow focus of this approach generally leads to pragmatic
advantages in the conduct of mechanistic studies addressing the neurobiological substrates of the specific behavior under study.
Furthermore, the study of putatively homologous behaviors in both human and nonhuman subjects effectively addresses and
bypasses the nonconstructive criticism that complex mental disorders cannot possibly be modeled in nonhuman animals. (See ref. 1
for a more extensive discussion comparing these two approaches to modeling.)

An illustrative example of this approach is provided by some of the models now being used to identify antipsychotic drugs, based on
the hypothesis that schizophrenia involves deficits in attentional filtering or gating (i.e., the psychological construct). Theoretically,
schizophrenia patients suffer from impairments in filtering or gating of sensory stimuli that lead to an inundation of information and
consequent cognitive fragmentation. The hypothetical construct of attentional filtering has been defined operationally and explored
in multiple paradigms and in both human and animal studies. For example, numerous studies of schizophrenia patients have
demonstrated deficits in behavioral habituation, which is a prerequisite to selective attention, prepulse inhibition (PPI) of startle, a
preattentional sensorimotor gating phenomenon, and the gating of auditory P50 event-related potentials (ERPs) (11 ,12 and 13 )
(see Chapter 51 ). Each of these operational measures is potentially relevant to the construct of deficient filtering of incoming
information, hypothesized to be a common element in the schizophrenia disorders (14 ,15 ). Each of these operational measures is
also amenable to cross-species studies of analogous or homologous behaviors. (See the following for a discussion of these terms.)
The fact that schizophrenia patients exhibit deficits in all three measures provides converging support for the hypothesis that
schizophrenia involves disturbances in the filtering of sensory and cognitive information (i.e., construct validity). Nevertheless, a
recent study explicitly testing the convergent validity of this hypothetical construct has prompted some further refinements in our
thinking. Specifically, in a group of normal subjects, P50 gating was strongly correlated with the amount of startle habituation and
only weakly with PPI (16 ), despite the fact that P50 gating appears to be more similar phenomenologically to PPI than habituation.
Similar findings have been reported in the parallel animal paradigms using the same operational measures (17 ). This situation
illustrates how phenomenologic similarity (i.e., face validity) can sometimes lead to erroneous conclusions until further detailed
behavioral investigations of the construct(s) are undertaken. Furthermore, as reviewed elsewhere (see Chapter 50 ), habituation,
PPI, and P50 gating exhibit some differences as well as similarities in their sensitivity to pharmacologic manipulations used to mimic
schizophrenia-like changes in animals. Of critical importance is the relationship, if any, between these experimental measures of
filtering deficits and clinical complaints of sensory overload or signs of thought disorder that prompted the original hypothetical
construct (i.e., extrapolation from animals to humans). Surprisingly, within a cohort of schizophrenia patients (19 ), those with
deficient P50 sensory gating reported fewer complaints of sensory overload than did patients with normal P50 gating (i.e., the
opposite of the predicted relationship). With regard to the PPI sensorimotor gating measure, however, significant correlations have
been observed between deficient PPI, and both distractibility (20 ) and measures of thought disorder based on an abstract problem-
solving task (21 ) in schizophrenia patients. Hence, it appears that the three main operational measures of deficient attentional
filtering do not all measure the same hypothetical construct. Thus, in parallel with the heterogeneous group of schizophrenia-like
disorders, the construct of deficient filtering may not be a unitary construct, although it could still represent a phenomenologically
common outcome of differing etiologies in different forms of schizophrenia. It is important to recognize that each of these measures
is demonstrably affected in (presumably heterogeneous) groups of schizophrenia patients and each has engendered animal models
that have varying degrees of predictive validity for the identification of antipsychotic treatments. It remains to be seen whether
different subgroups of schizophrenia patients will exhibit only one or another of these deficits. If so, the parsing of the original
hypothetical construct may lead to empirical distinctions among patient subgroups that could have important implications for the
application of specific treatment approaches.

Discovery of Novel Versus “Me-Too” Treatments

Another extreme approach to model development and use relates to the limited purpose of systematically and efficiently screening
and identifying potential therapeutic treatments without explicitly assessing the mode of action that leads to the therapeutic effect.
In such a case, the model may or may not mimic the actual psychiatric disorder.
 P.448

Rather, the model is only intended to reflect the efficacy of known therapeutic agents, and consequently lead to the discovery of
new pharmacotherapies. Thus, the principle guiding this approach has been termed “pharmacologic isomorphism” (2 ). As discussed
elsewhere (2 ,7 ), the fact that such models are developed and validated by reference to the effects of known therapeutic drugs
frequently limits their ability to identify new drugs having novel mechanisms of action. Accordingly, an inherent limitation of this
approach is that it is not designed to identify new therapeutics that may treat either the symptoms of the disorder that are
refractory to current treatments, or patient populations that are resistant to current treatments. An example of such a limitation is
found in the use of drug-discrimination paradigms used to identify new treatment compounds. In these paradigms, the animal is
trained to recognize the drug state induced by a prototypical drug. Typically, the animal is required to press either the right or left
of two levers, depending on whether it had been treated with the vehicle or training drug. Potential new therapeutics are then
identified by their ability to substitute for the prototypical drug on which the animal was trained. Because these paradigms rely only
on the subjective drug-induced cue to which each animal responds and not on an endpoint that can be validated by reference to
other behaviors in animals or humans, such procedures can only identify drugs having a similar effect on some unknowable
dimension. If the screening involves several paradigms, the profile of the drug can be compared qualitatively and quantitatively to
the profiles of known compounds. Such profiles, when combined with “a special kind of flair for the problem” (22 ), may lead to
reasonable predictions about the potential of the compound in the clinic. The ability to rapidly and efficiently identify treatments
that may be shown clinically to have some advantages over the older treatments is an advantage of this approach. Nevertheless,
these screening paradigms do not provide ways to predict whether the “me-too” drug will have any clinical advantages (e.g., fewer
side effects, treatment of refractory symptoms or patient populations) over the “prototypes,” other than in relation to potency.

Modeling Specific Aspects of Treatment Effects: Chronic Versus Acute Drug

Treatments
Because both the etiologies and core features of psychiatric disorders are still poorly understood, much research addressing the
neurobiology of these disorders has focused on the study of the mode of action of known therapeutics. The targets of clinically
effective therapeutics have provided excellent starting points in the investigation of the neurobiology of psychiatric disorders. When
taking this approach, it is important that specific aspects of the treatment effects are taken into consideration and incorporated
into the paradigms used. For example, it is recognized that chronic treatment with antidepressants is required before a therapeutic
effect is observed. This therapeutic delay is not only a severe limitation of current antidepressant treatments but also a hurdle in
determining the mechanisms through which antidepressants produce their beneficial effects. Because of this delay in the emergence
of the therapeutic effects, it is assumed that these effects are mediated by neuroadaptations that develop as a result of the chronic
drug administration. Much research has focused on these neuroadaptations in order to understand the neurobiology of depression;
because the therapeutic effect may be produced through “normalization” of the specific abnormalities characterizing depression. It
is possible, however, that the therapeutic effect may be produced by separate systems or mechanisms that counteract the
abnormalities that are etiologic in depression.

The preceding discussion is relevant, not only to approaches that may be taken in studying psychiatric disorders, but also to the
question of whether animal paradigms that demonstrate positive results after acute administration of an established antidepressant
are indeed valid models of depression rather than just screening paradigms. It could be argued that with acute drug administration
the mechanisms leading to the reversal of the behavioral deficit are not the same as the ones leading to the clinical therapeutic
effect. Such arguments certainly have merit. An animal paradigm that not only indicates therapeutic efficacy but also the time-
course of such effects is a powerful tool for both neurobiological investigations and drug discovery. The vast majority of animal
models of depression do not readily satisfy this criterion despite extensive efforts over decades. Thus, the question is how to best
design and interpret data from paradigms that appear to reveal primarily acute therapeutic effects. In many of the animal studies,
the acute drug doses are much higher than doses that would be tolerated by humans, especially on the first drug administration.
Higher doses may be more likely to produce an acute effect. This argument is supported by data with the forced swim model where
it was shown that either high doses of antidepressant drugs or chronic treatment with low doses of antidepressants, ineffective
when administered acutely, reversed immobility in the swim test (23 ). Further, it has been argued that antidepressants may
produce immediate improvement of some symptoms in humans, but this acute effect may be hard to detect statistically because the
initial improvement may be small and seen only in some, but not all, symptoms (23 ,24 ). Thus, it is possible that reversal of a
specific behavioral deficit in a model after acute treatment may indeed be consistent with the clinical reality about a specific
symptom. This experimental question is an example of a case in which preclinical animal data could guide the design of clinical
investigations that would help assess and improve animal models. Finally, animal models that can only detect acute effects, when
guided by good working hypotheses, can be used for target identification by investigating the mechanisms
 P.449

that lead to reversal or exacerbation of the deficit of interest.

Issues Regarding the Use of “Normal” (Healthy) Versus Perturbed Animals

Although most animal models rely on an explicit inducing manipulation, some models test the effects of putative therapeutic
compounds under baseline conditions, that is, without first inducing a deficit in the subjects. Even if such an animal model has
predictive validity, it may not be useful in furthering our understanding of the pathophysiology underlying the disorder if the effects
of therapeutic treatments depend on an interaction with the underlying pathophysiology. Specifically, the mechanisms through
which drugs produce their effects in “normal” versus perturbed animals may differ, even if the primary neurochemical effect may
be the same (25 ,26 ). Indeed, antidepressant drugs have been shown in some studies to have no effects in “normal” human or
nonhuman subjects, whereas inducing “therapeutic” effects in patients or perturbed subjects. For example, fluoxetine, a selective
serotonin reuptake inhibitor (SSRI) with antidepressant properties, does not induce euphoria or elevate mood in nondepressed
healthy individuals even after chronic treatment (27 ,28 ). Similarly, treatment with tricyclic antidepressants or SSRIs has no effect
on behaviors assessing reward function in unperturbed animals. More specifically, treatments with desmethylimipramine, a tricyclic
antidepressant, or low doses of fluoxetine typically have no effect on intracranial self-stimulation reward thresholds
(29 ,30 ,31 ,32 ,33 ,34 ,35 ,36 and 37 ), whereas reversing reward deficits observed after drug withdrawal or during chronic mild
stress (31 ,32 ,38 ). Moreover, recent findings indicated that the coadministration of fluoxetine together with a relatively selective
serotonin 1A receptor antagonist had opposite effects in “normal” rats (i.e., decreased reward), whereas reversing reward deficits
(i.e., increased reward) in perturbed animals in which a reward deficit had been induced (34 ). Recent clinical and preclinical
findings have suggested that the coadministration of a SSRI together with a serotonin 1A receptor antagonist leads to accelerated or
augmented antidepressant effects compared to those seen after treatment with the SSRI alone, presumably by enhancing
serotonergic neurotransmission to levels above those seen with the SSRI alone (39 ,40 and 41 ). In conclusion, the study of animals
that exhibit a deficit that is pathognomonic of depression, rather than “normal” healthy animals, may be critical to the study of
both the underlying pathophysiology and its treatment.

In other situations, however, it may be advantageous to utilize animal models that examine baseline behaviors. For example, known
antipsychotic drugs can be identified with reasonable predictive power using the conditioned avoidance response paradigm (42 ).
The conditioned avoidance response paradigm has then been applied to the testing of potentially novel mechanisms that may have
efficacy in the treatment of psychosis (43 ). Latent inhibition is another paradigm in which antipsychotics produce changes in
baseline behavior that are relevant to schizophrenia. Acutely ill schizophrenia patients exhibit deficits in latent inhibition that are
reduced by antipsychotic treatment (44 ). Similarly, when the appropriate testing parameters are used in the analogous animal
paradigm, both typical and atypical antipsychotics improve measures of latent inhibition under baseline conditions (45 ). In contrast
to pharmacologically induced models (see the following), models such as the conditioned avoidance response and latent inhibition,
in which known antipsychotics influence behaviors under baseline conditions, may be more effective in identifying new therapeutic
targets for antipsychotic effects. As discussed elsewhere (see Chapter 50 ), most of the schizophrenia animal models used
historically to identify antipsychotic agents have relied on the induction of abnormal behaviors by the administration of a
dopaminergic agonist and then define an antipsychotic as a drug that reverses the agonist effect. Hence, most such models are
primarily and perhaps exclusively sensitive to drugs that block dopamine receptors and may not detect novel mechanisms that could
have efficacy without involving dopamine receptor blockade. Ultimately, only further research with each class of psychiatric
treatments will determine the relative utility of models that use an explicit inducing condition versus models that rely on changes in
baseline behavior.

Inducing Conditions: Drug-Induced Versus Nonpharmacologic and Genetic Models

With the exception of paradigms assessing treatment effects in nonperturbed animals, the inducing manipulations constitute a
critical aspect of a model. The selection of inducing conditions for animal models of psychiatric disorders is difficult because the
etiologies of psychiatric disorders are generally unknown and are likely to be heterogeneous within each diagnostic category.
Inducing conditions could be environmental manipulations, drug manipulations, lesions, genetic manipulations, or combinations of
the preceding. Further, all of the preceding manipulations could be implemented during development or combined with
developmental manipulations or factors. An inducing condition may be selected: (a) based on theoretic arguments about the
environmental and/or neurobiological factors that lead to the disorder; (b) because it induces a deficit that is considered
pathognomonic of the disorder of interest, even though no theoretic arguments are made about the etiology of the disorder; or (c)
based on purely practical considerations about the predictive value of the model without theoretic arguments about either the
etiology of the disorder or the relevance of the dependent measure to aspects of the symptomatology characterizing the disorder.
The selection of
 P.450

each inducing condition has advantages and disadvantages and is often based on the following considerations.

Acute or chronic drug manipulations have the advantage of probing the function of a specific receptor or neurotransmitter system
that either is implicated in the etiology of the disorder or produces the desired deficit. The main disadvantage of an acute drug
manipulation, and often of chronic drug manipulations, is that it readily leads to “receptor” or “neurotransmitter tautology.” For
example, a deficit induced by a specific receptor agonist is very likely to be reversed most effectively by a receptor antagonist at
the same receptor, as in the case of dopamine agonist–antagonist interactions in most animal models of antipsychotic action. The
same applies to neurotransmitter systems. Nevertheless, reversal of a drug-induced deficit by a compound acting on a different
neurotransmitter system is a powerful indication of system interactions that may be relevant to the pathophysiology and/or
treatment of the disorder. Chronic drug manipulations offer additional advantages and disadvantages. Chronic drug administration is
likely to lead to compensatory adaptations to the acute effects of the drug that are likely to be longer lasting than the effects of a
single drug administration and to involve additional systems that are not involved in the acute drug effects. Nevertheless, the
resulting neuroadaptations may be irrelevant to the disorder unless there is a relationship between the deficits induced by the drug
and etiology of the psychiatric symptoms.

An example of a pharmacologic model is the use of the reward deficits seen during withdrawal from a variety of drugs of abuse as a
model of the core symptom of “diminished interest or pleasure” in rewarding stimuli that characterizes depression (46 ). In rats,
converging evidence indicates that withdrawal from psychostimulant drugs is associated with reward deficits expressed as elevations
in brain reward thresholds (33 ,47 ), decreased breaking-points under a progressive ratio schedule for a sucrose reinforcer (48 ), and
decrements in motivation for sexual reinforcement (49 ). The advantage of this model is the induction of deficits in reward and
motivational processes that are hypothesized to be, not only pathognomonic of depression, but also deficits expressed as negative
symptoms of schizophrenia. Thus, these paradigms focus on the study of a hypothetical construct that may have relevance to core
symptoms seen in multiple diagnostic categories. These deficits are most likely homologous to similar deficits seen in people abusing
these drugs because the etiology of the deficit is the same in both the animal model and humans. Nevertheless, it is not known
whether pharmacologically induced deficits in reward and motivational processes are homologous, or just analogous, to similar
deficits seen in nondrug abusing psychiatric populations. That treatments with clinically effective antidepressants reverse the drug-
induced reward deficits in both rats and humans suggests that the deficits may be homologous across species (32 ,34 ,38 ,50 ).

Environmental manipulations often induce only short-lasting deficits in healthy subjects because a healthy system is able to “bounce
back” readily once the inducing conditions have been removed. Potential interactions, however, between the environmental
manipulation and a genetic predisposition may lead to long-lasting behavioral or neurobiological changes having relevance to the
disorder of interest. Finally, environmental manipulations are important to use and incorporate into animal models because it
appears that psychiatric disorders often result from interactions between “nature” and “nurture” to a larger extent than most
nonpsychiatric diseases. Another advantage of environmental manipulations is that such manipulations are likely to affect integrated
brain functions rather than a single component of a system.

Lesion manipulations offer different advantages and disadvantages compared to environmental and drug manipulations. An
advantage of lesion manipulations over chronic drug manipulations is that lesions may lead to deficits and/or neuroadaptations in a
variety of brain systems rather than just the one or few affected by a drug. A disadvantage of traditional lesion manipulations is that
the initial lesion manipulation in most cases is a rather large insult to a specific brain site. Thus, the circuitry affected is dependent
on the interconnections of this specific brain site. Nevertheless, recent advances in genetic techniques are allowing very precise
“lesions” (knockouts) or increased expression (knockins) of specific proteins in selected brain sites in adult animals. Such
technological advances, when combined with more traditional behavioral and pharmacologic aspects of well-developed models, are
likely to advance our understanding of psychiatric disorders.

Developmental manipulations are gaining in popularity primarily because there is increased awareness that many psychiatric
disorders develop gradually through childhood and adolescence and are lifelong. In some cases, investigators combine one of the
previously discussed inducing manipulations with a developmental manipulation (e.g., applying the inducing manipulations during
development or in a genetically altered animal). For example, decreases in PPI of startle, an operational measure of sensorimotor
gating deficits that are evident in patients with schizophrenia, have been demonstrated to result from socially isolating rats from
weaning until after puberty (51 ). Social isolation of rats in early stages of development has been used to produce a variety of
behavioral abnormalities that have been related to both schizophrenia and depression. Recent studies have shown that 6 to 8 weeks
of social isolation during development, but not during adulthood (52 ), produces deficits in PPI that are at least partially reversible
by the administration of neuroleptic dopamine antagonists (51 ) or by clinically effective atypical antipsychotics having antagonist
activity at multiple receptors (53 ,54 ). Furthermore, postweaning isolation rearing of rats also results in deficits in the gating of the
N40 event-related potential, that are analogous to the deficits in P50 gating observed in schizophrenia (55 ).
 P.451

Because schizophrenia commonly emerges in early adulthood, developmental factors have provided the basis for some etiologic
hypotheses (56 ,57 ). Hence, further study of the gating deficits produced by isolation rearing of rats may establish a
nonpharmacologic and developmentally relevant animal model of the gating deficits observed in patients with schizophrenia.
Potentially, in contrast to the drug-induced models of gating deficits, such a model might have etiologic validity and might be
sensitive to antipsychotic drugs having novel mechanisms of action.

Genetic manipulations are popular because of the recent surge of interest in genetic contributions to psychiatric disorders. Such
interest promises to enable the development of a class of animal models based on hypothesized etiologic validity. As specific genes
and gene products become linked to specific disorders, molecular biologists will be able to generate mutant or transgenic animals
having genetic abnormalities that are potentially homologous to those seen in humans. Behavioral and pharmacologic studies of
these genetically engineered animals will then be important in identifying the phenotypic changes associated with the mutation,
testing hypotheses about the etiology of the disease, and exploring potential therapeutic treatments. The combination of genetic
and molecular biological approaches with behavioral and pharmacologic approaches may well revitalize interest in etiologically
based models of psychiatric disorders. It is important to recognize that genetic manipulations necessarily begin with the fetus and
often lead to compensatory adaptations throughout the course of development. Hence, developmental factors must be taken into
account and studied when working with such an early genetic alteration. The latter is an example of a case where a technological
limitation can lead to new creative ways of studying the function of a system and how it may contribute to our understanding of the
processes mediating a disease.

The increased use of strain differences and genetically engineered mutants in drug discovery programs will necessitate both
practical and conceptual modifications to the development and validation of animal models. Among the most fundamental
differences between these genetic models and most previous models involves the distinction between trait and state measures. Most
of the traditional models used to explore psychiatric treatments have relied on relatively short-term changes in the state of the
animal, as modified by inducing manipulations such as stressors or drugs. In contrast, by definition, genetically based models rely on
traits rather than states. For example, it has become commonplace to use approach/avoidance conflict tests to examine the
possibility that gene knockout mice exhibit alterations in what is called “anxiety.” Approach/avoidance conflict tests, such as the
elevated plus-maze or the light/dark box, have been widely used in rodent studies of anxiolytic drugs. Anxiolytic drugs increase
approach behavior in such paradigms, presumably because they reduce the anxiety that competes with the animal's tendency to
explore novel stimuli and environments. Such an observation with an unknown drug could as readily be interpreted as an increase in
novelty seeking (i.e., approach) rather than a decrease in anxiety (i.e., avoidance). The fact that known anxiolytic drugs increase
approach behavior has provided substantive validation of approach/avoidance conflict tests for the identification of changes in state
anxiety. Accordingly, such conflict tests are now being used widely in the characterization of mutant mice in attempts to identify
changes in trait anxiety. It should be recognized, however, that the validation of a measure as predictive of a change in state may
or may not validate the measure as reflective of a change in the conceptually related trait. That is, the observation of a shift in
approach/avoidance behavior in a mutant mouse that is similar to that produced by an anxiolytic drug cannot readily support the
conclusion that the mutant mouse exhibits low levels of trait anxiety rather than high levels of approach behavior, as in the trait of
high novelty seeking. Only by examining approach/avoidance behavior across a range of contexts can one determine which pole of
the approach/avoidance conflict is altered in the mutant animal (58 ).

Dependent Measures: Value of Analogous and Homologous Measures Across Species

As with the choice of inducing manipulations, the choice of dependent measures is not simple when developing animal models of
psychiatric disorders, primarily because the major and core features of human psychopathology are still poorly understood and still
debatable. Thus, what should be considered an adequate or appropriate endpoint for a model in psychiatry is not always clear.
Whenever possible, it is preferable to work with homologous rather than analogous endpoints. The terms analogy and homology
originated in comparative anatomy and refer to the morphology and function of a structure. Structures or behaviors across species
that are similar in origin (i.e., neurosubstrates), form, and function are termed homologous, whereas structures or behaviors that
have different origins or neurosubstrates, superficially similar form, and have similar function are termed analogous (59 ). Another
term that has been used to refer to analogous endpoints is isomorphism (60 ). Thus, in some sense, the terms homology and analogy
refer to both the symptomatology and the underlying substrates that relate to the etiology. Although homologous measures are
preferable, they are rare. Fortunately, analogous measures can also be valuable. It is because of the assumption of homology, or at
least analogy, among the physiological and behavioral characteristics of various species that extrapolations can be made from
nonhuman animals to humans (61 ). The establishment of multiple forms of validation for a particular model provides convergent
evidence in support of the postulate of cross-species homology.

When developing an animal model related to a psychiatric

 P.452

disorder, it is important to determine what features of the disorder(s) the experimental preparation is intended to model.
Investigators often begin by attempting to identify the core features of the particular disorder of interest. Nevertheless, it is clear
that appreciable diversity of both etiologies and symptom profiles exists within each of the major psychiatric diagnostic categories.
Furthermore, very few specific symptoms are unique to any specific diagnosis, but occur in multiple diagnostic categories. Hence, it
is most productive to focus on specific features observed in patients as endpoints for use in the development of animal models,
rather than on clusters of symptoms. A related implication of this reasoning is that multiple different animal models, in terms of the
endpoint used, may all be useful in parallel. Thus, it is advantageous to utilize an array of models rather than rely too heavily on any
one model. The endpoints used in such models could be in vivo behaviors, biological markers, or in vitro behaviors of biological
systems or preparations. Operational definitions, especially of in vivo behavioral measures, assist in determining the theoretic
relationship between the observable and the construct of interest (62 ,63 ). Finally, the observables should be measured objectively
and reliably.

Human Preclinical Models: Relationship between Animal and Human Phenomena

Human preclinical models can also contribute significantly to drug development. Unfortunately, such human models appear to be
underutilized, and relatively little effort is focused on the development of such human models. An advantage of using human
preclinical models is that one would not have to be concerned about cross-species generalizations. Nevertheless, even with human
models, questions regarding the etiology of the disorder or the relationship between the dependent measure and the symptoms still
need to be addressed using the same principles as when extrapolating across species. Relative to animal models, human preclinical
models are necessarily more constrained by the additional ethical considerations regarding the use of humans in research.

The most typical example of a human preclinical model is when a drug-induced state is used in healthy volunteers to mimic some
aspects of a disorder of interest. For example, the glutamate antagonist ketamine is used to induce a state that mimics some
aspects of acute schizophrenia in healthy volunteers (64 ,65 ). Then, using brain imaging, psychological assessments, and
pharmacologic interactions, the neurobiology of this drug-induced state can be studied to gain insight into the possible substrates
underlying the psychotic state in schizophrenia patients. Such a human preclinical model can also play an important role in assessing
novel treatments. For example, it has been found that the atypical antipsychotic clozapine reduces the exacerbation of symptoms in
schizophrenic patients given ketamine (66 ). In contrast, typical antipsychotics such as haloperidol are ineffective in treating
psychotic episodes induced by drugs such as ketamine or phencyclidine (PCP). Hence, studies of ketamine effects in either human or
animal preclinical models may aid in the identification of additional atypical antipsychotics having efficacy in the treatment of
patients who are nonresponsive to typical antipsychotics. Indeed, in the PPI models of schizophrenia, the disruptive effects of
glutamate antagonists on PPI of startle are reversed by atypical, but not by most typical, antipsychotics (67 ). Interestingly, this
effect of clozapine-like antipsychotics is mimicked by the putative antipsychotic M100907, a selective serotonin-2A antagonist (68 ).
In general, one goal of translational research is to utilize the knowledge gained from human preclinical and clinical studies to guide
invasive neurobiological studies in animals, which in turn can be translated back to the human clinical studies. In the present
example, this strategy would suggest that further studies could now determine whether M100907 reverses disruptions in PPI
produced by ketamine in healthy human volunteers. Furthermore, clinical trials of the efficacy of M100907 in schizophrenia could be
designed to test the hypothesis that only schizophrenic patients whose deficits in PPI are reversed acutely by M100907 would
respond clinically to prolonged treatment with M100907. This admittedly speculative example illustrates some of the potential
advantages derived from the use of homologous, or at least analogous, measures in animal and human preclinical models as well as
in clinical trials. Such translational research is needed in the field of psychiatric disorders in order to guide both the refinement of
the animal models and the development of new drugs.

DRUG DISCOVERY AND DEVELOPMENT: PRECLINICAL MODELS AND

CLINICAL TRIALS
Part of "33 - The Role of Preclinical Models in the Development of Psychotropic Drugs "

An emerging belief is that animal preclinical models represent a bottleneck in psychotropic drug discovery (69 ). Compared to high-
speed chemical synthesis, high-throughput screening of libraries of compounds, and rapid gene seeking and sequencing techniques,
the use of preclinical models as screening techniques appears slow. Nevertheless, such preclinical models of human psychopathology
are required to provide initial assessments of the functional effects of novel compounds in the integrated organism. Only such in
vivo functional measures can confirm predictions about the potential effects of psychotropic drugs in patients. It is unrealistic to
attempt to go from the “test tube” to the clinic when attempting to treat complex mental, cognitive, and emotional disturbances
that do not yet have clearly defined neurobiological substrates, or even correlates. The “relative paucity of preclinical behavioral
models predictive of clinical efficacy” (69 ) reflects the paucity of our quantitative measures of the human phenomena related to
psychiatric
 P.453

disorders, as well as the limited investment in the development of animal behavioral models over the past few decades. Despite the
excitement in the field of neuroscience about the recent progress made in understanding brain function (70 ), there is also an
appreciation of how little is known about the neurobiology of psychiatric disorders compared to advances in other fields of medicine
(71 ). Given the rapidity of techniques available to target discovery and drug screening efforts relative to the limited state of our
knowledge about psychiatric disorders, the role of in vivo preclinical models as the intermediary between these extremes needs to
be considered carefully. Some preclinical models, such as the tail suspension or swim tests for antidepressants and the prepulse
inhibition test for antipsychotics, are amenable to relatively rapid screening without knowledge or understanding of the compounds'
mechanism of action. Paradigms that are far more laborious can be used in the identification of new targets through the
investigation of the interacting systems that contribute to the disorder's symptomatology or the therapeutic effects of established
drug treatments. After identification of such novel targets, drug development efforts can be focused in identifying a compound with
the desired mechanism of action and other desired properties, such as no toxicity and limited actions at systems that would produce
side effects. Converging evidence from other basic research efforts would be crucial in such an undertaking. Even though the
previously described process is time consuming and requires well-integrated multidisciplinary research efforts, this process may lead
to the breakthroughs in psychiatric drug development that have been long awaited.

After a candidate drug has been identified through the use of both animal and human preclinical models and safety issues have been
addressed, then the therapeutic efficacy of the compound is tested in the clinical population. Unfortunately, such clinical trials
often do not have sufficient power, in the statistical and experimental design meaning of the term, to detect potentially beneficial
effects of novel candidate compounds. Because of the high cost of drug development, pharmaceutical companies are interested in
pursuing drugs that have the potential to be used in a large market that is often a broadly defined diagnostic category. This situation
is aggravated by the fact that diagnostic categories in psychiatry are still rather crudely defined by rating scales rather than by
objective and quantitative measures. For example, it is often assumed, at least implicitly, that there is diagnostic homogeneity
within a particular patient population. It is also assumed that the boundaries of psychiatric categories as currently defined are
rather absolute. In fact, most psychiatric disorders do not have clear pathological or biological markers and are defined as
constellations of symptoms that are on a continuum with normality (71 ). Even though such diagnostic issues are constantly discussed
and debated among psychiatrists, such issues are often put aside in clinical trials. Typically, the main focus in clinical trials is on the
global measures of remission that are acceptable to regulatory agencies. Unfortunately, the reliance on rating scales in clinical trials
provides little specific information that is useful in guiding either human or animal preclinical studies. Although understandable in
view of economic forces, the infrequent use of a selected battery of scientifically established objective measures even in the early
phases of clinical trials limits the further development of translational research involving cross-species comparisons and model
validation. Hence, clinical trials do not benefit sufficiently from the scientific information provided by academic research and
seldom provide the kind of empiric measures that are needed to adequately validate related animal models. Emphasis on multiple
biological or psychological measures of disease progression with or without treatment with the drug of interest could potentially
provide valuable information about the mechanisms that underlie various aspects or symptoms of the disease and thus lead to
pragmatic advances in our understanding of the neurobiology of psychiatric disorders. Communication from the clinic back to the
preclinical behavioral laboratory will enable the refinement of established models and the creation of new ones. In turn,
understanding of the disorder could benefit if clinical trials included measures suggested to be relevant from preclinical research in
either human or animal models. Another situation that limits progress in drug development is a recent movement to discourage
clinical trials that include a placebo control group. Instead, the new compounds are expected to show greater efficacy compared to
established therapeutics for the particular disorder. Overall, this state of affairs significantly limits the potential to identify new
drugs that may: (a) be more beneficial than established treatments to a subpopulation of patients; or (b) produce global
improvement through amelioration of symptoms that are not adequately assessed by the established measures of efficacy. Thus, it is
difficult to make real advances in the development of new drugs because the current system encourages a circular logic and
approach.

Another limitation of clinical trials that contributes to this circular approach is the absence of use of well validated, objective, and
reliable measures of psychopathology in addition to the available clinical measures. As with animal models, clinical trials also need
to incorporate measures that objectively and reliably assess specific psychological constructs or processes that appear to be altered
in the population of interest. The validation of any animal model can be only as sound as the information available in the relevant
preclinical human literature and the clinical literature (7 ). It is very fruitful when conceptually related experiments are undertaken
in both the relevant patient population and the putative preclinical human and animal models. That is, studies of appropriate
patients are needed to establish the operational definitions of the hypothetical construct, and the construct's relevance to the
particular disorder. In concert, parallel studies of the theoretically homologous construct,
 P.454

process, or dimension are required to determine the similarity of the animal model to the human phenomenon. Development of animal models
requires parallel development of clinical measures that allow meaningful comparisons. Clinical studies need to be informed by results from animal
studies as much as the reverse is true. An important and advantageous aspect of the approach described herein is that the validation of the
hypothetical construct and its cross-species homology can be established by studies of normal humans and animals, in addition to psychiatrically
disordered patients or experimentally manipulated animals. Thus, this approach adds to and benefits from the psychological and neurobiological
literature relevant to the hypothetical construct upon which the model is based. In a sense, this approach explicitly recognizes that the experimental
study of the disorder in humans involves as much of a modeling process as does the study of the disorder in an animal model. Thus, more
translational science is needed to relate animal findings to humans and vice versa.

ACKNOWLEDGMENTS
Part of "33 - The Role of Preclinical Models in the Development of Psychotropic Drugs "

This work was supported by National Institute of Health (NIH) grants DA02925 and MH52885, and the VISN 22 Mental Illness Research, Education, and
Clinical Center to MAG; and NIH grant DA11946, Tobacco-Related Disease Research Program Grant from the State of California 7RT-0004, and a
Novartis research grant to AM. The authors thank Dr. Daniel Hoyer for his comments and input, and Mike Arends for computer and library searches
and editorial assistance. This is publication 13601-NP of The Scripps Research Institute.

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34
Proof of Concept: Functional Models for Drug Development in
Humans
Dean F. Wong

William Z. Potter

James R. Brasic

Dean F. Wong and James R. Brasic: Department of Radiology, Johns Hopkins Medical Institution, Baltimore, Maryland.

William Z. Potter: Lilly Research Laboratories, Indianapolis, Indiana.

A drug developed for human use classically goes through a number of steps, including discovery, extensive preclinical studies for safety in experimental animals, and then
human safety and efficacy studies. The Food and Drug Administration (FDA) requires successful completion of all the above tasks before approval for therapeutic use in
human beings. The exciting advances in human genomics and combinatorial chemistry promise substantial applications to new drugs for human diseases; however, the
time and expense associated with the processes necessary to bring a new drug to market are rising exponentially. Thus, there is a great need for functional models of
disease progression, including animal models of human disease and biomarkers in human clinical trials.

 BIOMARKERS AND SURROGATE MARKERS

 ROLE OF SURROGATES FOR DRUG DEVELOPMENT
 PHARMACOKINETICS
 TOXICOKINETICS
 LIMITATIONS OF CURRENT SURROGATE MARKERS
 IMAGING STUDIES
 ESTIMATION OF DOPAMINE RECEPTOR OCCUPANCY BY ANTIPSYCHOTIC DRUGS
 MAGNETIC RESONANCE SPECTROSCOPY
 MAGNETIC RESONANCE IMAGING
 BIOMARKERS OF ALCOHOL ABUSE
 NEWER MARKERS FOR DRUG DEVELOPMENT
 CONCLUSION
 ACKNOWLEDGMENT

BIOMARKERS AND SURROGATE MARKERS

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

The monitoring of biologic disease processes increasingly employs biomarkers (Table 34.1 ). At a recent National Institutes of Health (NIH) and FDA conference (1 )
biomarkers for clinical efficacy were divided into several groups including natural history markers, biological activity markers, and surrogate markers.

TABLE 34.1.
ABBREVIATIONS
FREQUENTLY
ENCOUNTERED WITH
BIOMARKERS AND
SURROGATE MARKERS

A relevant issue in clinical trials is the selection of an appropriate endpoint. Endpoints that are less deleterious than death or onset of a disease are desirable. Surrogate
markers or endpoints are events of a more intermediate nature. These typically replace the final endpoints such as mortality. A surrogate marker is defined statistically as
a “response variable for which a test of the null hypothesis of no relationship to the treatment groups under comparison is also a valid test of the corresponding null
hypothesis based on the true endpoint” (2 ). A good biomarker, in contrast, provides information on the possible mechanisms of action of medications or the
pathophysiology involved in a disease process. Surrogate markers that are helpful in early drug development (e.g., FDA Phase I and II trials) provide a prognostic indication
of a clinical endpoint relevant to FDA Phase III trials.

Accelerated approval, which has become more common in FDA clinical trials, may occur based on surrogate marker effects although completion of longer-term clinical
outcome and clinical endpoints may eventually be required. Prentice (1989) suggested that a surrogate marker must be both prognostic of disease progression and
affected by treatment (2 ); the effect of the treatment on the surrogate marker should mediate the effect of the treatment on the clinical outcome or true outcome
measure. There are not yet any surrogates for neuropsychiatric disorders that fully meet these criteria.

ROLE OF SURROGATES FOR DRUG DEVELOPMENT

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

The value of surrogates and biomarkers for drug development is recognized by the pharmaceutical industry. The needs to reduce development costs, improve the practical
attainment of predictive outcomes, and expedite these endpoints are motivations for growing interest in this area. We present in the following an overview of the current
state-of-the art regarding utilization of various surrogate markers, in a much broader sense of the term, which includes biomarkers, in drug discovery and development.
We start with the most basic steps pertinent to initial studies in humans.

The integration of preclinical science and assessment of therapeutic potential in humans is done primarily through quantification of drug and active metabolites in
accessible specimens (blood, urine, and cerebrospinal fluid). This is an advance on the maximum tolerated dose (MTD) approach, which necessarily prevailed before
sufficiently sensitive assay
 P.458

methodology was available. In current drug development, upper doses for early human studies are based on toxicokinetic studies in
at least two animal species (most often rat and dog) whereby one does not exceed some predetermined ratio between the exposure
(i.e., plasma concentration) for the dose in humans and the exposure associated with toxicity in the most sensitive species (3 ). Thus,
depending on the ratio selected, exposures in humans may produce few side effects and be below those achieved in the older MTD
approach.

In practice, one often embarks on clinical studies with a constricted range of exposures and an inability to address the question of
whether therapeutic (or biochemical) effects are greater at the MTD until much later in development (4 ). If a compound does not
produce significant side effects within this original range, doses much higher than those used in initial Phase I studies can be tested
depending on the nature of the toxicity observed in animals at the limiting exposure. Even if a limiting dose is identified in
volunteer subjects, a greater dose may be tolerated in patients (5 ). Sramek and colleagues (1997) (6 ) have defined this transition
from dosing in healthy volunteers to patients to be “bridging.” For instance, the difference in tolerated dose for schizophrenic
patients compared with normal volunteers is often greater than 25-fold (5 ). Interestingly, before pharmacokinetic (PK) data were
routinely used to limit exposure, one explored an MTD relatively early and concluded that if response was not achieved by that dose,
one did not have a viable clinical candidate.

Most marketed and late in development psychopharmacologic agents do show limiting side effects above the recommended dose
range, especially if such doses are given initially. Starting with three or more times the lowest standard dose of a selective serotonin
reuptake inhibitor (SSRI) will produce far more marked nausea (and even vomiting) than after building up to the same dose over
several weeks, as is done for the treatment of obsessive–compulsive disorder (OCD). This may seem simply common sense but the
perceived market advantage of the starting and ultimate therapeutic dose being the same discourages exploration of higher doses
that require some sort of titration over time in order to be well tolerated. For instance, even though it was appreciated early on
that an antidepressant, venlafaxine, might be more efficacious at higher doses that were associated with
 P.459

more marked side effects (7 ), it was marketed at target lower doses (37.5 mg b.i.d.), which only produce effects consistent with
serotonin reuptake inhibitory activity. In the over 225 mg per day range (which requires some degree of titration to avoid
unacceptable side effects), venlafaxine produces effects consistent with norepinephrine (NE) as well as serotonin (5HT) uptake
inhibition (8 ,9 ) and is reported to be superior to SSRIs (10 ,11 ). Obviously, it would be beneficial if the relevant dose ranges of
such compounds could be better understood earlier rather than later in the life of a drug.

In this particular example, if valid markers of 5HT and NE uptake inhibition in the brain were investigated as early in development as
possible, then the doses necessary to achieve both effects and those achieving only one could have been systematically pursued
from the outset. Such markers would be “surrogate” markers for clinical doses, allowing one to test the widely held hypothesis that
in a big enough population of patients with depression, one will find those who respond better to combined NE/5HT uptake
inhibitors than to SSRIs alone. Table 34.2 shows examples of biochemical markers that may serve as surrogates.

TABLE 34.2. MEASURES THAT SERVE AS SURROGATES FOR PREDICTED

BIOCHEMICAL EFFECT OF DRUGS TARGETED TO MONOAMINE
NEUROTRANSMITTERS, TRANSPORTERS, RECEPTORS, AND DEGRADATIVE
ENZYMESa

In the section that follows we briefly review how traditional surrogate markers (in the broadest sense), which antedate the
application of brain imaging technology, are used.

PHARMACOKINETICS
Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

Analytical technology, especially that provided by much simpler to operate equipment, allows for precise quantification of very low
concentrations in blood so that rapid and accurate determination of a new compound’s pharmacokinetics (PK) is possible after much
lower doses than required formerly. If a half-life is too short or long or if nonlinear pharmacokinetics are marked (e.g., half-life
increases with doses in the expected therapeutic range), then development may go no further. Similarly, if a compound shows
unacceptably wide variations in clearance because its metabolism is dependent on a highly polymorphic isozyme of cytochrome P-
450 (e.g., CYP2D6), then it will be seen as of lower potential commercial value. Usually, in vitro tests using human hepatic
microsomal preparations or P-450 isozyme specific cloned systems are used to screen for this possibility prior to human use, but
these are not always
 P.460

fully predictive. Thus, concentrations measured over time in Phase I studies serve, at this most basic level, as a surrogate of
whether a compound has the potential to become a successful drug (12 ).

Drug interactions sometimes can constitute a serious safety risk (e.g., inhibition of terfenadine metabolism by CYP3A4 inhibitors
leading to fatal QTc prolongation in genetically susceptible individuals) and, almost always, a marketing disadvantage. The same
preclinical approaches referred to in the preceding are used to screen out compounds that depend for their metabolism on a
problematic pathway. In vivo PK (and sometimes drug interaction data) are also obtained in animals, but more to set dose and
frequency of administration to guarantee adequate concentrations for expensive chronic toxicology studies than to predict what the
PK characterization will be in humans.

Low bioavailability (i.e., concentration after oral versus intravenous doses) is a special problem at this stage of preclinical
development. First, for most compounds an oral form is seen as the ideal (if not only) option and low bioavailability almost always
means very high variability in exposure to parent compound for a given dose. For instance, 2% to 10% bioavailability means an
automatic fivefold range before individual variations seen in metabolic clearance after intravenous (IV) administration are taken
into account versus 60% to 80% bioavailability, which only entails a 11/3-fold variation. High variability in exposure per unit dose
makes clinical studies more difficult. Second, to reach the exposures required to define the concentration at which chronic toxicity
is observed, low bioavailability means that much more compound will be necessary. This is actually a far more significant issue than
is generally appreciated because amounts of material are limited early in development. It can be very costly to develop efficient
synthetic schemes; one does not want to invest too much in syntheses until one is fairly sure that a compound is safe at
pharmacologically active concentrations.

TOXICOKINETICS
Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

This introduces the critical role of toxicokinetics, the goal of which is to establish in animals (usually rats and dogs) the ratio
between the concentration that produces either unacceptable physiologic effects (e.g., QTc prolongation, seizures) or organ damage
(e.g., liver, bone marrow) and the one that produces the targeted biochemical or behavioral effect, a ratio of at least 2 to 1. As the
ratio approaches 1.0 it is increasingly unlikely that a compound will be taken into humans unless the observed unwanted effects are
readily reversible and one is dealing with a condition for which existing therapies are poor or nonexistent (Alzheimer’s disease [AD]
or amyotrophic lateral sclerosis [ALS]). There are instances in which humans ultimately prove tolerant to drugs much more than
would be predicted by these ratios, but this is only established after exposure to large numbers of subjects.

Although the ratios established in preclinical studies are interpreted as providing a reasonable estimate of any ultimate therapeutic
index in humans, they are specific for both species and mechanism of action. In other words, the concentration necessary to achieve
a behavioral or biochemical effect and produce toxicity can vary more than an order of magnitude across species. The relationship
among in vitro biochemical effects, activity in some in vivo model, and undesired effects is poorly understood even at the
preclinical level, particularly when one is investigating novel targets. Because efficacious in vivo concentrations in animals are
based either on activity in an animal model (e.g., anticonflict activity to identify a potential anxiolytic) or biochemical changes (e.g.,
changes in brain serotonin) that are not measured in humans, we do not even have validation for existing drugs relating preclinical
and clinical data (13 ). Not surprisingly, given the weakness of the link between an efficacious dose in rats and a therapeutic dose in
humans, the clinical development of central nervous system (CNS) drugs is notoriously inefficient to identify therapeutic
concentrations.

Measures of receptor occupancy or specific degree of enzyme inhibition do not drive the selection of an efficacious concentration. In
other words, the basic preclinical work that underlies any test of a specific hypothesis such as “X% of inhibition produces effect Y” is
rarely, if ever, done. Therefore, in toxicokinetic studies one hopes for very high ratios that provide a great deal of flexibility to
study a extremely wide range of concentrations in humans, especially with novel compounds.

Phase I
Select starting doses for healthy volunteers based on findings from the preceding toxicokinetic studies, which is often one-tenth of
the dose that produces a no-adverse-event level in the most sensitive species studied (4 ). Most marketed central nervous system
(CNS) drugs produce some sufficiently unpleasant (but not dangerous) side effect that sets the dose range for subsequent studies,
the so-called maximum tolerated dose (MTD). Multiple dose studies, administering various fixed doses over a 2- to 4-week period are
also part of the Phase I protocols to get a general idea of safety and tolerability under likely clinical conditions (14 ). The observed
steady-state concentration during repeated dosing, broadly speaking, is the first surrogate for the desired effect, the obvious but
often incorrect assumption being that a certain value assures a certain biochemical effect in the brain based on extrapolations from
preclinical data.

Once the initial safety studies are completed, there may be additional studies in healthy volunteers to assess efficacy. In the case of
sedatives and hypnotics, healthy volunteers can be used reliably with a combination of subjective reports, objective measures of
performance (looking for decrements), and the electroencephalogram (EEG) (15 ,16 ,17 ,18 and 19 ). These measures, however, are
direct ones, of a desired or undesired effect, not surrogates. On the other hand, the so-called
 P.461

pharmaco-EEG (P-EEG) is sometimes introduced as a surrogate measure of the therapeutic action a compound is likely to produce
(20 ,21 ), but is probably more usefully viewed as a direct measure that a compound has produced some change in surface brain
electrical activity. The P-EEG provides neither evidence of a direct effect in the brain nor proof of a hypothesized causative role of
specific pharmacologic activity of the molecule (22 ).

In the majority of CNS Phase I studies, aside from measures of drug concentration, the only usual objective measures are targeted to
the heart and other vital organs. Side effects are often simply recorded as spontaneous reports on a grid of prespecified subjective
descriptors; sometimes a formal checklist is used. Thus, quantitative surrogate measures of drug effect are sparse.

Phase 1B/2A (Proof of Concept) Studies

There is wide variation in practice as to what is done, both with healthy volunteers and patient populations, before going on to full
Phase II studies. These latter studies are powered to detect a clinically significant improvement, usually over placebo, often
utilizing multiple dose arms (each with at least 50 or more patients) and multiple clinical sites. In the case of a compound acting on
a novel target, the period before full Phase II trials is argued to be the phase during which exploratory work should be done (23 ).
For instance, if one had an ampakine such as CX-516 that improved learning in rats, then one might show enhancement of some
aspects of cognition in healthy volunteers after safe doses were established in Phase I studies (24 ). The improved performance in
healthy subjects would then be a surrogate for improvement in AD to identify the appropriate dose.

In the more usual case of a variation on an existing mechanism (e.g., SSRIs), this is the period to verify which dose produces the
desired biochemical effect so as to move as quickly as possible into Phase II trials with doses that one is confident will work (25 ,26 ).
In this latter instance, the direct accessible biochemical measure becomes a surrogate for an effect in brain that is hypothesized to
produce the clinical benefit.

We briefly review a range of specific measures that have been used in enough instances over the last two decades to qualify as
surrogate markers. There is no available literature as to the extent to which these measures actually drove decisions about what
doses to use in Phase II trials, but one can assume that they must have been influential in some cases. What is certain is that at least
in the United States, outside of polysomnography (for sleep-related disorders) and EEG (for seizures), the measures have had no
acknowledged regulatory impact. Assessments of decrements in performance (especially ability to concentrate, recall, and carry out
motor tasks) and measures of cognitive enhancement all seem to be better classified as direct measures of an intended or
unintended drug effect rather than surrogates. This leaves mainly biochemical measures, which can be broadly classified as
“neuroendocrine.”

Cerebrospinal Fluid
The monoamine hypotheses of depression and schizophrenia, which are, in turn, based on the pharmacology of antidepressants and
antipsychotics, generated an interest in measures of norepinephrine (NE), serotonin (5HT), and dopamine (DA) in humans. In most
instances these were initially studied in an attempt to distinguish patients from controls but came to be applied to validating
predicted effects of psychotropic agents (27 ).

Thus, NE reuptake inhibitors (NERIs) decrease NE turnover as measured by NE and its metabolites in urine or cerebrospinal fluid (CSF)
(28 ,29 ,30 and 31 ) and by increasing plasma NE (32 ) or, more precisely, its spillover rate (33 ,34 ). Another marker of NE reuptake
inhibition is based on the need for exogenously infused tyramine to be taken up into NE nerve endings to exert its effects, the so-
called tyramine pressor test (35 ) or, more recently, the dorsal hand vein constrictor test (36 ). These latter two procedures have
been recently employed to establish the dose at which venlafaxine produces NE reuptake inhibition (8 ,9 ,36 ). Because NE
metabolism varies according to whether it occurs inside or outside neurons (37 ), a more definitive and perhaps more sensitive
measure would be the ratio of extraneuronal to intraneuronal metabolites before and after treatment in an integrated pool (e.g., a
24-hour urine) (38 ) as shown for desipramine (30 ). However, all of these measures reflect functional changes in the peripheral
sympathetic nervous system; therefore, they are at best surrogates for effects in the brain.

Some of these same measures have been applied to study other compounds, which are predicted to affect NE function either
following acute or chronic administration. These include bupropion, clozapine, alpha 2 antagonists, and monoamine oxidase
inhibitors (MAOIs) (39 ,40 ,41 ,42 ,43 ,44 ,45 and 46 ). Interestingly, such studies are not yet available to support or refute claims
that the relatively new antidepressant mirtazapine produces alpha 2 antagonism in humans.

Most, if not all, drugs classified as SSRIs have been shown to inhibit platelet uptake of 5HT, most simply determined by investigating
5HT depletion in platelets following chronic administration (25 ,47 ,48 ,49 and 50 ). Some of these studies were clearly done prior
to Phase II trials so they can be inferred to have influenced the selection of dose. Doses of SSRIs known to inhibit platelet uptake in
humans have also been shown to reduce the turnover of 5HT in the CNS as reflected by reductions of its major metabolite 5-
hydroxyindoleacetic acid (5HIAA) in CSF (44 ,51 ). Similarly, MAOIs reduce both platelet MAO and 5-HIAA in CSF (51 ,52 ). There are
not, however, systematic dose response studies to compare the sensitivity of the peripheral and central measures. Moreover, 5HIAA
in CSF obtained by a lumbar puncture reflects a complicated process of all sources of formation and clearance of the metabolite.

DA has been studied almost exclusively in terms of its metabolite, homovanillic acid (HVA) in blood and CSF. There is a complex
relationship among administration of a
 P.462

dopamine type 2 (D2) antagonist, duration of treatment, clinical state, and HVA in either compartment (53 ,54 ,55 ,56 ,57 and 58 ).
Although changes usually can be explained as consistent with altered turnover as a function of receptor antagonism, time, and
presence or lack of clinical response, measures of HVA are not really useful as a surrogate measure of D2 antagonism. Decreased HVA
in CSF can be expected after mixed Types A and B MAO inhibition in the brain (52 ) and might be relevant to assessing DA uptake
inhibition. There is, however, no available positive control for the latter. Bupropion, the one compound studied in regard to possible
DA uptake inhibition, did not decrease HVA in the CSF (40 ,44 ).

Prolactin
Prolactin has the potential to be used as a marker for drugs affecting these systems, because either DA or 5HT can affect this
circulating hormone. In the simplest and most widespread instance, it has been used as an index of D2 antagonism. Unlike typical
neuroleptics, atypical neuroleptics produce no elevations of prolactin at therapeutic dosages (59 ,60 ). By extension, absence of
prolactin elevation after antipsychotics could be considered a surrogate of low D2 receptor occupancy in the striatum and, hence,
imply low to absent motor side effects (61 ,62 ).

Prolactin is also elevated following various pharmacologic challenges such as: (a) those that are predicted to increase extracellular
5HT in the brain including fenfluramine, clomipramine, l-tryptophan, and 5hydroxytrytophan; and (b) those that stimulate various
types of 5HT receptors, including meta-chlorophenylpiperazine (mCPP) and some, but not all, putatively selective 5HT1A agonists
(63 ,64 ,65 and 66 ). In all of these instances, prolactin increase becomes a surrogate of increased serotonergic transmission in one
or more regions of the brain. Blockade of this effect by appropriate 5HT receptor specific antagonists could serve, in turn, as a
surrogate of a compound’s ability to functionally antagonize the specified receptor in human brain (67 ,68 ,69 and 70 ).

Growth Hormone
There was a period in which plasma growth hormone (GH) was used as a surrogate for increased noradrenergic transmission in
human brain (presumably at the level of the hypothalamus) after, for instance, the α2 agonist, clonidine, or an NE uptake inhibitor
(71 ,72 ). One could, in turn, infer α2 antagonism by a test compound if it blocked the effect.

More recently, stimulation of plasma GH has been considered evidence of activation of both 5HT1A and 5HT1B/1D receptors (see Table
34.2 on page 459) (63 ). Sumatriptan increases GH, apparently through activation of the 5HT1B/1D receptors (73 ,74 ), with the most
recent evidence using the more brain penetrant, zolmitriptan, implicating 5HT1D postsynaptic receptors (75 ). It has been suggested
that stimulation of the 1B/1D receptors inhibits somatostatin release (76 ); however, an increasing volume of experimental research
indicates that 5HT can act directly on the adrenal gland and possibly on the anterior pituitary as well (77 ). This provides another
example of the same measure serving as a surrogate for very different CNS effects, the interpretation of which depends on knowing
the potential target for a compound in advance. It is doubtful that any substantial decisions concerning doses of compounds
affecting either noradrenergic or GABAergic or serotonergic systems have ever been made based on GH release stimulation or
inhibition.

ACTH/Cortisol
Stimulation of the hypothalamic–pituitary–adrenal axis as reflected by increases of ACTH and/or cortisol has also been used as a
marker of drug action in the CNS. This approach has been most extensively applied to evaluation of putative 5HT agonists,
sometimes coupled with pretreatment with whatever antagonists were available (e.g., ritanserin and pindolol) (63 ). Such studies
can generate evidence of apparent selectivity in postsynaptic receptor responses to indirect and direct agonists. ACTH release
induced by 5-hydroxy-L-tryptophan (5HTP) is argued to occur through indirect activation of 5HT2 receptors because it is antagonized
by ritanserin (78 ), whereas the cortisol response is not affected by doses of pindolol expected to produce 5HT1A antagonism (67 ).
Pindolol does, however, antagonize ACTH responses to a variety of agents classified as partial to full 5HT1A agonists (64 ,79 ,80 ).
Again, given the complexities of the ACTH/cortical response and the imperfect selectivity of the pharmacologic agents, quantitative
conclusions as to degree of specific receptor activation or antagonism are not possible.

Other Physiologic Measures

Sleep EEGs have already been referred to and may have utility as surrogates of, for instance, activation or antagonism of 5HT2C
receptors as reflected by decreases or increases of slow wave sleep, respectively (81 ,82 ).

Temperature decreases are consistently observed following 5HT1A agonists (63 ,83 ), and hence can serve as a surrogate marker of
5HT1A agonist effects in the CNS. Evaluating the ability of a novel compound to antagonize the hypothermia produced by a 5HT1A
agonist may be the easiest way to see if it antagonizes 5HT1A receptors in the human brain.

LIMITATIONS OF CURRENT SURROGATE MARKERS

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

As described, there is no validated link between the concentration of drug in blood (or even CSF) and a specific biochemical effect
in human brain (not to mention a specific brain region). Thus, even in the case of SSRIs, the most
 P.463

widely prescribed class of drugs in neuropsychopharmacology, we do not know how closely platelet 5HT depletion reflects 5HT
uptake inhibition in brain. Although known therapeutic doses of SSRIs invariably have been shown to deplete platelet 5HT, the
converse is not clear; that is, any dose that is effective in platelets will be therapeutic. Matters are even less certain when it comes
to using available surrogates of NE uptake inhibition to establish the dose of a drug. And, as already noted, primary dosing decisions
are not made on the basis of whether compounds affect prolactin, growth hormone, or ACTH/cortisol responses.

One could use the CSF concentration of a drug as a reasonable estimate of the free drug concentration in brain under true steady-
state conditions (84 ,85 and 86 ), and infer from one’s preclinical in vitro and in vivo studies that this will produce a specific effect
(87 ). The problem is that even in the most refined in vitro system, as represented by cloned human receptors expressed in some
vector, the relationship among receptor occupancy, drug effect, and free drug concentration may be extremely different from that
observed in vivo in humans thanks to multiple uncontrollable differences among these systems (13 ). Furthermore, in vivo animal
studies raise questions about species differences; therefore, how does one select the dose for clinical studies when testing a new
compound that is well tolerated with a wide range of safe concentrations that are predicted to be pharmacologically active by one
or more preclinical models? How does one know that the target in question has been blocked or stimulated so as be sure that one is
testing the hypothesis that such an effect produces therapeutic benefit? The answer is, one does not know with the surrogates
(discussed in the preceding). This brings us to a discussion of the promise of direct measures of drug effect in the brains of living
humans.

IMAGING STUDIES
Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

Measures of Substrate Metabolism

Regional cerebral blood flow (rCBF) is a well-established surrogate marker widely used for both clinical diagnostic procedures and
new drug development. rCBF is commonly assessed utilizing estimates of cerebral perfusion, such as single photon emission
computed tomography (SPECT) with radioligands labeled with 123I or 99mTc; for example, [123I]iodoamphetamine, [99mTc]ethylenediylbis-
L-cystein diethylester (ECD), and [99mTc]hexamethylpropyleneamine oxime (HMPAO). Although repeated assessments with multiple
radiotracer injections are usually impractical because of the long half-lives of both 123I and 99mTc, linearization techniques have been
developed to estimate sequential measurements (88 ). Furthermore, 99mTc is relatively available in practically every hospital with a
radiology or nuclear medicine department worldwide; therefore, SPECT procedures can be performed widely on a clinical basis.

By contrast, positron emission tomography (PET), another procedure to estimate rCBF, requires an onsite cyclotron, so it is not
available in many areas. Recent third-party camera reimbursement of some PET procedures (primarily [18F]fluorodeoxyglucose (FDG))
are making PET cameras more available, but cyclotrons are still relatively scarce. The two most widely used radioisotopes used in
PET are 11C with a half-life of 20 minutes and 18F with a half-life of 110 minutes. Additionally, 15O, with a half-life of 2 minutes is
primarily employed in brain perfusion studies. The most available PET radioligand is FDG, a tool to measure glucose metabolism. Its
advantages include ease of use, availability from commercial cyclotrons throughout much of the world, and sensitivity to a number
of studies to facilitate drug development and assess mechanism of action (89 ). Its disadvantage is lack of specificity.

An example of the value of PET studies to obtain potential surrogate markers for development of drugs for substance abuse is the
dysfunction of regional glucose metabolism in the limbic system and areas of working memory in cocaine euphoria and cocaine
craving (90 ). [15O] PET studies demonstrate increases in rCBF in the limbic system and decreases in the basal ganglia as
manifestations of craving for cocaine following exposure to videotapes suggesting cocaine use (91 ).

ESTIMATION OF DOPAMINE RECEPTOR OCCUPANCY BY

ANTIPSYCHOTIC DRUGS
Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

It has been hypothesized that positive symptoms of schizophrenia, such as hallucinations and delusions, result from increased
stimulation of postsynaptic dopamine (DA) receptors by DA (92 ). This DA hypothesis of schizophrenia is substantiated by the
observation that positive symptoms of schizophrenia abate when DA receptor blocking drugs, such as neuroleptics, occupy the
postsynaptic DA receptors. In 1988, Farde and colleagues demonstrated the concept that effective neuroleptic dosages for
schizophrenia correspond to 80% to 90% occupancy of DA type-2 receptors (D2Rs) by the drug (95 ). Thus, occupancy of 80% to 90% of
D2Rs may constitute a surrogate marker for the dosage of a D2 antagonist producing maximal beneficial effects with minimal adverse
and toxic effects. Furthermore, clinically equivalent doses of neuroleptics are estimated by comparing proportions of receptors
occupied by various psychotherapeutic agents (93 ,94 ). Several examples of this approach are given in the following.

Receptor Occupancy by Typical Neuroleptics

The occupancy of D2Rs by neuroleptics has been extensively evaluated by PET and SPECT. Historically, this began with the study of
typical neuroleptics, including haloperidol and
 P.464

fluphenazine, in healthy normal control subjects and patients with schizophrenia utilizing [11C]raclopride (95 ,96 ), spiperone
derivatives, including [11C]N-methyl-spiperone (NMSP) (97 ), [18F], or 76Br (98 ). This research has established that a therapeutic
response corresponds to occupancy of 65% to 90% of the D2Rs by the drug; however, occupancy of greater than 90% of the D2Rs is not
associated with a greater therapeutic effect. Therefore, occupancy of 65% to 90% of D2R2 is correlated with a therapeutic dose of
typical neuroleptics as well as other clinical manifestations of pharmacologic efficacy. For example, patients with acute
extrapyramidal side effects were found to have higher DA receptor occupancies (82%) than those without (74%) (99 ,100 ). Wolkin
and colleagues (1989) found a comparable occupancy of haloperidol in responders versus nonresponders indicating that treatment
nonresponse is not a function of insufficient CNS binding of the antipsychotic (100 ).

Kapur and colleagues recently confirmed that the D2 occupancy is an important mediator of beneficial and adverse effects (101 ) in a
study of first episode schizophrenia and haloperidol. Although the patients showed a wide range of D2R occupancy (38% to 87%), the
degree of D2R occupancy predicted clinical improvement, hyperprolactinemia, and extrapyramidal side effects. Also, Daskalakis and
associates (1998) (102 ) found a relationship between D2R occupancy and hyperprolactinemia.

Receptor Occupancy by Atypical Neuroleptics

D2R occupancy has been determined for humans treated with atypical neuroleptics. For example, receptors of people treated with
clozapine exhibit low (20% to 65%) D2R occupancy and high serotonin type 2A (5HT2A) receptor occupancy (94 ,99 ,103 ). In studies
directly comparing clozapine (450 mg/day) and haloperidol (5 mg/day), there was a reversal of receptor occupancy producing high
D2 blockade with haloperidol and high 5HT2A blockade with clozapine. Indeed PET imaging has been an important approach for
screening a number of new antipsychotics with an atypical profile. Risperidone has been shown to block 40% to 60% of D2 receptors
at only 1 mg (104 ) with even higher 5HT2A receptor occupancy. Indeed, in patients with schizophrenia, only 4 mg/day is needed for
70% to 80% occupancy with minimal risk of extrapyramidal side effects (105 ). After an oral dose of 6 mg risperidone, 75% to 80% of
D2Rs and 78% to 88% of 5HT2A receptors are occupied (106 ). Plasma levels of risperidone correlate with D2R occupancy (107 ). The
proportion of D2R occupancy has been estimated for other atypical neuroleptics; for example, olanzapine, has been shown to have D2
occupancy similar to risperidone and greater than clozapine (108 ). The usual clinical doses of olanzapine (10 to 20 mg/kg) produce
71% to 80% D2R occupancy. These doses usually do not result in the adverse effects of dyskinesias and prolactin elevation. On the
other hand, doses of greater than 30 mg/day olanzapine are associated with greater than 80% D2R occupancy as well as dyskinesias
and prolactin elevation. Olanzapine exhibits 59% to 63% D2Rs occupancy after single 10-mg dosing. Furthermore, olanzapine shows a
greater (68% to 84%) occupancy of D2Rs than clozapine (20% to 67%) after 10 to 20 mg daily dosing in patients (60 ). These
characteristics suggest that olanzapine differs from clozapine. Additionally, quetiapine is characterized by abatement of psychotic
symptoms in association with a transient increase in D2R occupancy (62 ).

Receptor Occupancy as a Surrogate Marker of Clinical Efficacy?

One of the most important questions about D2R and 5HT2A receptor occupancy is the prediction of doses yielding clinical efficacy. D2R
occupancy predicted the clinical improvement of 22 first episode schizophrenic patients randomly assigned to 1 or 2.5 mg/day of
haloperidol for 2 weeks (101 ). Thus, D2R occupancy is related to the clinical response to antipsychotics (99 ,109 ,110 ).

PET studies have helped demonstrate that high levels of D2R occupancy occur at very low haloperidol doses (111 ). Kapur (1996)
showed D2R occupancy of 53% to 74% at only 2 mg haloperidol in first-episode patients (114 ). Kapur and colleagues (1997) also
showed D2 occupancies between of 53% to 88% for haloperidol doses of 1 to 5 mg. Thus, the conventional therapeutic practice of
haloperidol doses of greater than 10 mg/day is too high for many schizophrenic patients because there is no increase in beneficial
effect, whereas the risk of adverse effects increases in proportion to the dose (112 ). Nevertheless, Volavka and colleagues (113 )
(1995) showed that antipsychotic efficacy of haloperidol increases with plasma levels up to 12 ng/mL, plasma levels that would
predict almost completely saturated D2 receptors according to the Kapur and associates (1997) data (112 ). Similarly, Wolkin found
D2 receptor occupancy increasing with haloperidol plasma levels up to 15 ng/mL and almost complete D2R occupancy saturation with
haloperidol plasma levels above 15 to 20 ng/mL (100 ). Thus, Wolkin’s (100 ) and Volavka’s (113 ) findings confirm each other and
contradict Kapur’s (112 ,114 ) studies. These differences could be explained partly by the differences in radioligands and patient
populations. Further research by other investigators with various populations is needed to resolve the controversy.

Another important implication of receptor occupancy with neuroleptics is the prediction of extrapyramidal side effects. The
probability of acute dyskinesias is directly proportional to the proportion of D2Rs occupied by the drug (99 ,110 ,115 ). Furthermore,
receptor imaging demonstrates
 P.465

a lower degree of D2 receptor occupancy during treatment with atypical neuroleptics. For example, PET has been used to obtain the
minimal effective dose of risperidone. The high D2R occupancy associated with 6 mg or more risperidone daily suggests a high risk of
acute dyskinesias. On the other hand, 4 mg risperidone daily results in 70% to 80% D2R occupancy and a lesser risk of acute
dyskinesias (116 ). An additional role for occupancy studies in drug development takes the form of the interpretation of the time
course of receptor occupancy following a single drug dose. Such studies help determine the appropriate dosing regimen for future
trials, such as once or twice a day dosing. For example, 70% to 90% of 5HT2A receptors are occupied during the 24 hours after a single
oral 20-mg dose of MDL100,907 (MDL), a selective serotonergic agent, whereas only 20% are occupied 24 hours after a 10-mg dose
(117 ). These results suggest that a 20-mg dose may be administered once daily, whereas a 10-mg dose requires administration twice
a day. Thus, occupancy studies constitute surrogate markers for the outcome variable and frequency of drug administration.

Another use of occupancy studies is the correlation of D2R occupancy with plasma levels of neuroleptics. This approach has been
successfully applied to estimate D2R occupancy by haloperidol in patients with low doses of haloperidol (118 ). These preliminary
results can be refined in future research with larger sample sizes.

In summary, receptor occupancies have helped establish the optimal dosage range of antipsychotic medications. These imaging
methods also have a role to determine in vivo occupancy of new neuroleptics with multiple sites for D2 and 5HT2A binding. The
studies probably have their greatest role in giving approximate dosage estimates for future clinical trials.

Other Roles for Neuroreceptor Imaging in Drug Development

Four major areas in which neuroreceptor imaging can assist in drug development are listed in Table 34.3 . The first and most well
developed area is in helping target rational drug dosing as in the neuroleptic studies described in the preceding. The second is the
elucidation of the biodistribution of the drug by radiolabeling the drug or a derivative of the drug. Examples of this application
include the characterization of MDL, which is the first selective 5HT2A antagonist developed primarily for schizophrenia. MDL has
been radiolabeled with [11C] in an isotopic form such that a stable carbon atom (atomic number 12C) is substituted with a radioactive
atom (11C) without a change in the pharmacology or chemistry. This procedure facilitates characterization of the biodistribution and
washout characteristics of the agent (117 ).

TABLE 34.3. COMPONENTS OF THE DRUG DEVELOPMENT PROCESS ACCOMPLISHED

BY NEURORECEPTOR IMAGING
The third application of neuroreceptor imaging to drug development is to better understand the mechanism of action of drugs. One
example of this is in the development of drugs for cocaine abuse. Unlike neuroleptics and antidepressants, drugs developed to
inhibit the action of cocaine have failed clinical trials. Although cocaine has been shown to affect multiple neurotransmitter systems,
current research efforts to develop effective treatment for cocaine dependence focus on the dopamine system. Cocaine is
hypothesized to produce euphoria by increasing the intrasynaptic concentration of dopamine. Cocaine has high affinity for the
dopamine transporter (DAT); therefore, contemporary research to treat cocaine dependence includes the development of
noncompetitive inhibitors of cocaine at this site without affecting dopamine transport. One example of potential treatments for
cocaine dependence is the development of GBR12909 (GBR), a potent DAT inhibitor. This pharmaceutical, originally developed in
Europe as an antidepressant, has found a potential new application as a prototypical drug for cocaine abuse. Prior studies have
shown that IV infusion of GBR to Rhesus monkeys selectively reduced (1 mg/kg) and eliminated (3 mg/kg) cocaine self-
administration (119 ). Villemagne and colleagues (120 ) tested the hypothesis that doses of GBR, which reduce self-administration,
also produce significant occupation of DAT. Doses of 1, 3, and 10 mg/kg demonstrated occupancy of 26%, 53%, and 72%, respectively,
in Papio anubis baboons (Fig. 34.1 and Fig. 34.2 ). These data suggest that doses that suppress cocaine administration also provide
high occupancy of the DAT. Preclinical research supports the hypothesis that elevations of mesolimbic DA mediate the addictive and
reinforcing effects of methamphetamine and amphetamine. In vivo rodent microdialysis has demonstrated that GBR attenuates
cocaine and amphetamine induced increases in mesolimbic DA. Utilizing PET scans of a continuous infusion of [11C]raclopride in
baboons, Villemagne and colleagues (120a ) also showed that GBR attenuates amphetamine induced striatal DA release by 74% (Fig.
34.3 and Fig. 34.4 ). Thus, GBR is a potentially effective agent to treat cocaine and methamphetamine dependence. This
experimental
 P.466
model is also being utilized to evaluate other forms of GBR such as the long-acting decanoate derivative.

FIGURE 34.1. Reductions in dopamine transporter occupancy are shown in transaxial

[11C]WIN35,428 images in Papio anubis baboons before (left) and after (right) administration
of three different doses of GBR. Each dose is given 90 minutes before [11C]WIN35, 428
injection. The illustrations represent average PET images at midstriatal level between 70 and
90 minutes after the injection of the radiotracer normalized to the injected radioactivity.
Modified from Villemagne V, Rothman RB, Yokoi F, et al. Doses of GBR12909 that suppress
cocaine self-administration in non-human primates substantially occupy dopamine
transporters as measured by [11C]WIN35, 428 PET scans. Synapse 1999;32:44–50. Copyright ©
1999, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. See color version of figure.

FIGURE 34.2. This histogram illustrates the percentage dopamine transporter (DAT)
occupancy by GBR 12909 (GBR) as measured by positron emission tomography imaging with
[11C]WIN35, 428. DAT occupancy is represented as the percentage mean ± standard error of
the mean differences between binding potentials at baseline and after GBR administration.
Percentage occupancy is calculated by the formula as follows: [(Baseline binding potential -
GBR binding potential)/Baseline binding potential] × 100. Inset: Relation between DAT
occupancy and GBR dose (120). Modified from Villemagne V, Rothman RB, Yokoi F, Rice KC,
Matecka D, Dannals RF, Wong DF. Doses of GBR12909 that suppress cocaine self-
administration in nonhuman primates substantially occupy dopamine transporters as measured
by [11C]WIN35, 428 PET scans. Synapse 1999;32:44–50. Copyright © 1999, Wiley-Liss, Inc., a
subsidiary of John Wiley & Sons, Inc.

FIGURE 34.3. These images illustrate the binding of [11C]raclopride to the basal ganglia of
Papio anubis baboons treated with saline (top row) and GBR (1 mg/kg) (bottom row) after the
administration of saline (3 mL/kg) (PRE AMP) (left column) or amphetamine (1 mg/kg) (POST
AMP) (right column). After the administration of saline (3 mg/kg) (top row) there is
prominent binding of [11C]raclopride to the basal ganglia at baseline (PRE AMP) (upper left)
and significant reduction after the administration of amphetamine (1 mg/kg) (POST AMP)
(upper right). After the administration of GBR (1 mg/kg) (bottom row) there is reduced
binding of [11C]raclopride to the basal ganglia at baseline (PRE AMP) (lower left) and minimal
reduction after the administration of amphetamine (1 mg/kg) (POST AMP) (lower right).
Modified from Villemagne VL, Wong DF, Yokoi F, Stephane M, Rice KC, Matecka D, Clough DJ,
Dannals RF, Rothman RB. GBR12909 attenuates amphetamine-induced striatal dopamine
release as measured by continuous infusion PET scans. Synapse 1999;33:268–273. Copyright ©
1999, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. See color version of figure.

FIGURE 34.4. These histograms contrast the release of DA after administration of

amphetamine (AMP) in Papio anubis baboons treated with saline (left) and GBR (1 mg/kg)
(right). For each histogram, the abscissa indicates the intravenous administration of saline (3
mL/kg) (PRE AMP) or amphetamine (1 mg/kg) (POST AMP) and the ordinate is the average
volume of distribution (Vh) and the standard error of the mean. The left histogram illustrates
a significant reduction in Vh consistent with the release of DA induced by amphetamine. The
right histogram demonstrates a reduced baseline (PRE AMP) Vh after administration of GBR
consistent with an increase in baseline extracellular intrasynaptic DA concentration (PRE AMP)
and the absence of a significant change in Vh after the administration of amphetamine (POST
AMP). Modified from Villemagne VL, Wong DF, Yokoi F, Stephane M, Rice KC, Matecka D,
Clough DJ, Dannals RF, Rothman RB. GBR12909 attenuates amphetamine-induced striatal
dopamine release as measured by [11C]raclopride continuous infusion PET scans. Synapse
1999;33:268–273. Copyright © 1999, Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

The fourth method in which neuroreceptor imaging can assist in drug development is the empirical evaluation of theories of disease, such as the DA hypothesis for
schizophrenia. For example, Grace (1991) (121 ) proposed that schizophrenia is characterized by intrasynaptic concentrations of DA that are abnormally low in the basal
tonic state and abnormally high in the simulated phasic state. This has been supported by numerous findings of elevated dopa decarboxylase measurements using
[18F]fluorodopa (122 ), elevated amphetamine induced dopamine release (123 ,124 and 125 ), and elevated D2Rs (97 ,126 ). There is also some potential evidence that
elevated intrasynaptic dopamine release is also found at baseline (126 ,127 and 128 ). In this example of the DA system, the combined strength of measuring presynaptic,
postsynaptic, and intrasynaptic DA, for example, provides converging evidence to test this hypothesis. Development of additional ligands such as those for glutamate,
glycine, and second messengers, will further expand the potential to evaluate the complex pathophysiology of schizophrenia.

MAGNETIC RESONANCE SPECTROSCOPY

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "
 P.467

Magnetic resonance spectroscopy (MRS) provides surrogate markers to determine clinical endpoints to evaluate new therapeutic
interventions for specific diseases. For example, choline metabolites estimated through photon MRS function as surrogate markers of
cognitive motor symptom severity in HIV-1 (129 ). Additionally, [19F]MRS has been explicitly employed to examine the
pharmacokinetics of psychotropic medications containing a fluorine group such as fluvoxamine (130 ). This allows direct comparison
of brain and plasma concentrations, and brain elimination half-lives (131 ), although the sensitivity allows only micromolar measures
in contrast to the nanomolar measures attained with PET. These methods have also been employed in animal models using
[19F]nuclear magnetic resonance (NMR) chemical shift imaging to study the cerebral distribution of general anesthetics in vivo (132 ).

MAGNETIC RESONANCE IMAGING

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

Magnetic resonance imaging (MRI) provides surrogate markers for disease progression to facilitate drug development. For example,
T2-weighted cerebral MRI functions as a surrogate marker in early stages of demyelinating disease to predict disease progression and
disability over the subsequent 10 years (132a and 132e ).

Another example of the use of MRI as a surrogate marker for drug development is in the diagnosis and treatment of subjects with AD.
Atrophy of the hippocampal formation has been correlated with memory and cognitive impairments. Reductions in the volume of the
hippocampus have been predictive for the individuals who later develop memory impairments consistent with AD (133 ). Another
marker of the vulnerability to develop AD is the measure of the apolipoprotein E (APOE) genotype. The relative risk for AD is
increased for people with the gene (134 ). The APOE gene is associated with loss of hippocampal volume (135 ). Cross-sectional
studies in 116 healthy volunteers, 59 to 85 years old, demonstrated significantly larger ventricular volumes and smaller gray and
white matter volumes in older compared to younger individuals and in men compared to women over a period of only 1 year. An
increase of over 1,500 mm3 in ventricular volume was demonstrated during this time but no detectable change in the total or
regional brain volumes. This suggests that determination of the pattern and rate of these changes longitudinally could be a future
predictor of cognitive declines and dementia (136 ).

Functional MRI (fMRI), a technique in which subjects are asked to perform particular mental or physical tasks while the MRI is
obtained, may provide biomarkers useful for drug development. For example, the rCBF of cocaine-dependent subjects administered
intravenous cocaine exhibited increases in the nucleus accumbens, subcallosal cortex, and hippocampus and decreases in rCBF in the
amygdala, temporal pole, and medial frontal cortex (137 ). Future studies utilizing this paradigm could additionally assess whether
another compound administered before cocaine can antagonize or amplify its affects.

BIOMARKERS OF ALCOHOL ABUSE

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

The synergistic combination of carbohydrate-deficient transferrin (CDT), gamma-glutamyl transferase (GGT),

 P.468

and mean red cell volume (MCV) functions as a possible biomarker for alcohol abuse (138 ).

NEWER MARKERS FOR DRUG DEVELOPMENT

Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "

Several techniques recently have been developed to provide the means to assess the efficacy of newly developed potential drugs.
These procedures assist clinicians in both the (a) diagnosis of neuropsychiatric disorders, and (b) monitoring of the course of disease
progression and the response to drug treatments and other therapeutic interventions. The application of the following novel
procedures to drug development is the result of a collaborative effort jointly of the National Institutes of Health (NIH), the United
States Food and Drug Administration (FDA), the Health Care Financing Administration (HCFA), and private industry (139 ). The
protocols described in this section are likely to be instrumental in drug development for neuropsychiatric disorders in the future.

External Imaging of Internal Bioluminescent and Fluorescent Signals

Biological processes, such as the propagation of cancer cells, are studied in animal models by the detection of bioluminescent (140 )
and fluorescent signals recorded externally by means of sensitive photon detection systems (141 ). For example, optical imaging
systems visualize near-infrared fluorescent molecular targets triggered by enzymes released by cancer in experimental animals
(142 ). Other examples include the evaluation of potential agents to treat human cancer and infection by assessing the behavior of
cells from patients in animals utilizing bioluminescent markers. The technique utilizes bioluminescent compounds bound to specific
enzymes that signal the proliferation of cancers and bacteria. Human subjects with cancer or infection are treated with agents to
alter the expression of genes that produce the enzymes needed for the growth of the cancer or bacteria. Then animals are injected
with (a) cells from the previously cancerous or infected tissue of treated humans, and (b) bioluminescent markers that respond to
the presence of the enzyme that was hopefully blocked in the humans. Detection of the bioluminescent markers in the animals
indicates the proliferation of the enzyme to be blocked in the treated human and, therefore, the failure of the therapeutic attempt.
This technique, which provides a surrogate for the clinical effects, is being applied to a variety of human diseases including cancer
and infection (http://www.xenogen.com/). By analogy, this procedure could be applied to assess drugs developed to treat
malignancy and infection of the CNS. For example, novel agents to alter the expression of the genes that code for the control of the
production of enzymes required by cancer and infection of the CNS could be administered as therapeutic agents to suitable patients
with malignancy and infection of the CNS. CSF from treated patients and bioluminescent compounds responsive to the presence of
the enzyme that should be absent could then be injected into experimental animals. Detection of the bioluminescence in the
experimental animal would then indicate failure of the new drug to prevent the growth of the tumor or infection. Thus, this
technique offers the potential of surrogate markers to monitor the presence of CNS tumors and infections.

Simultaneous Optical and Magnetic Resonance Microscopy

Simultaneous optical and magnetic resonance imaging (MRI) is being developed in experimental animals. MRI contrast agents (143 ),
such as fluorescently detectable magnetic resonance imaging agents, are utilized to permit light and magnetic resonance imaging
microscopy at the same time (144 ). These compounds can be detected deep in tissue, not merely at the surface as with simple
optical detection systems (e.g., for fluorescent dyes) (145 ). Although light microscopy can penetrate only 100 to 300 μm beneath
the surface of organisms, MRI microscopy can penetrate 1 to 6 mm into an organism (144 ). For example, agents that can be
simultaneously recognized by MRI microscopy and by fluorescent optical microscopy permit visualization of structures 1 to 6 mm
below the surface of an organism approaching cellular resolution (i.e., 10 μm) (143 ,144 ).

Diffusion-Based Optical Imaging

Procedures to measure light emitted into opaque structures have been termed medical optical imaging (MOI), medical optical
spectroscopy (MOS), near-infrared imaging (NIRI), and near-infrared optical spectroscopy (NIOS) (146 ). For example, three-
dimensional optical coherence tomography (OCT) is a technique to image nontransparent biologic tissue by recording and analyzing
light emitted into scattering media. OCT has been employed to visualize nerve fascicles in experimental animals for the
microsurgical anastomoses of vessels and nerves (147 ). An example of diffusion-based optical imaging is the use of optical
tomography to detect intraventricular hemorrhage in premature infants by external transmission of light emitted through the skull
(146 ). These techniques may be utilized to develop treatments for human disease, including infections and malignancies (146 ).
Hopefully, in the future they will be applied to CNS tumors and malignancies.

Magnetic Resonance Microscopy

Microscopic visualization of magnetic resonance images has detected transgene expression in experimental animals.
 P.469

Identification of pathologic processes, including the proliferation of tumor cells in clinical settings (148 ), may be facilitated by this
procedure. This technique offers the means to both detect the occurrence of malignancies and to monitor their growth (148 ). The
application of this procedure to human CNS malignancy is a goal to be attained in the future.

Electron Paramagnetic Resonance

Electron paramagnetic resonance (EPR) (149 ) imaging and spectroscopy are procedures to spatially map parameters of physiologic
importance by incorporating paramagnetic spin labels into the system of interest (150 ). This technique has been utilized to visualize
oxygen concentration in the tissues of experimental animals (150 ) and to measure oxygen free radical generation in human
endothelial cells exposed to anoxia and reoxygenation (151 ). EPR has been employed to estimate the production of nitric oxide in
biological systems (152 ), a process vital to the measurement of the progression of pathologic processes (153 ), including cerebral
ischemia (154 ) and malignancies.

CONCLUSION
Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "
Biomarkers and surrogate markers are tools currently utilized to develop new drugs. They provide evidence of the proof of concept
required for successful Phase 1B/2A studies submitted to the FDA. Currently drugs are being developed by the use of neuroendocrine
markers including CSF, prolactin, GH, ACTH, and cortisol. Imaging studies provide the means to estimate therapeutic dosages of new
drugs. Surrogate markers include a variety of neuroimaging techniques including MRI, MRS, PET, and SPECT. Newer techniques for
drug development are likely to include external imaging of internal bioluminescent and fluorescent signals, simultaneous optical and
MRM, diffusion-based optical imaging, and EPR.

ACKNOWLEDGMENT
Part of "34 - Proof of Concept: Functional Models for Drug Development in Humans "
We acknowledge grant support from United States Public Health Service Grants K24DA00412 (Wong), and from DA09482, DA11080,
NS38927, MH42821 (Wong/Brasic), the Rett Syndrome Research Foundation (RSRF) (Wong/Brasic), and from NARSAD and The Essel
Foundation (Brasic).

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35
Ethical Aspects of Neuropsychiatric Research with Human Subjects

Debra A. Pinals

Paul S. Appelbaum

Debra A. Pinals and Paul S. Appelbaum: University of Massachusetts Medical School, Worcester, Massachusetts

Considerable world attention in the last century has focused on the ethics of clinical research with human subjects. Coming to the
fore after World War II, with the Nuremberg War Crimes Trials, concerns were raised about the potential for abuse of nonvoluntary,
uninformed subjects who might be utilized in questionable research. Out of the trials came the Nuremberg Code (1 ), which
formalized ethical principles surrounding research with human subjects. When, in 1953, the United States opened the doors to the
Clinical Center of the National Institutes of Health, guiding principles regarding human subject research at that institution were in
place to greet the first subjects who enrolled in studies on site (2 ). By the following decade, in 1964, the World Medical Association
developed the Declaration of Helsinki, which was an attempt to modify and expand upon the Nuremberg Code (1 ). This document
classified research into clinical and nontherapeutic categories, and outlined the practice of consent that these types of research
would require. The Declaration has since gone through multiple revisions and continues to be a significant guideline for research
with human subjects, especially in Europe.

Despite these initial attempts to clarify the ethical principles and practices of human subject research, repeated abuses were widely
publicized in the ensuing years: the Tuskegee syphilis study (3 ), studies involving injection of live cancerous cells in patients
without their consent (4 ), and studies in which subjects were unknowingly exposed to radiation (5 ) among them. As each story was
exposed, it inspired international review of the ethics of human subject research. Minority populations and those who may be
vulnerable to exploitive research, such as the mentally ill, evoked particular concern. In the United States, federal commissions and
agencies were created to address the concerns. Among the outcomes of these initiatives were regulations affording the government
greater control over federally funded research. Institutions were required to develop Institutional Review Boards (IRBs) to review
research protocols to protect human subjects and ensure an adequate consent process.

Beginning in the 1990s public concern again grew, as research with patients with mental illness became a focus of media attention
(6 ,7 ,8 and 9 ). As an example, Hilts (6 ) in a widely publicized media report, described a study in which the use of
methylphenidate in research subjects “threw 60 per cent of them into severe psychotic episodes.” Another exposé (8 ) described
research at the University of California at Los Angeles (UCLA) involving outpatients with psychotic disorders who were withdrawn
from active antipsychotic medications and observed for signs of relapse over time. One patient ultimately committed suicide more
than a year after leaving the study, whereas a second had a significant exacerbation in psychotic symptoms, resulting in threats to
kill his parents. In 1994 the federal Office of Protection from Research Risks (OPRR) investigated allegations that the UCLA study's
research design and implementation had been unethical (10 ). Although the OPRR did not find unethical research practices, they
questioned the adequacy of the informed consent process for this potentially high-risk study (11 ).

As a result of the controversy in the United States, federal and state agencies have begun to take a closer look at ethical issues
raised in psychiatric research. One of the foci has been the process of informed consent in studies involving subjects who may have
impairments in their abilities to make decisions, such as patients with severe mental illnesses. A 1995 report of the Advisory
Committee on Human Radiation Experiments (ACHRE) found that approximately half of the studies it examined had “inadequate
explanations of risks and discomforts in their consent material and paid no attention to the question of how to deal with subjects
who might have impaired capacities to consent to research participation” (5 ).
 P.476

In 1995, President Clinton appointed the National Bioethics Advisory Commission (NBAC), in part to address these concerns. In
December of 1998, NBAC issued its report entitled Research Involving Persons with Mental Disorders That May Affect Decision-
making Capacity (12 ). Among other things, the report recommended that an independent professional should assess a potential
subject's capacity to provide informed consent for studies involving more than minimal risk. The report generated a swift and
critical response from many psychiatric professionals who expressed concern that the recommendations reflected the misconception
that all persons with mental illness have decision-making impairments. Thus, some considered the recommendations too restrictive
and stigmatizing of persons with illnesses very much in need of study (13 ). Charney (14 ), however, wrote on behalf of the
psychiatric research community that the NBAC report provided some valuable contributions to the ongoing debate, and
acknowledged that “there is a crisis in confidence in the ethics of psychiatric research” that needed to be addressed. NBAC
responded to the criticisms by stating that they envisioned their report as “part of a continuing societal conversation… about what
regulations and guidelines should govern research involving persons with mental disorders that may affect their decision-making
capacity” (15 ).

These developments highlighted numerous areas of ethical concern regarding research with human subjects with neuropsychiatric
disorders, including subject recruitment, confidentiality, data access, and conflicting roles of investigators acting also as treaters.
More recently, certain methodologic practices, such as placebo-controlled studies, drug withdrawal studies, and the so-called
“challenge” studies, have attracted particular attention. Related to concerns about methodology, the ability of patients with mental
illnesses to provide informed consent to research procedures has probably been one of the most controversial issues surrounding
psychiatric clinical research, as highlighted in the NBAC report.

 METHODOLOGIC CONTROVERSIES
 INFORMED CONSENT AND THE CAPACITY FOR CONSENT IN PERSONS WITH NEUROPSYCHIATRIC DISORDERS
 COMMENTARY
 ACKNOWLEDGMENT

METHODOLOGIC CONTROVERSIES
Part of "35 - Ethical Aspects of Neuropsychiatric Research with Human Subjects "

Placebo Studies
In the mid-1990s, controversy over the use of placebos in research was rekindled; some commentators (16 ,17 ) contended that
placebo use is unethical when standard effective treatments exist. Support for this limitation on the use of placebos stems, in part,
from the Declaration of Helsinki (1 ), which declares that human research subjects have a right to therapeutically proven methods
and treatments when available. Nevertheless, use of placebo agents is widespread throughout medical research (18 ,19 ).

Arguments in favor of the use of placebos in research, including psychiatric research, include the superior ability to assess
accurately the efficacy of an experimental medication through the use of double-blind, randomized, placebo-controlled studies. In
fact, this type of study design has been touted has “one of the major achievements of modern medicine” (20 ). The FDA, in
considering what constitutes “adequate and well-controlled studies” (required for approval of new medications) states that the
placebo-controlled experimental design has important scientific merit in establishing therapeutic efficacy as long as the objectives
and the rationale for placebo use are clear (1 ). However, the FDA cautions that placebos should not be used “where existing
treatment is life-prolonging” or if the placebo “exposes patients to a documented serious risk” (1 ).

Some authors have argued that findings of placebo research are misleading and deceptive (21 ), and that more or equally reliable
findings could be had using active control agents (16 ,22 ). A central argument against the use of placebos in research on serious
mental illnesses is that they are likely to contribute to a relapse of or failure to resolve psychiatric symptoms. One early report
suggested that psychosis in and of itself may be biologically toxic to the brain (23 ), and may lead to short- and long-term adverse
consequences. In addition to the potential risks associated with an exacerbation of primary symptoms, there are concerns about the
psychosocial effects of relapse on patients. Specifically, prolonged periods of significant psychological distress may be associated
with loss of interpersonal relationships, financial losses, and increased risk of suicide.

Despite the risks, by now it is relatively clear that the use of nonactive agents as a means of control has scientific merit when
effective treatments for a particular illness are not yet known. When effective treatments do exist, a placebo comparison may still
allow investigators to establish efficacy, learn more about the natural course of the illness, and compare side effect profiles of
active agents against nonactive compounds. Studies examining the efficacy of a known medication and an experimental medication
may—because of flaws in the studies’ design or implementation—not clearly differentiate between the two, and drugs with only
minimal potential may be seen as more worthwhile than they are (24 ). Moreover, historic controls are not an adequate substitute
for placebos because the apparent increase in the prevalence of experimental subjects who may already be resistant to treatment
with standard medications (25 ,26 ). Thus, the analysis of the complexities of psychiatric illness and decisions regarding the risks and
benefits of existing compounds compared with novel agents would be limited by only having the existing active comparison agent as
a reference point (20 ,27 ). Furthermore, it has been argued that brief placebo periods may be conducted safely, particularly for
inpatients (28 ), and do not appear to lead to long-term negative prognostic effects (29 ).
 P.477

Drug Withdrawal Studies

Medication discontinuation studies in psychiatric research have become another point of ethical contention. The scientific rationale
for drug discontinuation has included the desire to examine the pathophysiology and course of underlying illnesses when patients are
in an unmedicated state. Furthermore, assessment of the clinical and neurochemical effects of medications in some cases can be
more legitimately interpreted in a given individual after a period of drug washout, as the potential therapeutic or adverse effects of
the initial treatment may present a confounding variable, making interpretation difficult (20 ,30 ).

However, significant concern about the use of this approach has been raised both in the scientific community and in the lay press.
For example, recent literature suggests that chronic patients may have a poorer response to treatment or deleterious effects should
they be taken off medication and experience relapses (31 ,32 and 33 ). In patients with bipolar disorder, concern has been raised
that the clinical state following withdrawal of maintenance medication may actually be distinct from what it would have been had
the natural course of the illness progressed without treatment at all (34 ). Furthermore, the risk of relapse itself has been of
significant concern. In fact, a meta-analysis of the effects of drug discontinuation in schizophrenia demonstrated a relapse rate of
53% during an average 9.7-month follow-up (35 ) compared to a 16% relapse rate for those patients remaining on their medication.
Despite the greater relapse risk, patients who experienced a worsening of their symptoms when off medications were able to return
to baseline following reinitiation of treatment. Relapse risk may be particularly high when medications are discontinued abruptly
(36 ,37 ). Questions have been raised about whether inconsistent use of neuroleptics may result in a higher risk of tardive dyskinesia
(38 ,39 ).

Much as with placebos, the debate about the potential for neurotoxic damage as a result of experiencing psychosis itself (23 ,40 )
has raised further ethical questions about drug discontinuation studies. Although, the theoretic long- and short-term risks of
psychosis have been widely cited, others have argued that the data on the risks of brief psychosis occurring during research studies
are not clear (41 ,42 ). Furthermore, in the case of psychotic disorders, continuous treatment with neuroleptics is not without its
own risks, including some risk of relapse and the risk of serious side effects (38 ). About 30% of patients will have no significant
response to neuroleptics, and some patients can remain without relapse even after years of being off medications (43 ). The risks of
ongoing treatment and potential adverse sequelae of withdrawing medications must be weighed in all psychiatric research. In this
way, risks to human subjects may be minimized and drug withdrawal conducted when essential.

Challenge Studies
Another of the controversial research techniques that has undergone public scrutiny are provocation or “challenge” studies. These
terms refer to experiments in which patients and sometimes healthy control subjects are exposed to drugs that exacerbate or create
psychiatric symptoms. Provocation studies are not unique to psychiatry. In general clinical research, provocation studies have been
conducted to induce pain, nausea and/or vomiting, bronchoconstriction, tachycardia, cognitive impairment, and even sepsis (44 ).
These studies share the same basic goal of allowing investigators to learn more about symptom expression and potential therapeutic
interventions. Although widely used in medical research, their use in studies examining psychiatric illnesses seems to have captured
the interest of lay persons, advocacy groups, the media, and even policy makers. One theory is that these types of studies may be
more common in neurobiological research, where less is known about the diseases being studied and animal models are sparse (45 ).

One of the hottest debates most recently has involved the use of ketamine, an NMDA receptor antagonist, and an approved
anesthetic agent, to provoke psychotic symptom exacerbation in patients with schizophrenia and produce transient psychotic states
in well control subjects. Tishler and Gordon (46 ) expressed concern that giving a healthy control or nonpsychotic person ketamine
might present a risk of producing illness, given the “biological stressor of the experimental procedure and psychological stressor of
psychosis [induced by the pharmacologic challenge].” In a review of all North American schizophrenia subjects who underwent
ketamine challenge studies, Carpenter (47 ) concluded that the ketamine-induced increase in psychosis was mild to moderate and
brief, any anxiety induced was mild and brief, and there was no evidence of ongoing negative consequences for subjects. It is
noteworthy, as Carpenter points out, that the controversy surrounding the ketamine challenge study has been raised when results
with fewer than 50 patients have been published. The media outcry against this type of study has led to trepidation to continue this
novel avenue of scientific research. Yet, other authors have suggested that symptom provocation studies, beginning with early
research involving amphetamine loading and including the more recent symptom induction studies, have contributed significantly to
our understanding of psychiatric disease, at a cost of inducing only transient psychotic states with no long-term adverse effects
(48 ,49 ) or evidence of altered disease course (50 ).

Although the data suggesting the safety of current challenge studies are encouraging, ethical implementation of such studies is
complex because of the potential for negative consequences, even if transient or remote. It has been argued that these types of
studies might be ethically justifiable if the underlying scientific principle is sound, if the effects are
 P.478

not thought to be long-term or severe, and if subjects have the capacity to participate as “knowing, voluntary partners in the
research enterprise” (49 ).

INFORMED CONSENT AND THE CAPACITY FOR CONSENT IN PERSONS

WITH NEUROPSYCHIATRIC DISORDERS
Part of "35 - Ethical Aspects of Neuropsychiatric Research with Human Subjects "

Challenge, placebo, and drug withdrawal studies always raise questions of research ethics, but the controversy is heightened when
the subjects involved suffer from mental illnesses. At the heart of the debate is the concern that these subjects, more than other
human research subjects, have significant deficits in their abilities to provide informed consent, so that they may enter studies
without full understanding of the inherent risks. Unfortunately, although this has become the focus of political and media attention,
there is often a lack of understanding of what informed consent is, and what the literature shows regarding the capacity of mentally
ill subjects to give informed consent.

The doctrine of informed consent is built from a complex interrelationship of medicolegal and ethical principles. Generally,
informed consent, whether to research or treatment, is broken down into three parts: voluntariness, disclosure, and competence
(51 ). Voluntariness implies that research subjects must be acting of their own free will when they agree to participate in research.
Disclosure provides information on the basis of which potential subjects may make an informed choice. In research settings,
disclosures must generally include such details as the nature and purpose of the study, as well as the potential risks and benefits
involved in the study. Other information provided includes disclosure of the right to discontinue participation in the study, who will
have access to the data, the differences between participation in research and routine treatment, and the availability of
compensation should harm ensue as a result of the study.

Informed consent also requires task-specific competence. Competence consists of four separate elements (52 ). First, subjects must
be able to evidence a choice regarding the decision at hand. The choice need not be expressed verbally, but subjects must be able
to communicate their preference in some way. Also, the choice must be sustained over time. The inability to maintain a consistent
choice over time might reflect significant mental status deficits such as those seen in psychotic ambivalence or delirious states.

Additionally, subjects must have a factual understanding of the information that has been presented to them. The degree of factual
understanding required for competence is unclear, and there is no threshold value of how much information must be understood in
order to be considered to have “enough” factual understanding. Furthermore, acceptable levels of understanding may vary
depending on the risks involved in a proposed research study.

Subjects must also be able to rationally manipulate the information in a way that is not impaired by symptoms of their illness. They
must demonstrate ability to reason through the information presented to come up with a logical decision, which need not be the
decision that the person assessing competence would make. Patients with or without psychosis who have impairments in their
reasoning, in addition to their primary symptoms (e.g., cognitive deficits, concrete thinking, or inability to abstract), might have
difficulties in this regard.

Finally, subjects must have a realistic appreciation of their situation. Patients with schizophrenia, for example, who do not believe
they are ill will have a limited appreciation of why they are being enrolled in a study examining that particular illness. The
appreciation must include some awareness of the fact that the study involves research and not treatment, and so may be of no
direct benefit to the individual.

Understanding of the capacities of persons with mental illnesses to consent to research has historically relied on data gathered from
studies looking at competence to consent to treatment. Recent years have seen an expansion of the previously limited literature on
competence to consent in research settings.

With regard to the ability to communicate a choice, although sometimes taken for granted, studies have shown that a proportion of
patients will have difficulties in this area. In a study by Appelbaum, Mirkin, and Bateman, 9% of community mental health center
patients who were contacted to participate in a study were found to be mute or catatonic (53 ). Eighteen percent of primarily
depressed inpatients were unable to make a decision in vignettes that required some problem solving (54 ). The risk of simply
excluding these persons from studies is that their inability to communicate a choice may reflect a degree of illness that is worthy of
study, and their exclusion might skew the results of research based on altered group composition. Therefore, there may be value in
considering whether proxy decision makers might in certain circumstances and with appropriate safeguards, enter into research
those subjects who are unable to express a consistent choice.

Studies have also examined the capacity of patients to understand information. For example, Grossman and Summers (55 ) found
that patients with schizophrenia understood only about half the information presented to them regarding the risks and benefits of a
fictitious medication, and thus concluded that these patients may have difficulty providing true informed consent. The degree of
psychopathology may affect learning of new information in schizophrenics (56 ). Kleinman and colleagues (57 ) suggested that a
formalized informing process increased schizophrenic patient understanding of tardive dyskinesia. In a frequently cited study of 41
patients with affective disorders who were potential subjects of a sleep EEG study, Roth and colleagues (58 ) found that only about
50% of the subjects understood more than two-thirds of the information presented to them through a formal consent process,
whereas a significant minority
 P.479

of patients (about 25%) understood half or less of the information. Benson and associates (59 ) showed that patients with
schizophrenia demonstrated greater impairment in understanding specific psychiatric research purposes and methodology in
comparison to psychiatric patients with less severe psychopathology. Comparing the capacity of stable patients with schizophrenia
and healthy volunteers to understand a low-risk study involving a magnetic resonance imaging test for research purposes, Pinals and
co-workers (60 ) found no difference in understanding of consent forms between groups. Of note, neither group on average was able
to correctly answer 100% of the questions on a brief questionnaire related to information on the consent form. Another study using a
questionnaire relating to research protocols found that out of 49 patients with schizophrenia, 53% required a second trial at the
questionnaire after re-education about the protocols to achieve a score of 100%, and 37% of subjects required three or more trials
(61 ). The authors concluded that with an adequate informed consent process, research subjects with schizophrenia were able to
comprehend consent form information.

Impairment of the ability to appreciate the nature of one's situation and potential consequences may have particular relevance in
psychiatric disorders where insight into one's illness is often compromised. In a classic report, Soskis (62 ) found that 68% of
schizophrenic subjects did not recognize the reason they were receiving treatment compared to 13% of medically ill patients. In an
earlier study looking at patient appreciation of their participation in research, Appelbaum and associates (63 ) showed that more
than half of the psychiatric patients interviewed failed to comprehend the research nature of some component of the methodology
of the research in which they were participating. The authors called the subjects’ tendency to view research as a therapeutic
process, when in fact there may be no benefit to the subject at all, the “therapeutic misconception.”

With regard to the ability of psychiatric patients to rationally manipulate information pertaining to research, Stanley and colleagues
(64 ) reported that the degree of psychopathology in patients with mental illness did not appear to influence their willingness to
participate in hypothetical research compared to nonpsychiatrically ill subjects. In that study, patients tended to agree to low-
risk/high-benefit hypothetical studies more than high-risk/low-benefit studies. In a subsequent study, Stanley and associates (65 )
found that approximately one-third of patients with mixed psychiatric diagnoses refused low-risk/high-benefit hypothetical study
enrollment, whereas about 40% of patients agreed to participate in a hypothetical study of high risk/low benefit. Garety and
associates (66 ) found that subjects with schizophrenia or delusional disorder requested less information before reaching a decision
and were quicker to change their estimates of the likelihood of an adverse event compared to nondelusional psychiatric patients and
normal controls. In a study by Sachs and co-workers (67 ), persons with dementia were noted not to perform as well as nondemented
elderly subjects in providing logical reasons for their decisions to participate in hypothetical research protocols.

Probably the most extensive data examining competency to make treatment decisions was reported by Grisso and Appelbaum
(68 ,69 ) from the MacArthur Treatment Competence Study. This study utilized standardized instruments designed to assess
capacities to make treatment decisions, and involved the assessment of multiple components of competence (understanding,
appreciation, and reasoning) and the use of several subject groups. Deficits were most pronounced in patients with schizophrenia,
and slightly more patients with depression were likely to have deficits than controls. Because the majority of all subjects performed
well on measures of competence, the study underscored the notion that subjects cannot be presumed incompetent by virtue of
mental illness alone.

Carpenter and associates (70 ) recently reported their findings examining how psychopathology and cognition affect decisional
capacity. They used a modification of the MacArthur study instruments (MacCAT-CR: MacArthur Competency Assessment Tool-
Clinical Research) (71 ) to examine making decision abilities relevant to research. In this study, 30 research subjects with
schizophrenia did not perform as well as healthy controls in decision making, and performance was strongly related to cognitive
impairments and somewhat related to symptomatology. However, the study found that a weeklong educational intervention that
provided information regarding the hypothetical study led to improved decisional capacity such that scores of schizophrenic subjects
were not significantly different from the well control group. In another recently published study, Appelbaum and associates (72 )
assessed the capacities of depressed patients to consent to research utilizing the MacCAT-CR. In this study, female outpatients with
major depression did not show impairments in their decision-making capacities related to research. This study further demonstrated
the utility of instruments such as the MacCAT-CR as a means of assessing decisional capacity as part of the broader informed consent
process in an actual research study.

COMMENTARY
Part of "35 - Ethical Aspects of Neuropsychiatric Research with Human Subjects "

Although ethics in human subject research has long been the focus of attention, awareness of the ethical dilemmas has been
heightened in recent years. Despite calls for a moratorium on all nontherapeutic, “high-risk” experiments, including drug washout
and challenge studies (73 ), adverse events appear to be much less common than the public may have been led to believe. What can
be gleaned from the current debate is that researchers must attend to the concerns raised, both to maintain public trust and ensure
the
 P.480

ethical integrity of research itself. As Bonnie (74 ) noted, the challenge is to create generally accepted guidelines on safeguards for
subjects without compromising the pursuit of important knowledge or threatening the integral partnership of mental health
advocates, persons with mental illness, and researchers.

The research community has made several efforts to tackle these issues. The American College of Neuropsychopharmacology (ACNP)
has developed guidelines on ethical practices related to neuropsychopharmacologic research. Highlighted are the needs to: (a)
ensure appropriateness of the study and its design; (b) minimize risk to subjects and maximize benefit to subjects or to the
population of patients with the illnesses under study; (c) ensure informed consent, while paying particular attention to the needs of
those subjects who may have decision-making impairments; and (d) protect confidentiality (75 ). The NIMH has established new rules
for “high-risk” studies, including the creation of a special Human Subject Research Workgroup of the National Advisory Mental
Health Council (NAMHC), which will review study protocols involving challenge methodology or drug withdrawal studies (76 ). After
several meetings with representatives of the NIMH, in 1995 the National Alliance for the Mentally Ill (NAMI) adopted “Policies on
Strengthened Standards for Protection of Individual with Severe Mental Illnesses who Participate in Human Subjects Research” (77 ).
Among these policies are a recognition of the “critical necessity” of human subject research, and recommendations for protection of
persons with cognitive impairments, clearer standards for consent protocols, and specialized training for members of IRBs that
review studies involving neuropsychiatric disorders. Measures to ensure that ethical issues are addressed have been developed,
including the Research Protocol Ethics Assessment Tool (RePEAT), which may assist in the planning of experimental protocols (78 ).

In addition to these efforts, there is a growing consensus on mechanisms for the ethical conduct of human subject research. It has
been suggested that not pursuing placebo and drug withdrawal studies would be unethical, given all there is to learn from them
regarding the pathophysiology, natural course, and treatment of severe mental illness (79 ). That said, it also is clear that specific
approaches can be utilized in order to ensure that this research is conducted safely and ethically. For example, there may be some
studies of pathophysiology in which subjects may be maintained on a low but effective neuroleptic dose, without interfering with
the acquisition of valid data (38 ). For neuroleptic withdrawal in patients with schizophrenia, slow rather than abrupt tapering with
careful ongoing monitoring may mitigate potential for bad outcomes (40 ). Drug-free phases may best be conducted while subjects
are in an inpatient setting, or while they are very closely monitored as outpatients (38 ). In this way, if symptoms begin to re-
emerge, subjects may be quickly and effectively treated before a bad outcome ensues. “Exit criteria” should be established a priori
to determine when patients will be restarted on their medications (41 ). In addition, alternative treatments (such as adjuvant
medications, psychotherapy, and rehabilitation treatments) during the placebo phase may be beneficial without compromising
research design. Patients at known risk of catastrophic responses to relapse should be excluded from the subject pool. Finally, study
subjects must be given the opportunity to provide informed consent, pose questions, and withdraw from study participation at any
time. Those patients with initial decision-making deficits and those who may become decisionally impaired during the study will
require special measures of protection, addressed in the following.

When challenge studies are proposed, again the scientific merit of the protocol must be weighed against its potential risks. Several
suggestions have been made that may offer protections to subjects. For example, Tishler and Gordon (46 ) have suggested a careful
recruitment process that would include detailed disclosure of the inherent risks, review of compensation for participation, and
screening prospective normal subjects for the presence of or vulnerability to develop psychiatric illness. Miller and Rosenstein (49 )
indicated that: (a) the study should have clear scientific merit, (b) subjects with specific clinical vulnerabilities may need to be
excluded from participation, (c) selected methods should minimize risks, (d) subjects should have access to careful monitoring and
follow-up, and (e) informed consent disclosure should make clear that the challenge study is distinct from other studies in which the
subject may be enrolled (80 ).

With regard to the consent process and the potential for decision-making impairments of mentally ill research subjects, existing
literature provides only rudimentary guidance in identifying groups at high risk of impairment. A substantial number of persons with
severe neuropsychiatric illnesses may have impairments in their decision-making ability related to research consent. Yet, the data
also have shown that many of these persons will retain abilities to make decisions that affect their lives, and thus it is misleading to
presume them incompetent by virtue of their diagnoses without adequate assessment.

Although policies will need to offer protection for those who may have decision-making impairments, excessive burdens must be
avoided if advancement of knowledge is to continue. By thwarting attempts to conduct bold and novel studies, society runs the risk
of limiting knowledge of the very populations who may be most in need of such research. The many subjects who have participated
in neurobiological research willingly, even when the risk is high and the potential for benefit is low, testify to the desperation that
some of these patients may feel regarding their illnesses. Brody has similarly commented on the justification for use of mentally
infirm adults in nontherapeutic research, even if the research presents greater than minimal risk (1 ), because of the need
 P.481

to study these complex illnesses. With these caveats, areas worthy of further consideration include disclosure practices,
identification and assessment of subject competence, and questions of threshold levels of competence (81 ,82 ).

In all of these arenas, existing IRBs seem to be in a strong position to provide the scrutiny required. Unfortunately, many people
believe that IRBs have become little more than clearinghouses for consent forms, rather than committees designed for careful
review of all aspects of research ethics (83 ). In an attempt to deal with this concern, the NBAC report proposed the establishment
of a special standing panel to review certain protocols that way present a greater risk to subjects (12 ). There are, of course,
negative aspects of a shift from currently accepted local IRB authority to a federal agency far removed from where the study would
take place (84 ). Regardless of the reviewing body, if the methodology appears questionable, persons with specialized knowledge in
these areas should be consulted to address the questions raised. Attention to the minimization of potential risks of studies is also an
important part of the mission of an IRB. With regard to the consent process, the IRB, in addition to reviewing consent forms, should
be able to monitor investigator disclosure and determine the level of required subject competence based on a standardized
evaluation of the risks and potential benefits involved in a proposed study.

Investigators may have other ways of advancing our current approaches to consent to research. For example, current literature has
demonstrated that a modification of disclosure procedures may facilitate subject understanding and enhanced learning
(57 ,59 ,60 ,70 ,85 ,86 ,87 ,88 and 89 ). Even with such efforts, however, there will always be potential subjects who will lack
capacity, in one or more of its realms, to provide valid informed consent to participate in research. When patients are participating
in studies of greater risk, a higher standard of competence should be required. The investigator and the IRB could work together to
decide when formal capacity assessments are indicated (90 ).

After the inherent risks and competence needs are determined, a sliding scale of options regarding capacity assessment might be
implemented. For example, in a low-risk study, one might consider a straightforward consent form and clinical assessment of
competence, perhaps aided by a questionnaire specifically geared to the study at hand. As the stakes increase, formalized
assessment instruments, such as the MacCAT-CR, might be adapted to the study in question. Oldham and colleagues (13 ), in their
response to the NBAC report, suggested that “formal capacity assessments should be required for subjects when there is reason to
believe that a mental or emotional state or a primary or secondary brain dysfunction may interfere with decision making.” They also
suggest that, given the inherent potential for investigator bias, for research that presents more than a minor increase over minimal
risk, independent evaluators, who function separately from the research team, could ascertain subject capacities. It is unclear at
this time how feasible such an approach would be, but it may merit exploration.

Once subjects are identified who clearly lack capacity to consent or who may come to lack capacity as research progresses (such as
patients with Alzheimer's disease or patients with schizophrenia enrolled in placebo-controlled studies), additional protections might
be implemented to allow such persons to participate in research. Pursuit of a legal determination of incompetence and the
appointment of a guardian to make decisions for the subject appears to be utilized rarely, in part because of the impracticalities
and cost involved (51 ). The use of a durable power of attorney or advance directive might, however, allow a substitute decision
maker to make decisions that the patient would have made during periods of greater competence (91 ,92 ,93 and 94 ).

Human subject research will always require careful scrutiny. Our history has shown that even well intentioned investigators may not
be able to assess ethical aspects of the research they are undertaking objectively. Additionally, potential research subjects may
enroll in studies for a variety of reasons, conscious and unconscious, without a full awareness or appreciation of the risks they are
undertaking. Nevertheless, the current focus on ethical issues related to research should serve to heighten the awareness of the
research team, including both investigators and subjects, regarding measures that can be taken to allow scientific advancement
while protecting potentially vulnerable populations.

ACKNOWLEDGMENT
Part of "35 - Ethical Aspects of Neuropsychiatric Research with Human Subjects "

Dr. Pinals has served on a speaker's bureau for Janssen Pharmaceutica.

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54. Radford MH, Mann L, Kalucy RS. Psychiatric disturbance and decision-making. Aust NZ J Psychiatry 1986;20:210–217.

55. Grossman L, Summers F. A study of the capacity of schizophrenic patients to give informed consent. Hosp Commun Psychiatry 1980;31:205–206.

56. Schacter D, Kleinman I, et al. The Effect of psychopathology on the ability of schizophrenic patients to give informed consent. J Nerv Ment Dis 1994;182:360–362.

57. Kleinman I, Schacter D, Koritar E. Informed consent and tardive dyskinesia. Am J Psychiatry 1989;146:902–904.

58. Roth LH, Lidz CW, Meisel A, et al. Competency to decide about treatment or research: an overview of some empirical data. Int J Law Psychiatry 1982;5:29–50.
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59. Benson PR, Roth LH, Appelbaum PS, et al. Information disclosure, subject understanding, and informed consent in psychiatric research. Law Hum Behav 1988;12:455–476.

60. Pinals DA, Malhotra AK, Breier A, et al. Informed consent in schizophrenia research. Psychiatric Serv 1998;49:244.

61. Wirshing DA, Wirshing WC, Marder SR, et al. Informed consent: assessment of comprehension. Am J Psychiatry 1998;155:1508–1511.

62. Soskis DA. Schizophrenic and medical inpatients as informed drug consumers. Arch Gen Psychiatry 1978;35:645–647.

63. Appelbaum PS, Roth LH, Lidz CW, et al. False hopes and best data: consent to research and the therapeutic misconception. Hastings Center Report 1987;17:20–24.

64. Stanley B, Stanley M, Latin A, et al. Preliminary findings on psychiatric patients as research participants: a population at risk? Am J Psychiatry 1981;138:669–671.

65. Stanley B, Stanley M, Peselow E, et al. The effects of psychotropic drugs on informed consent. Psychopharmacol Bull 1982;18:102–104.

66. Garety PA, Hemsley DR, Wessely S. Reasoning in deluded schizophrenic and paranoid patients: biases in performance on a probabilistic inference task. J Nerv Mental Dis
1991;179:194–201.

67. Sachs GA, Stocking CB, Stern R, et al. Ethical aspects of dementia research: informed consent and proxy consent. Clin Res 1994;42:403–412.

68. Grisso T, Appelbaum PS. A comparison of standards for assessing patients’ capacities to make treatment decisions. Am J Psychiatry 1995;152:1033–1037.

69. Grisso T, Appelbaum PS. The MacArthur treatment competence study. III. Abilities of patients to consent to psychiatric and medical treatments. Law Hum Behav
1995;19:149–174.

70. Carpenter WT, Gold JM, Lahti AC, et al. Decisional capacity for informed consent in schizophrenia research. Arch Gen Psychiatry 2000;57:533–538.

71. Appelbaum PS, Grisso T. The MacArthur Competence Assessment Tool. Clinical Research, 1996.

72. Appelbaum PS, Grisso T, Frank E, et al. Competence of depressed patients for consent to research. Am J Psychiatry 1999;156:1380–1384.

73. Shamoo AE. The Unethical use of persons with mental illness in high risk research experiments: testimony to Human Subjects Subcommittee of the National Bioethics
Advisory Commission United States Government, July 15, 1997.

74. Bonnie R. Research with cognitively impaired subjects: unfinished business in the regulation of human research. Arch Gen Psychiatry 1997;54:105–111.

75. Kupfer DJ. A statement of principles of ethical conduct for neuropsychopharmacologic research in human subjects. Draft Document of ethical guidelines for
neuropsychopharmacologic research with human subjects performed by members of the American College of Neuropsychopharmacology (ACNP). April 26, 1999.

76. New NIMH rules for high-risk studies. Psychiatric News April 16, 1999, p 12.

77. Shamoo AE, Johnson JR, Honbeg R, et al. NAMI's standards for protection of individuals with severe mental illnesses who participate as human subjects in research. In:
Shamoo AE, ed. Ethics in neurobiological research with human subjects: the Baltimore Conference on Ethics. The Netherlands: Gordon and Breach, 1997.

78. Roberts LW. Ethical dimensions of psychiatric research: A constructive, criterion-based approach to protocol preparation. The Research Protocol Ethics Assessment Tool
(RePEAT). Biol Psychiatry 1999;46:1106–1119.

79. Neylan TC, Wright BA, Shelton MD, et al. More on ethics of drug discontinuation studies in schizophrenia. Arch Gen Psychiatry 1990;47:192.

80. Rosenstein DL. IRB Review of psychiatric medication discontinuation and symptom-provoking studies. Biol Psychiatry 1999;46:1039–1043.

81. Berg JW, Appelbaum PS. Subjects’ capacity to consent to neurobiological research. In: Pincus HA, Lieberman JA, Ferris S, eds. Ethics in psychiatric research: a resource
manual for human subjects protection. Washington, DC: American Psychiatric Association, 1999.

82. Appelbaum PS. Missing the boat: competence and consent in psychiatric research. Am J Psychiatry 1998;155:1486–1488.

83. Appelbaum PS. Rethinking the conduct of psychiatric research. Arch Gen Psychiatry 1997;54:117–120.

84. Michels R. Are research ethics bad for our mental health? N Engl J Med 1999;340:1427–1430.

85. Munetz MR, Roth LH, Cornes CL. Tardive dyskinesia and informed consent: myths and realities. Bull Am Acad Psychiatry Law 1982;10:77–88.

86. Silva MC, Sorrell JM. Enhancing comprehension of information for informed consent: a review of empirical research. IRB: Rev Human Subjects Res 1988;10:1–5.

87. Tymchuk AJ, Ouslander JG, Rahbar B, et al. Medical decision-making among elderly people in long-term care. Gerontologist 1988;28:59–63.

88. Miller R, Willner HS. The two-part consent form: a suggestion for promoting free and informed consent. N Engl J Med 1974;290:964–965.

89. Wager E, Tooley PJH, Emanuel MB, et al. How to do it: get patients’ consent to enter clinical trials. Br Med J 1995;311:734–737.

90. Dresser R. Mentally disabled research subjects: the enduring policy issues. JAMA 1996;276:67–72.

91. Sunderland T, Dukoff R. Surrogate decision-making and advanced directives with cognitively impaired research subjects. In: Pincus HA, Lieberman JA, Ferris S (Des). Ethics
in psychiatric research: a resource manual for human subjects protection. Washington, DC: American Psychiatric Association, 1999.

92. Sachs GA. Advance consent for dementia research. Alzheimer Dis Assoc Dis 1994;4:19–27.

93. DeRenzo E. Surrogate decision-making for severely cognitively impaired research subjects: the continuing debate. Cambridge Quart Health Ethics 1994;3:539–548.

94. Kapp MB. Proxy decision-making in Alzheimer disease research: durable powers of attorney, guardianship, and other alternatives. Alzheimer Dis Assoc Dis 1994;4:28–37.
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 P.485

36
Regulatory Issues

Paul Leber

Paul Leber: Neuro-Pharm Group, LLC, Potomac, Maryland.

For over 60 years, the United States has relied primarily on a federal system of premarket drug product clearance to ensure the
quality of the nation’s drug supply. When the premarket clearance system was first introduced in 1938 in the aftermath of the Elixir
of Sulfanilamide tragedy in which over a hundred patients needlessly died because of a drug manufacturer’s carelessness (1 ),
federal law required only that new drugs be tested and shown, prior to marketing, to be “safe for use.” Since 1962, however, the
law requires that new drug products also be shown to be “effective in use” under the conditions of use recommended in their
proposed labeling.

This chapter considers how these two fundamental requirements of the Federal Food, Drug and Cosmetic Act (FFDCA), our national
drug regulatory law, are currently interpreted and applied by the Food and Drug Administration (FDA) in its evaluation of new drug
products. Issues that are singularly important to the evaluation of products intended for use in the management of psychiatric
conditions are identified and explicated.

 THE US FEDERAL DRUG REGULATORY SYSTEM: LEGAL BASIS, STRUCTURE, AND MODE OF OPERATION
 THE INVESTIGATIONAL NEW DRUG APPLICATION
 WHAT AN APPROVABLE NDA MUST DEMONSTRATE
 HOW THE AGENCY INTERPRETS THE ACT’S GENERIC REQUIREMENTS
 THE EVALUATION OF DRUG PRODUCTS INTENDED FOR USE IN THE MANAGEMENT OF PSYCHIATRIC CONDITIONS
 THE FUTURE OF DRUG REGULATION

THE US FEDERAL DRUG REGULATORY SYSTEM: LEGAL BASIS,

STRUCTURE, AND MODE OF OPERATION
Part of "36 - Regulatory Issues "

The basic structure and operation of the US federal drug regulatory system are established under the provisions of the FFDCA.

The FFDCA makes it unlawful “…to introduce or deliver for introduction into interstate commerce any new drug, unless an approval
of an application… is effective with respect to such drug.”

The application to which the Act refers is a New Drug Application (NDA). By law, authority to approve NDAs resides with the
Secretary of the Department of Health and Human Services, but the Secretary delegates the actual authority to review and approve
NDAs to the Food and Drug Administration (FDA, the agency).

The Act instructs anyone (i.e., a sponsor) seeking to market a new drug product to submit and gain FDA’s approval of an NDA for the
product prior to marketing it. Importantly, although it is not widely appreciated, NDAs are not approved for drug substances (i.e.,
chemical entities), per se, but for one or more specific “claimed” uses of a specific drug product (i.e., a specific formulation of the
drug substance) under a specific set of conditions of use recommended (i.e., described) in the product’s proposed labeling.

The Act describes, albeit in rather general terms, the information and reports that each NDA must contain. The details need not
concern us, however. Suffice it to say that a sponsor’s NDA is required to provide all the information necessary to allow the FDA to
determine whether or not the drug product that is the subject of the application meets the standards set out in the Act for a
lawfully marketed drug product. These standards address not only matters bearing on the product’s safety and effectiveness in
clinical use, but on its method of manufacture; chemical identity, purity, and strength; pharmaceutical performance; bioavailability;
and proposed labeling.

The Act requires the FDA, in turn, to “file” (i.e., accept for review) any NDA that appears on initial inspection to provide, in a
reasonably organized and coherent format, full reports of all the tests necessary to evaluate whether or not the drug product meets
the requirements just cited.

Finally, the Act instructs the FDA to review and to approve a sponsor’s NDA within 180 days of its submission, unless, on review, the
agency determines that the reports it contains fail to establish that the drug product identified in the application fully complies
with the Act’s requirements.

THE INVESTIGATIONAL NEW DRUG APPLICATION

Part of "36 - Regulatory Issues "

Because the Act forbids the introduction into interstate commerce of new drugs unless they are the subjects of an approved NDA,
the lawful clinical testing of unapproved new drug products would be a practical impossibility if the Act did not provide for an
exemption to this ban.

The original FFDCA (1938), accordingly, provided for

 P.486

precisely such an exemption, known then as a “Notice of Claimed Investigational Exemption for a New Drug.” The exemption is still
available, but it is now officially known as an Investigational New Drug (IND) application.

Initially, an investigational exemption could be obtained largely for the asking. Between 1938 and 1963, the sponsor of an IND had
only to agree to keep records and clearly label its new drug as to its status as an unapproved investigational new drug, but little else.

With the passage of the Kefauver and Harris amendments of 1962, however, the IND requirements were extensively revised and
expanded. Congress was led to alter the requirements for investigational use because of yet another public health disaster involving
a drug product. In this case, fortunately, the drug thalidomide, although widely marketed in Europe, was not marketed in the United
States. However, the potent teratogen thalidomide was widely distributed under INDs in the United States; worse, when its
teratogenicity was recognized and efforts were undertaken to recall the supplies of it that had been distributed, the extent of
domestic distribution was not easily determined.

Although very few American women who had received thalidomide under an IND bore children with limb reduction defects, the
episode raised substantial concerns about the safety of human research subjects (2 ,3 ). Thus, Congress amended the Act so as to
give the FDA the authority to monitor and control the conduct of clinical drug research within the United States.

Under the 1962 amendments, the agency gained explicit authority not only to establish mandatory prerequisites for the granting of
INDs, but also the power to prevent the initiation and/or suspend the conduct of a clinical investigation (i.e., impose a “clinical
hold”) being carried out under an IND if and when the agency concludes that an investigation poses an unreasonable or unnecessary
risk to human subjects. In 1997, with the passage of the Food and Drug Modernization Act (FDAMA), FDA’s authority under the Act’s
IND provisions was clarified and explicated in more detail, but not substantively modified.

In sum, since 1962, the IND serves not only as a license sponsors must obtain to allow them lawfully to ship unapproved new drugs in
interstate commerce, but also the device through which the agency monitors and maintains control over the way in which clinical
research with new drugs is conducted within the United States.

WHAT AN APPROVABLE NDA MUST DEMONSTRATE

Part of "36 - Regulatory Issues "

The text of the Act, at least insofar as safety and effectiveness standards are concerned, speaks almost entirely to broad goals and
generic principles. Responsibility for interpreting the Act and developing, revising, and promulgating the regulations and policies
necessary to secure the aims Congress had in mind in drafting the Act are delegated to the FDA.

Safety
Insofar as safety is concerned, the Act demands that a sponsor provide full reports of all tests necessary to establish that its product
will be safe for use. The Act instructs the agency to reject a sponsor’s application, if, on review, it determines that the drug has
been inadequately tested, or, if tested adequately, the findings of the tests conducted are inadequate to show that the drug, as
recommended for use, is safe for use, or show that the drug, again as recommended for use, is unsafe for use.

Efficacy
The Act instructs the FDA to approve an NDA unless, on review of the reports submitted, it concludes there is a lack of “substantial
evidence” that the drug is effective as claimed in its proposed labeling.

HOW THE AGENCY INTERPRETS THE ACT’S GENERIC REQUIREMENTS

Part of "36 - Regulatory Issues "

Safety for Use

No pharmacologically active drug substance is ever likely to be entirely free of risk. Accordingly, the agency maintains that a
regulatory determination that a drug is “safe for use” is, in actuality, a favorable “risk-benefit” determination (i.e., a conclusion,
based on the information evaluated, that the risks imposed by the use of the drug are acceptable in light of the benefits it provides).

Risk-benefit assessments, however, are hardly straightforward undertakings. To begin, their reliability is in large part a function of
the extent and quality of the information on which they are based. Unfortunately, the information ordinarily available to inform a
regulatory risk-benefit assessment is limited in scope; a typical NDA, for example, is approved based on experience gained with a
drug product in perhaps 1,000 to 2,000 human subjects in toto.

The information that is available, moreover, is in many respects marginal in regard to its aptness; the individuals who participate in
drug development programs, although they are reasonably representative of the patient population to which the drug will be
administered when marketed, are not fully representative of it. Accordingly, risks uniquely, or more likely to be, associated with the
use of a drug in various subgroups of the population, particularly when those subgroups are rare and/or under-represented in the
samples studied during the drug’s development, are almost never appreciated, let alone factored into the regulatory risk-benefit
determination.
 P.487

Data bearing on the risks of a drug are collected during premarket testing under conditions of use (e.g., dose, regimen of
administration, duration of use, restricted use of concomitant medications, etc.) that vary substantively from those under which a
marketed drug is likely to be used. This is of especial concern where duration of use is concerned.

Ordinarily, the bulk of premarketing data are obtained in relatively short-term clinical trials (weeks or months) although the product
under development typically will be used, once marketed, over much longer intervals (months to years). As a consequence, a typical
drug development program has little, if any, chance of detecting untoward effects of a drug that emerge only after an extended
period of exposure.

Regulatory officials are well aware of these limitations, but for practical (i.e., a politic word for economic and political) reasons,
must tolerate them. The International Conference on Harmonization (ICH) in which the US participates, for example, has issued a
guideline (4 ) that states that it is ordinarily sufficient to evaluate a new drug, prior to marketing, in no more than 300 to 600
patients for 6 months and 100 patients for a year.

Anyone familiar with the arithmetic of risk estimation will recognize that an experience of “safe passage” on a drug gained in such
limited numbers of patients is not very reassuring. The failure to see even one catastrophic or fatal event in a sample of 300
patients only reduces to 5% the chance that the drug investigated causes such unobserved events at a rate no greater than in one of
every 100 patients exposed to it (5 ).

(The notion of “safe passage” in drug safety assessment is the author’s invention; it likens regulatory premarket clinical trials to
journeys undertaken on uncharted waters. Much as early seafarers determined which of several routes between two points was safer
by comparing the risks of one with another, society determines whether or not a new drug is safe for use from the proportion of
patients exposed to it who enjoy safe passage.)

Whatever one’s personal sense of the size of such a risk, it is truly enormous from a public health perspective. Just imagine the
horror if a new antidepressant drug product caused a fatality in one in every thousand patients exposed to it. Yet, our society, like
those of other developed nations, is seemingly content to market drugs without being able to confidently exclude a 10-fold greater
risk.

In recent years, highly publicized withdrawals of new drugs shortly after their approval for marketing because of previously
unrecognized risks of use have focused public attention on the limitations of premarket drug safety assessment. Both the agency and
regulated industry have, consequently, been urged to develop new and better methods and paradigms to predict the likelihood of
new drugs to cause injury (6 ).

No doubt, both the agency and regulated industry would be delighted to do so if they could. It is somewhat difficult to fathom,
however, what kinds of methods will make it possible to identify drug-induced injuries in advance of their occurrence caused by
pathogenetic mechanisms that have yet to be recognized, let alone characterized.

On the other hand, it is difficult to deny that the systematic study of a drug product’s capacity to cause effects that have in the past
been associated with an increased risk of untoward effects should sometimes be useful. The risk of pharmacokinetic interactions, for
example, should be predictable if the major metabolic pathways involved in the elimination of a new drug, its metabolites, and the
pathways of elimination of other drug products likely to be coadministered with the new drug are identified and adequately
characterized. Knowledge of a drug product’s metabolism also makes it possible to identify individuals in the population that might
be at unique risk of suffering injury because of their diminished capacity to metabolize the drug (e.g., 6% to 8% of the white
population are “poor” metabolizers of drugs that are CYP 450 2D6 substrates). Presumably, as our knowledge of the human genome
expands, our ability to predict drug-induced risks on such grounds will grow.

Efforts to screen drugs prior to marketing for specific properties that predict drug-associated harms are still largely in their infancy,
however. Moreover, such approaches have inherent limitations. Their utility is typically predicated on the assumption that the
indicator of risk employed (e.g., a capacity to prolong the QTc interval on the surface ECG) reliably and consistently predicts a
drug’s capacity to cause harm. As is the case with almost all surrogate indicators, however, there is always a possibility that the
association between the surrogate and harm found in one set of circumstances will not hold in another.

In contemplating the development of new approaches to premarket safety assessment, it is important to be mindful that many of
our expectations may be unrealistic, even magical. Congress, for example, did not intend that premarket testing would successfully
identify every unsafe or unfit drug product. If it had, it would not have authorized the agency (Section 505(e) of the FFDCA), to
withdraw approval of any NDA for a drug if new information, not available at the time of approval, becomes available which shows
the drug is unsafe for use as labeled. Moreover, it is evident that Congress not only anticipated that new risks would be recognized
after a drug’s approval for marketing, but expected that postmarketing surveillance would detect them. Specifically, to ensure that
adverse information and reports bearing on the safety of marketed drug products would be collected and made available for
evaluation, the Act (Section 505(k)) requires the sponsors of marketed drugs “…to establish and maintain… records, and make…
reports to the Secretary, of data relating to clinical experience and other data or information, received or otherwise obtained by
[the sponsor]… with respect to… [its drug]… to enable the Secretary to determine, or facilitate a determination,
 P.488

whether there is or may be ground for… [withdrawing approval of the NDA].”

In actuality, however, only a relatively small proportion of marketed drug products are withdrawn from the market on grounds of
being unsafe. This fact is frequently overlooked in the midst of the sensationalist publicity and second-guessing that so often
accompanies product withdrawals. In the vast majority of instances, in fact, after their evaluation, adverse reports received on a
drug from postmarketing surveillance sources lead, at most, to revisions being made in the product’s approved labeling.

The nature of the labeling changes made, and the publicity given to them, is a function of the severity, estimated frequency,
potential reversibility, and likelihood of mitigation or avoidance of each newly appreciated risk. From a regulatory perspective, a
labeling change is a sufficient legal remedy, even for relatively serious newly appreciated risks, provided it remains possible for the
agency to sustain its earlier conclusion, albeit under the newly revised labeling, that the drug product is “safe for use” as labeled.

The foregoing discussion reveals just how subjective and tenuous drug safety assessments actually are. Indeed, even if the risks
associated with the use of a drug product were known exhaustively and in detail, its risk-benefit assessment would likely remain
arguable. Paradoxically, the crux of disputes about risk-benefit determinations less often concerns the seriousness of the harms a
drug causes, than the value of the benefits its use provides.

There is good reason for this. Except in those few instances in medical therapeutics were the use of a drug can be shown to reduce
the absolute incidence of death or a serious, otherwise irreversible, injury, a substantively meaningful numerical estimate of the
value of the benefit provided by a drug lies beyond reach. This occurs because there is a substantive distinction between a drug with
an effect and an effective drug treatment.

Given the proper choice of experimental design, an appropriate patient sample, and the use of validated outcome measures, a
competently executed clinical experiment can, of course, generate a numerical estimate of the magnitude of a drug’s effect on
some assessment instrument or scale of measurement. What such a trial cannot do, at least in a way that can be understood in a
public sense, is to provide a meaningful measure of the value of the drug’s clinical benefit. Unfortunately, it is the clinical benefit
provided, and not the numerical estimate of the magnitude of a drug’s effect as measured on some rating instrument that must be
considered in a substantively meaningful risk-benefit assessment.

Thus, in areas of medicine, like psychiatry, where the beneficial effects of a treatment are not ordinarily measured by counts of
cures or numbers of actual deaths prevented, but in the degree of symptomatic relief afforded by treatment, it is ordinarily
impossible to obtain a publicly meaningful, let alone quantitative, estimate of a drug’s value.

It is to be acknowledged that there are statistical estimates of “effect size” available (7 ), but these are intended only to gauge the
relative magnitudes of a measured effect and the natural variation of that measured effect among individuals in the population
being investigated. Although such “effect size” estimates are necessary to make informed “guesses” about the numbers of patients
that must be admitted to a controlled clinical trial to obtain a statistically significant result, they say nothing whatsoever about the
value of the effect being measured.

In sum, although a regulatory risk-benefit determination is widely represented to be a reasonable and responsibly considered
judgment that derives from a disinterested weighing of the gains and harms known to be associated with the use of a drug, it is
much more accurately depicted as a gestalt informed by an inchoate process that mixes, in undetermined proportions, evidence,
sentiment, and personal values.

Nonetheless, because the Act requires that marketed drugs be shown to be safe for use prior to marketing, the FDA must take the
information and reports presented in a sponsor’s NDA, and with the assistance of its consultants and advisors, determine whether a
reasonably qualified and informed expert, in possession of the data that are available, could conclude, fairly and responsibly, that
the drug is “safe for use” as labeled for use within the meaning of the Act.

Effectiveness in Use
Effectiveness determinations under the Act are, at least in comparison to those involving safety, relatively straightforward, provided,
however, that a modicum of agreement exists within the community of qualified medical experts as to: (a) what constitutes a
beneficial treatment effect in the area of therapeutics involved, and (b) how that therapeutic effect is to be measured.

Given agreement on these two matters, it is a relatively simple, although not always an easy or quick, task for a sponsor to procure
the evidence necessary to meet the Act’s substantial evidence standard, provided, of course, that the sponsor’s product is truly an
effective one. If controversies are extant within the community concerning the condition that is the target of treatment (e.g., its
features, diagnostic boundaries, cardinal manifestations, etc.), as they often are in the field of psychiatry, the task of
demonstrating a drug’s effectiveness in use becomes considerably more complicated but still possible.

The key question that remains to be addressed in this section then, is what constitutes substantial evidence within the meaning of
the Act.

Between 1962 and 1997, Section 505[d] of the Act offered the following definition of substantial evidence:

… the term “substantial evidence” means evidence consisting of adequate and well-controlled
investigations, including clinical
 P.489

investigations, by experts qualified by scientific training and experience to evaluate the effectiveness
the drug involved, on the basis of which it could fairly and responsibly be concluded by such experts
that the drug will have the effect it purports or is represented to have under the conditions of use
prescribed, recommended, or suggested in the labeling or proposed labeling thereof.

A number of points about the agency’s traditional interpretation of the substantial evidence standard are noteworthy.

The agency has long held that “substantial” evidence must derive, at least in part, from the findings of valid clinical experiments.
This presumably reflects the view that the value of drugs intended for the treatment of a human disease or condition can only be
evaluated meaningfully in tests conducted in human subjects actually afflicted with or at risk of developing that disease or condition.

Agency regulations make clear that for evidence to be deemed substantial, it must, in part, be adduced in scientifically valid
experiments (i.e., adequate and well-controlled investigations). Inferences based on scientific theory alone, or on the findings of
uncontrolled clinical studies or observations (e.g., case reports, case series, etc.) will not suffice (e.g., see 21 CFR 314.126 (e)).

Neither can clinical judgment or professional opinion, per se, contribute to the body of evidence required to meet the substantial
evidence burden. In light of the importance seemingly given to expert opinion in the statutory definition of substantial evidence,
this assertion may seem at odds with the Act’s requirements. A careful reading of the statutory definition reveals, however, that
substantial evidence does not include what experts believe. To the contrary, the reference to experts in the definition of substantial
evidence serves only to describe the character of the evidence that can be deemed substantial. (The evidence must be of a kind,
quality, and quantity that would allow a disinterested and informed expert to conclude, “fairly and responsibly,” from the evidence
that the drug will have the effect its sponsors claims it has.)

It is also important to be mindful that “substantial” is an arcane legal term. It is frequently, and incorrectly, taken to mean
definitive, even compelling, but substantial does not have that connotation within the meaning of the Act. Authorities on the
legislative history of the Act (8 ) note that Congress considered, but decided not to employ, a “preponderant” standard of evidence,
choosing, instead, the legally less demanding “substantial” evidence standard. Congress concluded, evidently, that the public health
would be better served if the Act allowed the marketing of a drug even if only a minority of qualified experts agreed that it had
been shown to be effective. It may help, therefore, to think of substantial evidence as the quanta of evidence sufficient to persuade
at least a sizable number, but by no means a majority, of disinterested experts that a drug has been shown to be effective as
claimed.

Among all the agency’s interpretations of the substantial evidence standard, none, perhaps, has caused more concern and enduring
complaint than its determination that positive findings from more than one adequate and well-controlled clinical investigation are
ordinarily required to establish a drug product’s effectiveness in use.

Although the requirement that experimental findings be independently substantiated prior to their formal acceptance is fully
consonant with common scientific practice and epistemological principle, the agency’s interpretation of the statute in this fashion
proved so controversial and so politically vexatious that it eventually led Congress (Food and Drug Administration Modernization Act
of 1997 [FDAMA]) to add the following sentence to the Act’s definition of substantial evidence.

If the Secretary determines, based on relevant science, that data from one adequate and well-
controlled clinical investigation and confirmatory evidence (obtained prior to or after such
investigation) are sufficient to establish effectiveness, the Secretary may consider such data and
evidence to constitute substantial evidence for purposes of the preceding sentence.

FDA authorities contend (circa 1999 to 2000) that the revised definition does no more than confirm FDA’s long standing authority (its
regulatory discretion) to interpret the Act’s efficacy provision flexibly and responsibly so as to secure the “aims of Congress” and
advance the interests of the public health. In fact, before the Act was amended in 1997, the agency had, on occasion, taken
exception to its traditional interpretation of the effectiveness standard, approving an NDA on the basis of findings from but a single
adequate and well-controlled clinical study (9 ).

Agency spokespersons assert, further, that in all but highly unusual circumstances corroborating positive findings from more than
one adequate and well-controlled clinical investigation will continue to represent the minimal quanta of evidence sufficient to
satisfy the Act’s substantial evidence requirement.

Evidently, because Congress failed to explain clearly what it intended by the terms “relevant science” and “confirmatory evidence”
in drafting FDAMA, agency officials seemingly enjoy a degree of latitude as to whether and when to rely on the findings of a single
controlled clinical investigation. Whether FDA officials will elect to or be able to retain this flexibility in the long run is far from
certain, however.

THE EVALUATION OF DRUG PRODUCTS INTENDED FOR USE IN THE

MANAGEMENT OF PSYCHIATRIC CONDITIONS
Part of "36 - Regulatory Issues "

The Division of Neuropharmacological Drug Products (DNDP) is the organizational unit within the FDA’s Center for Drug Evaluation
and Research (CDER) that is responsible for the monitoring of INDs and the evaluation of NDAs for psychiatric drug products.
 P.490

It is the Division’s obligation to review an application, determine whether or not it meets the requirements of the FFDCA as
interpreted under FDA’s prevailing regulations and policies, and, based on that review, take appropriate regulatory action.

For IND issues, signatory authority on most matters is at the level of the Division Director. Signatory authority is divided where NDAs
are concerned. NDAs for new chemical entities (NCEs) are approved or disapproved at the Office level (Office of Drug Evaluation 1);
supplemental NDAs (those involving new claimed uses of already marketed drug products) are approved at the Division level.

It is important to be mindful that Divisional policies evolve over time. A reader who wishes to understand whether, and if so, how a
particular policy applies to a specific problem or drug product would be prudent, therefore, to seek fresh guidance on the subject
directly from an appropriate Division representative.

From a regulatory perspective, the fact that a drug product is intended for the management of a psychiatric, sign, symptom,
condition, or disease presents no unique problem.

A drug claim can be advanced for virtually any effect on the structure or function of the body of humans, for the cure or
management of a disease or condition, or relief of a sign or symptom. The Act makes no distinction between the value of
symptomatic treatments and those that are advanced as cures for a disease. Almost any claim can be made, provided that it can be
presented in product labeling in a way that does not make the product’s labeling, “false or misleading” in any particular.

In psychiatry, as in most other therapeutic areas, the effectiveness of a new chemical entity will almost always have to be
demonstrated in more than one adequate and well controlled clinical investigation. It is possible, however, when an application is
submitted for a claim closely related to one for which the drug product is already marketed, that a single controlled clinical study
with robust and internally consistent findings might suffice. Because reliance on a single study is an exception to ordinary practice,
however, the decision whether or not to take this approach will invariably be made on an ad hoc basis.

The agency’s regulations (21 CFR 314.126) enumerate five control conditions that may be suitable for the evaluation of the
effectiveness of new drug products. In light of the variability in course and outcome among samples of patients assigned the same
psychiatric diagnosis, it is highly unlikely that either the historic control or the no control designs would ever be deemed acceptable
for the evaluation of a drug for a psychiatric indication.

A randomized controlled and blinded trial employing any one or combination of the three other enumerated control conditions
(placebo, graded dose, and standard active drug), provided it produces a statistically significant (P ≤ 0.05, two-tail) difference
favoring the investigational drug over a control condition, will invariably serve as one source of evidence contributing toward the
quanta required to establish substantial evidence of a psychiatric drug product’s effectiveness in use.

Among all the designs conforming to the requirements enumerated in 21 CFR 314.126, however, one that includes both a placebo
and a standard drug control is especially attractive. Not only does such a design allow an estimate of what might have been had no
treatment been administered (i.e., the response among placebo-assigned subjects) (10 ), but it provides a test of the capacity of the
sample of patients participating in the experiment to respond to a treatment of established effectiveness (i.e., the response among
patients randomized to the active control)

The three- (or more) arm design just described provides an internal means to assess what Modell and Houde (11 ) describe as an
experiment’s “assay sensitivity,” its capacity to discriminate an inert substance from placebo and one level of an active drug
substance from another.

Knowledge of an experiment’s “assay sensitivity,” or lack thereof, is especially important in the evaluation of psychiatric drug
products because a sizable proportion of psychiatric drug trials (e.g., close to 50% or so of antidepressant trials) (12–14) fail to
discriminate between active and inert treatments. Obviously, the trial’s failure can be discounted if the failure of a study to find a
drug placebo difference is owing to the inability of the sample of patients randomized in the study to respond to drug. On the other
hand, if the sample of patients enrolled can respond to standard treatment, but not the investigational drug, the trial must be
viewed as a source of evidence that speaks against the effectiveness of the investigational drug.

Why the agency encourages the use of an active control arm in a placebo-controlled study, incidentally, is often misunderstood. It
bears emphasis that the active control is not included to obtain an estimate of the standard drug’s performance relative to that of
the investigational drug, but solely to gauge whether or not the experiment has “assay sensitivity.”

When an investigational drug appears to have a relatively low therapeutic ratio, the use of fixed-dose graded designs is
advantageous because it provides the surest means to identify the dose or dose range most likely to be acceptable for a typical
patient. The agency is mindful, of course, that a single dose is unlikely to be the best choice for all patients; nonetheless, for dose
evaluation purposes, a fixed-dose design is more likely to be interpretable than a dose titration design. Clinicians often find this
assertion counterintuitive, but designs allowing up-titration for therapeutic nonresponse commonly produce an inverted dose-
response relationship (i.e., treatment resistant subjects who show poor response are given the highest doses of drug).

Another vexing issue regularly confronting regulators is how best to extrapolate the results of clinical investigations of new
psychiatric drug products to labeling claims.
 P.491

Although the agency’s regulations require that the sample of patients evaluated in controlled effectiveness trials be “reasonably
representative” of the population of patients for which a drug will be recommended for use, the patients recruited in typical
commercially sponsored drug trials are never truly representative of the population, at least not in any formal statistical sampling
sense. To the contrary, the choice of patient subject is almost always based on the sponsor’s convenience and its desire to maximize
the statistical efficiency of its study (15 ). This sampling strategy is not objectionable from a regulatory perspective when the
primary goal of a clinical study is to establish that the investigational drug product has the effect claimed for it in at least some
patients with the condition for which the treatment will be marketed.

What claimed uses should be granted to a sponsor of a drug based on the results of such studies is yet another arguable matter.
During the author’s tenure as DNDP’s director, for example, clinical trials conducted in acutely exacerbated schizophrenic patients
were deemed sufficient to support a claim for the use of a neuroleptic drug product in the “management of the manifestations of
psychotic disorders.” In a good faith attempt to adhere to the Act’s requirement that product labeling not be false or misleading in
any particular, the text of the Indications and Usage section also briefly described the clinical investigations that supported the
“antipsychotic” claim, including the nature of the patient samples that had been employed in them and whether or not long-term
maintenance trials had been conducted. The strategy employed was intended to reserve for practitioners the right to determine the
extent to which the results of a sponsor’s effectiveness trials applied to psychotic conditions other than schizophrenia. DNDP’s
current leadership takes a different view. At DNDP’s annual morning session at the NIMH’s annual NCDEU meeting (June 2, 2000)
held in Boca Raton, Florida, Tom Laughren (group leader for DNDP’s Psychopharmacology Unit) announced that henceforth claimed
uses for psychiatric drug products would be more narrowly defined (i.e., ordinarily limited to the population of patients actually
studied). Thus, drugs shown to be effective in studies enrolling schizophrenic patients will get claims for use in schizophrenia, not
psychosis. Incidentally, the newly announced approach to product labeling is perfectly reasonable and certainly consistent with the
requirements of law, although it is obviously not the one that the author prefers.

Difficulties arise in the extrapolation of study results in many other areas as well. Drugs are administered to individuals, not
diagnoses. There is advantage in knowing, therefore, whether, and if so how and to what extent, various individual patient
characteristics (sex, age, race, severity of illness, etc.) and the interactions among them affect response to a drug. Offsetting the
interest in obtaining the data necessary to address these issues are the difficulties and costs (both time and money) encountered in
obtaining them; recruiting representative patients from even the more important of the patient subpopulations (e.g., children, the
elderly, the very ill, etc.) is often exceedingly difficult, and sometimes, just not feasible.

For decades, the FDA took a relatively passive stance in regard to the demands it made on the regulated industry for data that might
better inform the use of prescription drug products in children. Groups interested in the welfare of children have lobbied long and
hard for making the study of investigational drugs in children a premarket obligation. FDAMA attempted to encourage sponsors to
conduct remedial pediatric studies by offering the incentive of a 6-month extension on the patent life of certain drug products. In
1998, however, the agency issued new regulations (21 CFR 314.55 and 21 CFR 201.23), asserting its authority to require sponsors to
evaluate new drugs in clinical trials enrolling subjects of pediatric age.

It seems unlikely to the author that the agency would actually refuse to approve an otherwise safe and effective new psychiatric
drug product on the grounds that the effects of the drug had not been adequately studied in children, unless, of course, the primary
use of the product was likely to be in children (i.e., the drug is intended as a treatment for ADHD).

The fact that the official psychiatric nosology is continuously evolving further complicates the extrapolation of study results to
product labeling claims. For example, prior to the promulgation of DSM-III in 1980, sedatives, as they were then known, were
granted broad and nonspecific claims for anxiety, anxiety neurosis, etc. As a result of DSM-III, a distinction had to be made between
generalized anxiety disorder and panic disorder. In recent years, claims have been further expanded to include not only long-
established entities such as obsessive–compulsive disorder (OCD), but also new entities such as social anxiety.

On each occasion that the psychiatric nosology expands or changes, the agency is confronted with a set of new problems including
not only how to evaluate claims for newly created entities, but how to conform existing drug product claims to fit with the revised
nosology. In dealing with these issues, the agency has to consider whether or not already approved older claims subsume the new
entities. (Is a new claim simply a re-expression of a previous one, a claim for a subset of the patients covered by the previously
approved claimed use, or an entirely new claim for a previously nonexistent entity?)

The agency also must decide how broad a claim or set of related claims may be. For example, sponsors often seek to define a
claimed use in a way that will allow the unique promotion of their drug product (i.e., distinguish their drug from others within the
same therapeutic class). When a claim links some diagnostic entity or subtype of entity to a drug’s effect and that linkage is
irrelevant to the expression of the effect, the claim is considered, “pseudospecific.” (The author early in his tenure as DNDP
director coined the
 P.492

term “pseudospecificity”; it was first applied in connection with claims advanced for the use of benzodiazepines in anxious patients
suffering from specific medical conditions [e.g., the anxiety of heart disease, cancer, etc.].) Such claims, moreover, are misleading
because they seek to promote a distinction without meaning; consequently, they can be rejected by the agency because they can be
held to be a violation of the Act’s requirement that a product’s labeling not be false or misleading in any particular.

A claim that a marketed antibiotic is effective for the pneumonia of dementia, for example, even if based on empirical evidence
that the drug is effective in curing pneumonias in patients with dementia, is pseudospecific because the linkage between the
pneumonia and the diagnosis of the patients treated is of no pharmacologic or biological importance, existing solely because of the
sponsor’s decision to select demented patients with pneumonia as subjects for study. A legitimate (i.e., nonpseudospecific) disease-
related claim requires a demonstration that the effect of the drug is in some way conditioned on the presence of the diagnosis (i.e.,
the diagnosis of the disease controls to what extent, if any, the effect of the drug is expressed).

The distinction made by the Division between pseudospecific and legitimate disease-related claims has often proved to be both
unpopular and a source of continuing controversy. Claims advanced by sponsors for the use of marketed antipsychotic drug products
in the management of psychotic demented patients are a case in point. During the author’s tenure as Director of DNDP, these claims
were regularly deemed to be pseudospecific, in the absence of evidence to prove the contrary.

Based on testimony and discussion at a Psychiatric Drug Products Advisory Committee (PDAC) held in March, 2000 on treatments for
the management of behavior in dementia, however, it appears that the Division is now inclined to accept “the psychosis of
Alzheimer’s disease” as a bona fide entity for which drug product claims may be made.

The seeming reversal in the Division’s prior position, as far as the author can determine, came about because the current
membership of the PDAC endorsed the psychosis of Alzheimer’s disease as an entity sui generis. Support for the existence of this
entity derived primarily from the testimony of psychiatrists who treat demented patients as to what they believe is the true state of
nature. The fact that a diagnostic algorithm for the capture of patients with the “psychosis of Alzheimer’s disease” had been
recently proposed, seemingly gave further support to the reality of the putative entity. Although there is little doubt that the
algorithm endorsed by the PDAC does capture demented individuals who exhibit behaviors that can be deemed psychotic, the fact
that it does so in no way establishes that there is, in nature, a unique psychosis of Alzheimer’s disease. Indeed, what the discourse
at March meeting of the PDAC revealed is that when a diagnostic nosology is based on a taxonomic system controlled by
authoritative figures (i.e., “opinion leaders” in the terminology of marketing departments within the regulated industry), the
existence of a diagnostic entity may owe more to politics than biology.

Incidentally, the taxonomic nature of our official psychiatric nosology not only complicates the task of drafting of accurate product
labeling, but also contributes to the high failure rate of studies intended to document the effectiveness of psychiatric drug products.

It bears note that DSM-III was developed, at least in part, in the hope that phenotypic similarities among patients would reduce
within diagnostic category genotypic variability and, thereby, make psychiatric diagnoses more predictive of course, outcome, and
treatment response than were the diagnostic categories based on the pseudoexplanatory, dynamic systems employed in DSM-II (16 ).

Unfortunately, this goal of DSM-III has yet to be achieved, in part, perhaps, because the effort to improve psychiatric nosology has
been confounded by issues and interests that have little to do directly with biologic classification (e.g., a focus on new entities that
expand the size of a drug’s potential market, or that advance a professional career, or a political interest, etc.).

Whatever the explanation may ultimately be, however, sample-to-sample variation in both drug and placebo response rates among
samples of patients assigned identical psychiatric diagnoses documents that our current psychiatric nosology has poor predictive
power, at least insofar as drug responsiveness is concerned. As a consequence, to demonstrate that their products are effective in
use, sponsors have had little option but to conduct trials using relatively large sample sizes.

Some critics have complained that the FDA, by allowing sponsors to conduct these “overpowered” trials, has turned the drug
regulatory system into an institution that certifies as valuable drug products that provide little, if any, more benefit than placebo
(17 ). Perhaps there is some truth in these allegations, but, then again, who is to say what drug effect is so small as to be dismissible?
As discussed, from a strictly regulatory perspective such criticisms are irrelevant. It is important to recall that Congress could have
set out other and/or more demanding standards for establishing drug effectiveness, but it did not.

The agency does have an obligation under the law, however, to ensure that its effectiveness determinations are based on fair and
reliable estimates of a drug’s measured effects. Accordingly, a large part of the effort expended in the review of an NDA is devoted
to excluding the possibility that bias, rather than a true drug effect, accounts for the positive study reports submitted in the
application.

Great pains are thus taken to ensure that a sponsor’s claims are supported by valid statistical analyses of outcome measures
prospectively identified in each study’s protocol. Each study report is scrutinized for lapses in the execution of a study that might
have introduced bias into the estimates
 P.493

of drug effect it provides. Reviewers search for evidence that the randomization process failed, the treatment mask was penetrated,
subjects failed to comply with protocol requirements, etc. The extent, pattern, and timing of premature discontinuations are
carefully analyzed in an effort to determine whether the censoring process has created a biased estimate of the drug’s effect.

The immediate goal of these efforts is to ensure that the comparison between the investigational and control treatments can be
made “ceteris paribus” (all other things being equal).

The overarching aim is to determine whether or not the evidence submitted meets the Act’s substantial standard fairly and
responsibly.

THE FUTURE OF DRUG REGULATION

Part of "36 - Regulatory Issues "

These are, at once, the best of times and the worst of times for medical therapeutics. On one hand, we have access to more
information about the function and body of humans than we have ever had; therefore, we have good reason to hold sanguine
expectations for the future in regard to the discovery and development of effective new treatments. On the other hand, the
armamentarium stands at increasing risk of being polluted by worthless, even unsafe drugs. Signs of this potential risk abound.

The public is gullible and uncritical where therapeutic claims are concerned, exhibiting not only unrealistic expectations for the
countless potential treatments touted as promising by the medical establishment and the pharmaceutical industry, but a willingness
to spend billions of dollars on unproved, perhaps unsafe, remedies and nostrums.

Although the FDA’s substantive powers under the FFDCA to regulate the drug supply, even after the passage of FDAMA, have been
constrained to only a relatively minor extent, FDA’s mission has noticeably changed.

Today’s agency is expected to work as a “partner” of the regulated industry in a joint effort to expedite the development of new
and promising treatments, not merely to keep unfit drugs off the market. It is arguable whether such dual expectations truly
advance the interests of the public health, however. As the former head of the FAA noted when she resigned in the wake the
ValueJet crash in Florida’s Everglades, it is difficult for a federal regulatory agency to serve simultaneously and well the interests of
both the consumer and the industry it regulates.

Congress has limited FDA’s authority in less obvious ways as well. An egregious and illuminating example of its behavior is the
Dietary Supplement Health and Education Act (DSHEA) of 1994. DSHEA has, by fiat, simply removed whole classes of drugs from
FDA’s jurisdiction and oversight.

By what is as close to an Orwellian 1984-like maneuver as one is likely to find, the Congress simply declared whole classes of drugs
to be something else: nutriceuticals, botanicals, food supplements, etc. These substances escape the premarket drug clearance
requirements of the FDDCA because they are deemed by DSHEA not to be drugs.

Some may consider the legerdemain of DSHEA to constitute a relatively minor threat to the public health. Vitamins, for example,
have long escaped the premarket clearance requirements of the Act, and have not proved dangerous, except perhaps when some
are taken at excessive doses. Subsumed within the ill-described mix of botanicals, nutriceuticals, food supplements, and other
materials deemed to be “nondrugs” under DSHEA, however, are large numbers of incompletely characterized, pharmacologically
active drug substances that have yet to be fully and reliably evaluated for their safety and efficacy.

The lack of data bearing on the safety and efficacy of these drug substances did not deter the Congressional Leadership, however; to
the contrary, when Senator Orin Hatch, a sponsor of DSHEA, introduced the bill he asserted that…dietary supplements can help
promote health and prevent certain diseases, a fact substantiated by an ever-growing body of scientific studies and other evidence.
In my own state of Utah, healthy life-styles, coupled with common use of dietary supplements have made a real difference. Our
state is one of the healthiest in the nation and we enjoy one of the lowest incidence rates for cancer and heart disease. In Utah, the
use of herbs is a well-accepted practice that has passed from generation to generation (18 ).

What is chilling here is not Senator Hatch’s espousal of his state’s virtues and the benefits of herbs, but his seeming willingness to
approach an important public health issue in his nonscientific manner. Even more alarming is the inference congressional passage of
DSHEA allows about the general state of our society’s respect for science and the scientific method. Evidently, a majority of our
elected representatives believes the benefits of scientific progress are attainable without the use of sound scientific methods.

If the armamentarium is to be kept reasonably free of unsafe and ineffective drugs, the antidrug regulatory drift of our national
politics must be reversed. The FDA has demonstrated that it can, with the limited authority it has under the FFDCA, serve as an
effective guardian of the drug supply. It will continue to serve effectively in this role, however, only if it obtains the necessary
political support.

Given the current antiregulatory political environment, and the unrealistic expectations of the body politic for near magical
breakthroughs in therapeutics, that support will not be forthcoming without the full support of the medical, scientific, and health
care communities.

REFERENCES
1. Jackson CO. Doctor Massengill’s elixir. In: Food and drug legislation in the new deal. Princeton, NJ: Princeton University
Press, 1970:151–174.

2. Silverman M, Lee PR. Drug efficacy: the case of the emperor’s old drugs. In: Pills, profits and politics. Berkeley: University
of California Press, 1974:107–137.
 P.494

3. Silverman M, Lee PR. The impossible dream: search for the absolutely safe drug. In: Pills, profits and politics. Berkeley:
University of California Press, 1974:81–106.

4. Anonymous. The extent of population exposure to assess clinical safety: for drugs intended for long-term treatment of non-
life-threatening conditions. Fed Reg 1995;60:11270.

5. Leber P. Postmarketing surveillance of adverse drug effects. In: Lieberman J, Kane J, eds. Adverse effects of psychotropic
drugs. New York: Guilford Press, 1992:3–12.

6. Wood AAJ. Thrombotic thrombocytopenic purpura and Clopidogrel—a need for new approaches to drug safety. NEJM
2000;342:1824–1826.

7. Cohen J. Statistical power analysis for the behavioral sciences, rev ed. Orlando, FL: Academic Press, 1977.

8. Hoffman JE. Administrative procedures of the FDA. In: Adams DG, Cooper FM, Kahan JS, eds. Fundamentals of law and
regulations, vol. 2. Washington, DC: Food and Drug Law Institute, 1977:13–53.

9. Anonymous. Guidance for industry: providing clinical evidence of effectiveness for human drugs and biological products.
Rockville, MD: Food and Drug Administration, 1998.

10. Leber P. The use of placebo control groups in the assessment of psychiatric drugs: an historical context. Biol Psychol
2000;47:699–706.

11. Modell W, Houde RW. Factors influencing clinical evaluation of drugs with special reference to the double-blind technique.
JAMA 1958;167:2190–2199.

12. Leber PD. Hazards of inference: the active control investigation. Epilepsia 1989;30:S57–S63.

13. Leber PD. Is there an alternative to the randomized controlled trial? Psychopharmacol Bull 1991;27:3–8.

14. Khan A, Warner H, Brown W. Symptom reduction and suicide risk in patients treated with placebo in antidepressant clinical
trials: an analysis of the FDA database. Arch Gen Psychiatry 2000;57:311–317.

15. Leber PD, Davis CS. Threats to the validity of clinical trials employing enrichment strategies for sample selection.
Controlled Clin Trials 1998;19:178–187.

16. Wilson M. DSM-III and the transformation of American psychiatry: a history. Am J Psychiatry 1993;150:399–410.

17. Fisher S, Greenberg RP, eds. From placebo to panacea: putting psychiatric drugs to the test. New York: John Wiley, 1997.

18. Statement of Sen. Hatch on Introduction of S. 784/April 7, 1993, Appendix 1: Section H. In: Bass IS, Young AL. Dietary
Supplement Health and Education Act: a legislative history and analysis. Washington, DC: Food and Drug Law Institute, 1996.
 P.495

37
Pharmacogenomics and Personalized Therapeutics in Psychiatry

Vural Özdemir

Vincenzo S. Basile

Mario Masellis

Pierandrea Muglia

James L. Kennedy

Vural Özdemir: Department of Psychiatry and Pharmacology, Center for Addiction and Health, University of Toronto, Toronto,
Ontario, Canada.

Vincenzo S. Basile, Mario Masellis, Pierandrea Muglia, and James L. Kennedy: Department of Psychiatry, Center for Addiction
and Mental Health, University of Toronto, Toronto, Ontario, Canada.

The advances in molecular medicine are taking place at a hitherto unprecedented pace. Genetics has come of age and will greatly
influence the future of health care and therapeutics in the twenty-first century, in much the same way the breakthroughs in
quantum physics and chemistry shaped science and society during the early phases of the twentieth century.

The field of pharmacogenetics was introduced more than 40 years ago to emphasize the role of heredity in person-to-person
differences in drug response (1 ,2 ). The focus of pharmacogenetic investigations has traditionally been unusual and extreme drug
responses resulting from a single gene effect. Pharmacogenomics is a recently introduced concept that attempts to explain the
hereditary basis of both monogenic as well as subtler and continuous variations in drug responses that are under multigenic control
(3 ). Although the two terms are often used interchangeably, the scope of pharmacogenomic investigations follows a genome-wide
approach and also aims to identify novel biological targets for drug discovery, with use of the new affordable high-throughput
molecular genetic technologies (4 ). In theory, pharmacogenomics can assist in clinical decision making to choose the most
appropriate medication and dose titration regimen for individual patients. Moreover, the principles of pharmacogenomics are not
limited to therapeutics. They can be applied to understand the hereditary basis of differences in sensitivity or resistance to any
foreign chemical (xenobiotics) or environmental factor including foodstuffs, pesticides, infectious diseases, and ionizing radiation
(5 ,6 and 7 ).

The Human Genome Project has already provided a draft nucleotide sequence of the human genome by mid-2000 and the nearly
complete sequence is projected to be available by 2003. In addition, nucleotide sequence variations among individuals, populations,
and species will be available in the near future. It is clear that these advances will soon lead to identification of many genes causing
common complex diseases, thereby creating numerous new potential drug targets. This will also present a bioinformatics and data
analysis challenge for lead optimization among numerous new chemical entities (NCE) directed to such disease targets for
therapeutic purposes. Pharmacogenomics is a hybrid research field that bridges the knowledge gained from the Human Genome
Project with existing principles of population genetics, pharmacokinetics, pharmacodynamics, cell physiology, proteomics, and
bioinformatics. It is expected that pharmacogenomics will importantly contribute to development of guidelines for rational and
personalized drug treatment; it should also expedite the drug discovery, development, and approval process in the pharmaceutical
industry (8 ).

The purpose of this chapter is to introduce pharmacogenomics to those from a clinical psychiatry perspective, and discuss the future
research challenges for those who may have prior experience in the field.

 HISTORICAL OVERVIEW AND CONCEPTUAL FRAMEWORK FOR PERSONALIZED THERAPEUTICS

 HUMAN GENETIC VARIATION AND SINGLE NUCLEOTIDE POLYMORPHISMS
 GENETIC VARIABILITY IN DRUG METABOLISM: CONTRIBUTION TO PHARMACOKINETIC VARIABILITY
 CYTOCHROME P450 2D6 GENETIC POLYMORPHISM: A COMMON AND CLASSICAL EXAMPLE OF A MONOGENIC VARIATION IN DRUG
METABOLISM
 CYP3A4: A NONPOLYMORPHIC VARIATION IN DRUG METABOLISM
 GENETIC VARIABILITY IN RECEPTORS AND DRUG TRANSPORTERS: CONTRIBUTION TO PHARMACODYNAMIC VARIABILITY
 PHARMACOGENOMICS AND DRUG DISCOVERY
 PHARMACOGENOMICS AND DRUG DEVELOPMENT
 ETHICAL AND HEALTH POLICY CONSIDERATIONS
 CONCLUSION
 ACKNOWLEDGMENTS

HISTORICAL OVERVIEW AND CONCEPTUAL FRAMEWORK FOR

PERSONALIZED THERAPEUTICS
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "

The marked interindividual variability in psychotropic drug effects was recognized long ago (9 ,10 ). For example, the
 P.496

same dose of an antidepressant medication may cause toxicity, efficacious treatment, lack of efficacy, or qualitatively different
drug effects among patients and populations (11 ). Understanding the sources of such variability in dose–response relationships is
central to individualized dosage and choice of drugs for therapeutic purposes (12 ). Interestingly, it has been a commonly held
viewpoint that genetics is important mainly for the permanent characteristics of an individual (e.g., stature) or predisposition to
certain diseases, rather than variations in drug effects (13 ). In 1932, Snyder documented one of the first known interactions
between heredity and response to xenobiotics: the ability to sense the bitter taste of phenylthiourea, which is under strong genetic
control (14 ). In 1962 Werner Kalow published the first monograph on pharmacogenetics (1 ). Some argue that the field of
pharmacogenetic inquiry dates as early as 510 BC, when Pythagorus in Croton, southern Italy, warned about the “…dangers of some,
but not other, individuals who eat the fava bean” (5 ). The molecular basis of this historic observation was later documented to be
hemolytic anemia owing to glucose-6-phosphate dehydrogenase deficiency.

The interest in personalized therapeutics was further fueled by the thalidomide disaster in 1960s. Subsequent observational studies
found that drug–drug interactions, and hepatic and renal insufficiency importantly contribute to the risk for adverse drug reactions
(15 ). A series of studies in monozygotic and dizygotic twins firmly established that genetic factors play an important role in
metabolism of many drugs, and not only in a few cases of unusual adverse
 P.497

drug reactions (16 ). This led to the publication of systematic guidelines on individualization of drug therapy by the American College of
Physicians, based on genetic, environmental, and disease-related determinants of person-to-person differences in dose–effect relationships (17 ).

The pharmacologic drug response is a complex trait and is likely under polygenic control, rather than simple monogenic regulation (18 ).
However, in comparison to disease-related complex traits, there is a well-established theoretic working model describing the relationship
between the prescribed drug dosage and clinical drug effects (19 ). The conceptual framework developed by the seminal works of Sheiner and
others allows one to target candidate genes with potential mechanistic relevance to partition the variability in any drug effect into
pharmacokinetic and pharmacodynamic components (Fig. 37.1 ) (20 ,21 ,22 ,23 ,24 and 25 ).

FIGURE 37.1. Pharmacological cascade describing the dose:

clinical outcome relationship. Modified from Koch-Weser J. Drug
therapy. Serum drug concentrations as therapeutic guides. N Engl
J Med 1972;287:227–231.

The term pharmacokinetics describes the “drug concentration versus time” relationships in an organism by mathematical formulations of drug
absorption from the site of administration, distribution, and elimination by metabolism and/or excretion (25 ). The pharmacodynamics explains
the “drug concentration versus response” relationships and the related biological covariates (e.g., receptors, second messenger systems) (25 ).
At present, pharmacogenomics is extending the early pharmacogenetic studies of drug metabolism to a broader context, to dissect the genetic
control at multiple levels of the pharmacokinetic and pharmacodynamic pharmacologic cascade, from drug absorption and transport to drug–
receptor interface and beyond. The progress made by pharmacogenomics, in many ways, is akin to developments in the computer industry. The
potential benefits of high speed and efficient computing was self-evident early in the days of cumbersome mainframe computers, but it was not
until the development of low-cost and proficient microcomputers that computerized information processing could be applied in daily life
(analogous to contemporary ultrahigh throughput microarray genotyping technologies).

HUMAN GENETIC VARIATION AND SINGLE NUCLEOTIDE

POLYMORPHISMS
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "

The biallelic single nucleotide polymorphisms (SNPs) represent the most common DNA sequence variation in the human genome. It is thought that
the complete human sequence including the coding regions, introns, and promoters will contain approximately one million SNPs (26 ). SNPs often
result in predictable changes in amino acid sequence and contribute to diversity in protein function. SNPs are valuable biomarkers to elucidate
the genetic basis of common complex diseases and pharmacologic traits. In the near term, the new genomic technologies will allow large-scale
genetic association studies between numerous SNPs and drug response phenotype(s) in large samples of patients during routine drug treatment
and clinical trials.

A pharmacogenetic polymorphism refers to a “…Mendelian or monogenic trait that exists in the population in at least two phenotypes (and
presumably at least two genotypes), neither of which is rare; that is, neither of which occurs with a frequency of less than 1 to 2%…” (27 ). The
definition of the minimum frequency threshold (i.e., 1% to 2%) is arbitrary and aims to emphasize that pharmacogenetic polymorphisms are not
rare and different from those owing to recurrent spontaneous mutations occurring at much lower frequencies. A characteristic feature of
pharmacogenetic polymorphisms is that they are usually biologically silent and do not present an evolutionary disadvantage or result in disease.
This allows the maintenance of the less frequent phenotype at or above the 1% to 2% frequency level. Their clinical manifestations occur only
when exposed to drugs or other xenobiotics, which target the polymorphic gene products.

Evident in the definition of pharmacogenetic polymorphism described in the preceding is the emphasis on phenotype (14 ). Alternative
definitions of pharmacogenetic polymorphisms based on allele frequency also have been suggested (28 ,29 ). For example, Harris (1980) proposed
that a genetic polymorphism occurs if the “…commonest identifiable allele (p) has a frequency no greater than 0.99…” (29 ). It was pointed out
earlier that if the goal of pharmacogenetic studies is to investigate the clinical relevance of pharmacogenetic polymorphisms, phenotype-based
definition of polymorphisms might be more applicable (28 ). On the other hand, if the goal is to use genetic polymorphisms as anthropological
tools to study the evolution of the species and differences in response to xenobiotics between populations, a definition incorporating both allelic
variations and phenotype may be more appropriate to allow understanding at a molecular and mechanistic level (28 ).

GENETIC VARIABILITY IN DRUG METABOLISM: CONTRIBUTION TO

PHARMACOKINETIC VARIABILITY
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "
Drug metabolism is one of the pivotal factors, which contribute to variability in pharmacokinetics. Drug metabolism is generally
divided into two phases. Phase 1 reactions involve oxidative, reductive, and hydrolytic reactions, which unmask or introduce a
functional group (e.g., a hydroxyl-moiety) to the parent compound. This often results in an increase in polarity of the drug. Phase 2
reactions involve conjugation (e.g., with glucuronic acid) of the metabolite produced in phase 1 reactions, or the parent compound,
to more hydrophilic metabolites (30 ). Although drug metabolism is necessary for the elimination of lipophilic drugs (e.g.,
psychotropics), it may also be crucial for activation of prodrugs (e.g., codeine).
 P.498

Phase 1 reactions are mediated, to a large extent, by the CYP enzymes that are mostly found attached to the smooth endoplasmic
reticulum of the hepatocytes and other drug metabolizing cells (e.g., enterocyte in the gut) (31 ). A recent analysis of over 300
drugs from diverse therapeutic classes such as psychotropics, analgesics, and anti-infectious agents found that 56% of them primarily
depend on CYPs for their metabolic clearance (32 ). Among CYPs, the largest contributions are made by CYP3A4 (50%), CYP2D6 (20%),
CYP2C9, and CYP2C19 (15%) (32 ). Some of the drug metabolizing enzymes (e.g., CYP1A2 and CYP2D6) are also expressed in the
brain and may potentially play a role in local disposition of psychotropics at the site of action (33 ,34 ).

CYTOCHROME P450 2D6 GENETIC POLYMORPHISM: A COMMON AND

CLASSICAL EXAMPLE OF A MONOGENIC VARIATION IN DRUG
METABOLISM
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "
CYP2D6 genetic polymorphism is one of the most intensively studied autosomal recessive monogenic defects in drug metabolism.
Many psychotropics, including most typical (e.g., perphenazine) and some atypical antipsychotics (e.g., risperidone), tricyclic
antidepressants (e.g., nortriptyline), drugs of abuse, some of the serotonin reuptake inhibitors (SSRIs) (e.g., paroxetine), and
codeine are metabolized by CYP2D6 (35 ,36 and 37 ).
Among Whites, approximately 7% of the population are poor metabolizers (PMs), whereas the rest are extensive metabolizers (EMs)
for CYP2D6 substrates (35 ). The prevalence of PMs and the distribution of enzyme activity appear to be fairly consistent across the
Western European and North American Whites. On the other hand, the frequency and type of CYP2D6 alleles vary considerably
among different ethnic groups. In Asians, the prevalence of PMs is only 1%, owing to almost complete absence of the nonfunctional
alleles (e.g., CYP2D6*4) found in Whites (35 ). However, an often overlooked point in comparisons of pharmacogenetic
polymorphisms between populations (e.g., Asians versus Whites) is that the key dependent variable is not only the prevalence of PMs
but also the distribution of enzyme activity within EMs (36 ). Asian EMs (i.e., 99% of the population) display a significant shift in the
distribution of CYP2D6 activity toward lower levels. The molecular basis of a lower CYP2D6 activity in Asian EMs is owing to a C188→T
base change in exon 1 which leads to Pro34→Ser amino acid substitution in a highly conserved region (Pro-Pro-Gly-Pro) characteristic
of CYP1 and CYP2 families (37 ). This allele was named CYP2D6*10 (51% allele frequency in Chinese) and leads to a 10-fold decrease
in activity in vivo (35 ). Thus, there may be discrete interindividual differences in disposition and therapeutic/adverse effects of
psychotropics within Asians, depending on the gene-dose for the CYP2D6*10 allele.
Another novel allele with reduced catalytic function, CYP2D6*17, occurs at high frequency in many black African populations and
African-Americans. However, there appears to be considerable heterogeneity in the CYP2D6 locus in Black populations. For example,
in Ethiopia, only 1.8% were PMs of debrisoquine, 16% carried the CYP2D6*10B allele characteristic of Asian populations, and the
CYP2D6*17 was present in 18% of the subjects (38 ). Importantly, 29% of the Ethiopians carried alleles with duplicated and
multiduplicated CYP2D6 genes associated with ultrarapid metabolism of substrates (38 ). Also, a high percentage of gene duplication
and ultrarapid metabolism was found in Saudi Arabia and Spain, presumably owing to the genetic admixture during the earlier
Islamic migration originating from some of the North African populations with ultrarapid CYP2D6 activity (36 ,37 ). The clinical
significance of reduced catalytic function associated with the CYP2D6*17 allele requires further research in patients with African
ancestry.
At present, more than 50 CYP2D6 alleles were described that encode an enzyme with inactive, decreased, increased, or normal
catalytic function. The number of functional CYP2D6 genes correlates with drug and metabolite concentrations in the plasma, as
aptly documented using nortriptyline as a model substrate (Fig. 37.2 ) (39 ). In general, PMs are at risk for drug toxicity on
treatment with medications predominantly inactivated by metabolism via CYP2D6. On the other hand, in prodrugs, which need to be
converted to their active form by CYP2D6, opposite clinical consequences may occur in PMs. For example, codeine does not produce
analgesic effects in PMs or after treatment of EMs with CYP2D6 inhibitors such as quinidine. Among EMs, those with duplicated or
multiduplicated CYP2D6 genes and ultrarapid metabolism may develop subtherapeutic plasma concentrations and inadequate
clinical response (40 ). Although high doses would be necessary in such patients, an alternative strategy would be to use low
subtherapeutic doses of the CYP2D6 inhibitor quinidine, especially for drugs with high acquisition costs, to attain therapeutic plasma
concentrations (41 ). Overall, routine genotyping for CYP2D6 may be useful to avoid drug toxicity in PMs, to ascertain the
pharmacokinetic mechanism of resistance to some psychotropics, as well as for differential diagnosis of noncompliance versus
ultrarapid CYP2D6 activity. In addition, the markedly increased metabolite formation in patients with multiduplicated CYP2D6 genes
may potentially lead to qualitatively different and unexpected drug effects and toxicity (40 ).

FIGURE 37.2. Average plasma concentrations of nortriptyline and 10-hydroxynortriptyline after a 25-mg single oral dose in
White healthy volunteers with 0, 1, 2, 3, and 13 functional copies of the CYP2D6 gene. Note that the concentration of
nortriptyline and its metabolite 10-hydroxynortriptyline are inversely affected by the number of functional CYP2D6 gene.
Reprinted with permission from Dalén P, Dahl ML, Ruiz ML et al. 10-Hydroxylation of nortriptyline in white persons with 0, 1, 2,
3, and 13 functional CYP2D6 genes. Clin Pharmacol Ther 1998;63:444–452.

It is noteworthy that CYP2D6 with identical pharmacologic and molecular properties was identified in microsomal fractions in the
brain. Hence, CYP2D6 may potentially contribute to local clearance of psychotropics at the site of action (42 ). Moreover, CYP2D6 in
the brain is functionally associated with the dopamine transporter and shares similarities
 P.499

in substrates and inhibitors (e.g., d-amphetamine), suggesting a role in dopaminergic neurotransmission (42 ). Differences in
personality traits between EMs and PMs were noted in both Swedish and Spanish healthy White subjects, also suggesting that there
may be an endogenous substrate for CYP2D6 in the brain (42 ).

The common polymorphic drug metabolizing enzymes in humans and their major variant alleles are presented in Table 37.1 (37 ). A
worldwide web page with detailed descriptions of new alleles, nomenclature and useful references can be found at
(http://www.imm.ki.se/CYPalleles/).

TABLE 37.1. HUMAN POLYMORPHIC CYTOCHROME P450 ENZYMES AND THE

GLOBAL DISTRIBUTION OF THEIR MAJOR VARIANT ALLELES

CYP3A4: A NONPOLYMORPHIC VARIATION IN DRUG METABOLISM

Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "

The term “polymorphic metabolism” is often perceived as an alarming indication of marked variability in drug disposition. Although
this assertion is correct to a certain extent, it does not imply that a nonpolymorphic drug-metabolizing enzyme is associated with
reduced variability. For example, CYP3A4 is the most abundant CYP isoform in the adult human liver with large interindividual
variability in its expression. In vivo, CYP3A4 activity displays at least 20-fold
 P.500

difference in the population (43 ). Yet, the distribution of CYP3A4 catalytic activity is unimodal and nonpolymorphic in many
populations.
CYP3A4 contributes to disposition of more than 60 frequently prescribed therapeutic agents with diverse chemical structures
including antilipidemics, benzodiazepines, HIV protease inhibitors, immunosuppressants, and macrolide antibiotics (43 ,44 ). CYP3A4
also plays an important role for the metabolism of endogenous steroids (e.g., testosterone) as well as activation of dietary
mycotoxins (e.g., aflatoxin B1) (43 ). The prediction of CYP3A4-mediated drug metabolism is complicated by the presence of at least
two distinct pools of the CYP3A4 protein, in the liver and intestine, whose expressions appear to be regulated independently. The
appreciation of marked variability in CYP3A4 activity is critical for individualized treatment with CYP3A4 substrates, to forecast
drug–drug interactions mediated by CYP3A4, and to identify the factors predisposing to long-term toxicity (e.g., prostate and liver
cancer) associated with variable metabolism of steroid hormones and procarcinogens (44 ).
CYP3A4 expression can be markedly induced in vivo during chronic treatment with drugs such as the antibiotic rifampicin,
anticonvulsant carbamazepine, and glucocorticoid dexamethasone (43 ). Conversely, CYP3A4 catalytic activity can be inhibited
potently by commonly used drugs including the azole antifungal agents (e.g., ketoconazole) and the macrolide antibiotics (e.g.,
erythromycin), or by foodstuffs such as grapefruit juice (44 ). For example, excessive sedation or psychomotor impairment can occur
after oral administration of benzodiazepines with a low bioavailability (e.g., triazolam) or some nonbenzodiazepine (e.g., buspirone)
hypnosedatives together with grapefruit juice (44 ). Many patients with a mental health problem also use nonpsychotropic
medications. In such cases, clinically significant hypotension may be observed during treatment with dihydropyridine calcium
channel antagonists (e.g., nifedipine) and CYP3A4 inhibitors.
Studies in monozygotic and dizygotic twins indicate a high heritability (H2 = 0.88) of CYP3A4 activity (45 ); however, there has been
relatively little progress in identification of the molecular genetic underpinnings of heterogeneity in CYP3A4 expression. Recently, a
novel allele, CYP3A4*2, causing a Ser222Pro change was found in Whites at a frequency of 2.7%, but this allele was absent in Black
and Chinese subjects (46 ). The CYP3A4*2 displays a substrate-dependent diminished metabolic clearance; for instance, nifedipine
(but not testosterone) intrinsic clearance is impaired (46 ). Because functional polymorphisms in the promoter or the coding region
of CYP3A4 do not appear to be very common, it is likely that CYP3A4 activity represents a complex trait regulated by multiple
interacting genetic loci in the genome (47 ).

GENETIC VARIABILITY IN RECEPTORS AND DRUG TRANSPORTERS:

CONTRIBUTION TO PHARMACODYNAMIC VARIABILITY
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "
Historically, the pharmacogenetic factors related to drug efficacy and safety were mainly studied in the context of drug metabolism
(48 ). In the past decade, the increasing application of mathematical models for “concentration versus effect” relationships during
routine drug development clearly documented the marked interindividual variability in pharmacodynamics (23 ). It is estimated that
approximately 30,000 proteins with diverse structures are expressed in the human brain, many of which may serve as potential drug
targets (49 ). Recent studies of SNPs in genes relevant for psychotropic pharmacodynamics indicate that human genetic variation in
drug receptors and transporters may significantly contribute to overall variance in response to drugs.
Dopamine D3 receptor (DRD3) gene is expressed in the basal ganglia and is thought to play a role in locomotion. Three independent
studies found that the Ser9Gly polymorphism in the N-terminal extracellular domain of the DRD3 is associated with an increased
propensity to develop tardive dyskinesia in patients treated with typical antipsychotics (Fig. 37.3 ) (50 ,51 and 52 ).
Pharmacogenetic polymorphisms in the dopamine D4 receptor (DRD4) gene, especially the hypervariable exon III 48 bp variable
number of tandem repeat (VNTR), have been studied intensively in relation to antipsychotic response to clozapine (53 ). Although
the exon III 48 bp VNTR in DRD4 does not appear to be a major contributor to clinical outcome during clozapine treatment, it is
possible that haplotype analyses incorporating several variants in different locations would be necessary before definitive
conclusions can be drawn for the importance of DRD4 in antipsychotic response. Moreover, studies involving the serotonin receptor
gene polymorphisms suggest an association between the 5-HT2A receptor and response to clozapine (53 ,54 and 55 ). Other
neurotransmitters such as norepinephrine, acetylcholine, and glutamate may also contribute to antipsychotic drug effects, but
genetic variation in these receptors has not been investigated following a pharmacogenomic perspective (18 ). A detailed review of
polymorphisms in dopamine and serotonin receptor genes, their relevance for response to clozapine and other atypical
antipsychotics, and methodologic considerations for application of molecular approaches to psychiatric genetics are available
elsewhere (53 ).

FIGURE 37.3. Average Abnormal Involuntary Movement Scale (AIMS) scores in 112 schizophrenic patients previously treated
with typical antipsychotics and genotyped for the serine to glycine polymorphism in the N-terminal extracellular domain of the
dopamine D3 (DRD3) receptor. A post hoc Student-Newman-Keuls test revealed a higher average AIMS score in patients
homozygous for the glycine allele of the DRD3 gene, compared to those with a heterozygous or homozygous genotype for the
serine allele. The analysis of variance results were corrected for age, gender, ethnicity and pairwise comparisons (F = 8.25, df
= 2, P < 0.0005 [P < 0.0015, Bonferroni corrected]). Reprinted with permission from Basile VS, Masellis M, Badri F, et al.
Association of the MscI polymorphism of the dopamine D3 receptor gene with tardive dyskinesia in schizophrenia.
Neuropsychopharmacology 1999;21:17–27.

Because the first report more than 60 years ago on the use of amphetamine in children with attention deficit hyperactivity disorder
(ADHD), it became clear that approximately 75% of ADHD cases show clinically significant improvement after d-amphetamine or
methylphenidate treatment. Although the precise mechanism of action of these stimulant agents still remains elusive, their
interaction with the dopamine transporter may contribute to their therapeutic
 P.501

effects in ADHD. The VNTR polymorphism of the dopamine transporter gene appears to influence the response to methylphenidate,
based on a preliminary study in 30 African-American children with ADHD (56 ). These limited data, however, do not allow
generalizations on genetic determinants of response to pharmacologic interventions in ADHD at the present time.

The marked temporal delay in therapeutic effects is a well-known phenomenon with antidepressant agents. Therefore, it is
advantageous to identify beforehand the subpopulation of patients who are unlikely to respond to a given medication so that various
augmentation efforts can be initiated promptly. The high-affinity serotonin transporter (5-HTT) is a prime target for the serotonin
reuptake inhibitor antidepressants (SSRIs). A functional polymorphic variant of the 5-HTT gene characterized by a 44-bp insertion in
its promoter region leads to differences in the amount of 5-HTT transcript and the extent of 5-HT reuptake (57 ). Clinical studies
suggest that the 44-bp insertion polymorphism of the 5-HTT gene influences the antidepressant response to SSRIs including
fluvoxamine and paroxetine (57 ). Further studies with other SSRIs and classical tricyclic antidepressants are called for to assess the
overall clinical significance of 5-HTT promotor polymorphism(s).

P-glycoprotein encoded by the MDR1 gene is another drug transporter that affects transmembrane efflux and intracellular or tissue
availability of numerous drugs. For example, amitriptyline (but not fluoxetine) can penetrate the brain more readily in knockout
mice that do not express p-glycoprotein (58 ). Hence, differences in MDR1 expression owing to genetic polymorphisms or secondary
to chronic antidepressant treatment may explain treatment-resistance to amitriptyline in patients who otherwise attain therapeutic
plasma drug concentrations.

The study of pharmacogenetic polymorphisms in drug targets is a relatively new but rapidly expanding research area. It is likely that
molecular genetic profiling of patients for SNPs or other types of human genetic variation in both pharmacokinetic and
pharmacodynamic targets will bring psychiatric genetics and clinical pharmacology one step closer to achieve the ultimate goal of
individualized therapeutics.

PHARMACOGENOMICS AND DRUG DISCOVERY

Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "

The drug discovery in psychiatry was initially based on serendipity. The identification of lithium in 1949 and chlorpromazine in 1950s
are two well-known examples where putative mechanisms of action were elucidated after the drugs were shown to be efficacious.
The newer drug discovery paradigms have depended on the synthesis and identification of novel compounds through combinatorial
chemistry
 P.502

and screening for biological activity against known receptors or other biological targets with established endogenous ligands or
substrates (59 ,60 ). With the Human Genome Project approaching to its completion, essentially all human genes will be available as
potential drug targets. The challenge in drug discovery will then be to discern the function and therapeutic utility of these genes
and their expressed products.

The experimental paradigms used by pharmacogenomics borrow substantially from the field of population genetics and the
methodology used in earlier genetic studies of common complex diseases (60 ,61 ). For example, linkage and association studies are
two well-known strategies to identify the genes causing a specific disease or variability in drug effects. The linkage design was
traditionally used to test the relationship between inheritance of a complex disease phenotype within family members and
microsatellite markers comprised of five or less short tandem repeats of DNA. The increasing availability of SNP markers and the
ability to genotype the entire genome of large segments of patient populations with ultrahigh throughput methods such as the DNA
microarrays, often referred to as “DNA chips,” now allow the application of genetic linkage or association designs to elucidate the
genes responsible for variations in therapeutic response and toxicity. On the other hand, the obvious difficulties in administering
drugs to different family members and obtaining relevant data on drug response phenotype may pose a constraint on application of
linkage design to pharmacogenomics.

DNA microarray is an emerging powerful technological breakthrough that enables the study of global gene expression patterns and
sequence variations at a genome level (62 ). In essence, DNA microarray is an extension of the Southern blot procedure and is
comprised of different cDNAs or oligonucleotides etched systematically on a solid surface such as silica or glass plate. Each DNA
species on the array represents a specific gene or expressed sequence tag, which is used to identify different SNPs or transcripts by
hybridization and fluorescence detection. Microarrays with 10,000 or more genes are now available for use in clinical research or
trials. An important application of microarrays is monitoring of temporal changes in gene expression during drug treatment or
patients versus healthy individuals. The premise in these studies is that patterns of gene expression may serve as indirect clues
about disease-causing genes or drug targets. Moreover, the effects of drugs with established efficacy on global gene expression
patterns may provide a guidepost, or a “genetic signature,” against which the new drug candidates can be validated (63 ). Other
new genomic technologies such as genotyping by mass spectrometry also are being developed. Collectively, pharmacogenomics adds
another dimension to contemporary drug discovery efforts because it aims to identify novel drug targets in the entire human
genome without a priori assumptions on disease pathogenesis or drug targets, thereby presenting an opportunity to unlock
unprecedented novel mechanisms of drug action (4 ).

PHARMACOGENOMICS AND DRUG DEVELOPMENT

Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "

After the discovery of an NCE with therapeutic potential, the next step involves clinical testing in healthy volunteers and relevant
target patient populations. For every clinician, an appreciation of the drug development process is important to make evidence-
based choices among therapeutic alternatives and to be aware of the shortcomings of the data presented to support the efficacy and
safety of new medications.

The drug discovery and development is a high-risk venture. Typically, it takes 8 to 12 years to introduce a new drug from discovery
to clinical practice, with costs often approaching $100 to $300 million. On the other hand, it is estimated that approximately 90% or
more of NCEs under development fail to meet the regulatory approval for clinical use (64 ). It is well known to most pharmaceutical
scientists that the art of timely and cost-effective drug development rests on early identification and removal of drug candidates
with poor efficacy and safety. It is conceivable that some of these NCEs may in fact have a favorable efficacy and safety profile in
certain genetically determined subpopulations. Through proper design of clinical trials and using low-cost high-throughput genetic
analyses, pharmacogenomics eventually can allow patenting of such “failed” NCEs in discrete patient populations and reinstate their
market potential. In addition, a genetic test predicting drug effects would be considered another pharmaceutical product and an
additional financial incentive for drug developers. For example, clozapine was recognized as a potential antipsychotic drug in early
1970s. The occurrence of agranulocytosis in several cases caused the termination of further development of clozapine in treatment-
resistant patient populations until the late 1980s. The presence of genetic or other predictors of agranulocytosis would have
expedited the development of clozapine and prevented the inconvenience of periodical hematologic monitoring.

Pharmacogenomics is also relevant for better use of medications that are already in routine clinical use (phase 4 drug development).
A recent meta-analysis of prospective studies from 1966 to 1996 found that the incidence of serious and fatal adverse drug reactions
in United States was 6.7% and 0.32%, respectively; ranking between the fourth to sixth leading cause of death, ahead of pneumonia
and diabetes (65 ). Importantly, the adverse drug reactions in the latter study occurred during treatment with usual doses of drugs
that already met the regulatory requirements for clinical use, and excluded cases owing to intentional or accidental overdose, errors
in drug administration, or noncompliance.
 P.503

It is likely that the proportion of such patients who are inadequately treated may further increase after accounting for therapeutic
failures secondary to ultrarapid drug metabolism, for instance, and mismatches between the pharmacodynamic attributes of
medications and drug targets in individual patients (23 ,37 ). Evidently, the existing pharmacotherapy system based on the
traditional trial-and-error approach is unable to deliver personalized drug treatment and health care.
From the perspective of patients, healthcare providers, and managed care organizations, an increased probability of therapeutic
response through genetic testing would reduce duration of inpatient hospital care, frequency and inconvenience of repeated
physician visits owing to treatment resistance, and thus, easily offset the costs of pharmacogenomic-based drug development. For
NCEs that readily meet the regulatory requirements with large efficacy margins (i.e., “blockbuster drugs”) over placebo or the
existing standard treatment modalities, there may be less financial incentive—on the manufacturers’ part—to identify different
subpopulations with differing drug effects, because this may potentially decrease the market share of their newly introduced
medication. Therefore, although pharmacogenomics provides a clear rationale for improved drug discovery and personalized
therapeutics, it will likely need enforcement by regulatory agencies before it can be utilized in routine clinical practice and
pharmaceutical industry. This may in turn require amendments to existing regulatory policies for drug development. Also, as a result
of the global harmonization attempts to standardize drug development and approval process, the settings of future clinical trials will
not only be limited to Western society; therefore, it would be critical and advantageous to plan such policy amendments at a
multinational level.

ETHICAL AND HEALTH POLICY CONSIDERATIONS

Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "
Pharmacogenomics arrived at the heels of the molecular biology revolution in 1980s and early 1990s. Although there is much
optimism for a more efficient drug discovery and development process, there is also increasing concern about the implementation of
genetic testing at various levels of the medical practice and its repercussions for health policy and managed care organizations.
Confidentiality of the genetic test results is critical because it has important bearings on finding employment and obtaining life,
health, or disability insurance. There is an urgent need to amend the existing medical curriculum to educate future clinical
personnel for genetic counseling and fundamentals of molecular medicine. About 5% of the budget for the Human Genome Project is
reserved to address these social and ethical issues.

CONCLUSION
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "
Personalized therapeutics has been a preoccupation in clinical psychiatry for many decades. The traditional gene-by-gene approach
to explain variability in drug response has been a mainstay in most pharmacogenetic investigations to date. However, the biological
underpinnings of drug response are complex and often involve contributions by multiple genes, environmental factors, drug–drug and
drug–food interactions, to mention a few (Fig. 37.4 ) (18 ,66 ,67 ,68 ,69 ,70 and 71 ). Clearly, a genome-wide approach will be an
important advance in understanding the variability in drug efficacy and safety. To this end, sequence variations in the genome are
only the first level of complexity. More intriguing and challenging is establishing the significance of differences in global gene
expression patterns in relation to drug effects and targets. High throughput and genome-wide transcript profiling for differentially
regulated mRNA species in disease, normal physiology, and after drug treatment offer an additional dynamic perspective for drug
discovery. The development of protein chips may permit further explorations of functional genomics in the context of
psychopharmacology. We may soon be surprised that the mechanism of action of some psychotropics may in fact rest on targets
entirely different than what the conventional pharmacologic wisdom suggests (e.g., the monoamine hypothesis for antidepressant
drug effects).

FIGURE 37.4. The interaction of genetic and

environmental factors that may influence drug
response in humans. Reprinted with permission
from Vesell ES. On the significance of host
factors that affect drug disposition. Clin
Pharmacol Ther 1982;31:1–7.

At the present time, however, it is not clear whether and to what extent the genomic hypotheses can be tested within the
framework of the available clinical trial methodology. For example, the sample size in most phase 3 clinical trials does not usually
exceed 3,000 to 4,000 patients. Genome-wide association studies and statistical correction for multiple testing will require sample
sizes well beyond the current resources of any single pharmaceutical company or an academic laboratory. Ideally,
pharmacogenomics should be used for the prospective design of phase 3 clinical trials and not to salvage an NCE that proved to be
ineffective or unsafe at the end of phase 3 investigations. Care should be taken for adequate representation of each subpopulation
identified by genetic markers. The information obtained by genomic methods should ultimately be translated into discrete product
labeling information. Otherwise, it is uncertain whether the off-label data available in the form of scientific publications will
transform routine clinical practice and lead to personalized therapeutics. Also, genomic data are fundamentally different than the
traditional covariates (e.g., weight, age) that have been used to explain variability in drug efficacy and safety, and thus require
special considerations. Clear regulatory guidelines and new collaborations between academic institutions and the pharmaceutical
industry, both at the level of basic and clinical research, are called for to implement pharmacogenomics in the drug development
process and evaluate its significance for achieving drug safety, efficacy and effectiveness (72 ,73 and 74 ).
 P.504

Pharmacogenomics emerged in late 1990s by coalescence of traditional methodologies used in human genetics, common complex
diseases and pharmacogenetics, together with the impetus provided by novel genomic technologies developed as part of the Human
Genome Project. The collection of genomic data is being more feasible by increasing accessibility and decreasing costs of molecular
genetic analyses. Pharmacogenomics has far-reaching implications in medicine and biology and can be applied to various facets of
therapeutics from drug discovery and neuroimaging to drug–drug and drug–food interactions (5 ,6 ,75 ). The road from
pharmacogenomics to personalized therapeutics is arduous and challenging but the technology is now in place to validate the utility
of pharmacogenomics in routine clinical practice and pharmacotherapy (76 ).

ACKNOWLEDGMENTS
Part of "37 - Pharmacogenomics and Personalized Therapeutics in Psychiatry "

V. Özdemir is the recipient of a postdoctoral fellowship from the Ontario Mental Health Foundation and a NARSAD young investigator
award. M. Masellis is the recipient of a research studentship from the Faculty of Medicine, University of Toronto. P. Muglia is
supported by a postdoctoral fellowship from the Canadian Institutes of Health Research and the Schizophrenia Society of Canada. J.L.
Kennedy is supported by grants-in-aid from the Canadian Institutes of Health Research and a NARSAD independent investigator
award. The authors thank Professors Werner Kalow and Laszlo Endrenyi for many insightful discussions and continuing support and
encouragement.

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38
Pharmacokinetics, Pharmacodynamics, and Drug Disposition

David J. Greenblatt

Lisa L. Von Moltke

Jerold S. Harmatz

Richard I. Shader

D. J. Greenblatt, L. L. von Moltke, J. S. Harmatz, and R. I. Shader: Department of Pharmacology and Experimental Therapeutics,
Tufts University School of Medicine, and Division of Clinical Pharmacology, New England Medical Center, Boston, Massachusetts.

During the last decade, the application of pharmacokinetic and pharmacodynamic modeling techniques has become an increasingly
important aspect of contemporary clinical psychopharmacology (1 ,2 ,3 ,4 and 5 ). These techniques have been applied during the
process of development of new drug entities as well as for the improved understanding of the clinical actions of drugs that are
already marketed. Techniques for the study of drug metabolism in vitro have advanced substantially during the last decade, and
now are an integral component of preclinical drug development and the link to subsequent clinical studies of drug metabolism and
disposition. Kinetic-dynamic modeling techniques have been combined with in vitro metabolism procedures and in vitro–in vivo
mathematical scaling models to provide insight into the general problem of pharmacokinetic drug interactions in clinical
psychopharmacology (6 ,7 ,8 and 9 ).

This chapter reviews some advances in pharmacokinetics, pharmacodynamics, and drug metabolism, along with methodologic
applications to selected problems in clinical psychopharmacology.

 POPULATION PHARMACOKINETICS
 KINETIC-DYNAMIC MODELING
 CYTOCHROMES P-450 IN PSYCHOPHARMACOLOGY: THE IMPORTANCE OF P-450-3A ISOFORMS
 DRUG INTERACTIONS IN PSYCHOPHARMACOLOGY
 COMMENT
 ACKNOWLEDGMENTS

POPULATION PHARMACOKINETICS
Part of "38 - Pharmacokinetics, Pharmacodynamics, and Drug Disposition "

Principles
Pharmacokinetic studies based on a traditional intensive-design model are usually conducted using carefully selected volunteer
subjects, a controlled experimental design, and collection of multiple blood samples. After measurement of drug and metabolite
concentrations in all samples, pharmacokinetic models are applied to determine parameters such as elimination half-life, volume of
distribution, and clearance. During the new drug development process, a series of pharmacokinetic studies are conducted to
determine the influence of major disease states or experimental conditions hypothesized to affect drug disposition. Such factors
might include age, gender, body weight, ethnicity, hepatic and renal disease, coadministration of food, and various drug
interactions. Classical pharmacokinetic studies can quantitate the effects of anticipated influences on drug disposition under
controlled circumstances, but cannot identify the unexpected factors affecting pharmacokinetics. A number of examples of altered
drug pharmacokinetics became apparent in the patient care setting only in the postmarketing phase of extensive clinical use.
Examples include the digoxin-quinidine interaction, altered drug metabolism due to cimetidine, and the ketoconazole-terfenadine
interaction.

Population pharmacokinetic methodology has developed as an approach to detect and quantify unexpected influences on drug
pharmacokinetics (10 ,11 ,12 ,13 ,14 ,15 ,16 ,17 and 18 ). Population pharmacokinetic studies, in contrast to classical or traditional
pharmacokinetic studies, focus on the central tendency of a pharmacokinetic parameter across an entire population, and identify
deviations from that central tendency in a subgroup of individual patients. One software program widely applied to population
pharmacokinetic problems is the nonlinear mixed-effects model (NONMEM). Analysis of clinical data using a population approach
allows pharmacokinetic parameters to be determined directly in patient populations of interest and allows evaluation of the
influence of various patient characteristics on pharmacokinetics. Because the number of blood samples that need to be collected
per subject is small, this approach is often suitable for patient groups unable to participate in traditional pharmacokinetic studies
requiring multiple blood samples (e.g., neonates,
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children, critically ill patients, or individuals who are not able to provide informed consent) (19 ). In many cases the population
approach has yielded pharmacokinetic parameter estimates similar to those delineated in classical pharmacokinetic studies of the
same drug.

Application: Methylphenidate Pharmacokinetics

The population approach is illustrated in a study of methylphenidate (MP) pharmacokinetics in children (20 ). This is a patient group
for whom the multiple-sample pharmacokinetic study design may not be appropriate for ethical and practical reasons. Participating
subjects were 273 children aged 5 to 18 years having a primary diagnosis of attention-deficit/hyperactivity disorder (ADHD). They
had been receiving MP at a fixed dosage level for at least 4 weeks, and were under treatment for at least 3 months. The treating
physician for each patient judged MP to be clinically effective.

Children meeting the eligibility criteria had an initial screening visit, at which one parent or a legal guardian provided written
informed consent, and the child provided assent. Demographic characteristics were recorded, including the dosage of MP, the usual
times for individual doses, and the duration of treatment.

The second visit, which followed shortly, was a blood-sampling day. Each child, accompanied by parent or guardian, arrived at the
investigator’s office 30 to 60 minutes prior to blood sampling. The time and size of the last MP dose, and of any other medication
received that day or during the prior 2 weeks, were recorded. A 5-mL whole blood sample was obtained by venipuncture. This
sample was immediately centrifuged, and a 2-mL aliquot of plasma was removed for subsequent determination of MP concentrations
by a liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS) assay.

Analysis of Data
The identified independent variables were age, sex, body weight, size of each dose, and time of sample relative to the most recent
dose. Since only single samples were available for all but 16 of these children, the contribution of within-subject variance to overall
variability in outcome could not be assessed. The pharmacokinetic model was a one-compartment model with first-order absorption
and first-order elimination, under the assumption that all subjects were at steady state (Fig. 38.1 ).

FIGURE 38.1. Population pharmacokinetic model for methylphenidate (MP). A series of data points, each consisting of the time
(t) after the first dose of the day and the plasma MP concentration (C) at that time, was available from 273 subjects (one data
point per subject). Each of these was linked to that subject’s individual dose schedule, size of each dose, interval between
doses, and body weight. These variables were entered into a one-compartment pharmacokinetic model with first-order
absorption and first-order elimination, as shown. Using nonlinear regression, the process yielded “typical” population values of
clearance per kilogram body weight, the elimination rate constant (Ke), and the absorption rate constant (Ka).

The overall model was specifically modified for each of the 273 subjects to incorporate the individually applicable independent
variables, as well as the dosage schedule (b.i.d. or t.i.d.). Individual values of continuous variables (t = time sample taken relative
to the first dose; C = plasma MP concentration) were fitted to a single set of iterated variables using unweighted nonlinear
regression (Fig. 38.1 ). When the time between first and second doses, or between second and third doses, was not available, the
mean value was assigned based on cases in which the data were available. For the b.i.d. dosage, the mean interval was 4.3 hours.
For the t.i.d. dosage, the mean intervals were 4.1 and 3.7 hours, respectively. As is customary, clearance was assumed to be
proportional to body weight.

Results
The total daily dose of MP was significantly lower in subjects receiving MP b.i.d. (n = 109) compared to subjects on a t.i.d. schedule
(n = 164); the mean total daily dosages in the two groups were 25 and 39.3 mg, respectively (p <.001). Within each group, clinicians’
choices of total daily dosages were influenced by body weight, as mean total daily dose increased significantly with higher body
weights. However, the association of body weight with mean plasma concentration was not significant for the b.i.d. dosage group,
and of only borderline significance (.05< p <.1) for the t.i.d group. This finding is consistent with the underlying assumption that
clearance is proportional to body weight.

Age was significantly correlated with body weight (r2 = 0.54, p <.001) and with height (r2 = 0.77). Height and body weight also were
significantly correlated (r2 = 0.77).

An acceptable estimate of absorption rate constant could be derived only for the b.i.d. dosing data. The iterated parameter
 P.509

estimate was 1.192/h, corresponding to an absorption half-life of 34.9 minutes. This estimate was then fixed, and the entire data
set analyzed to determine clearance per kilogram of body weight, and the first-order elimination rate constant. The iterated
estimates were 0.154/h for elimination rate constant, corresponding to an elimination half-life of 4.5 hours (relative standard error:
23%). For clearance, the estimate was 90.7 mL/min/kg (relative standard error: 9%). The overall r-square was 0.43 (Fig. 38.2 ).
There were no evident differences in pharmacokinetics attributable to gender. Figure 38.3 shows predicted plasma MP concentration
curves for b.i.d. and t.i.d. dosage schedules, based on the population estimates.

FIGURE 38.3. Predicted plasma methylphenidate concentration curves for b.i.d. and t.i.d. dosage schedules, based on
parameter estimates from the population analysis, together with mean values of input variables (body weight, size of doses,
intervals between doses). (From Shader RI, Harmatz JS, Oesterheld JR, et al. Population pharmacokinetics of methylphenidate
in children with attention-deficit hyperactivity disorder. J Clin Pharmacol 1999;39:775–785, with permission).

FIGURE 38.2. Overall relation of observed and predicted plasma methylphenidate concentrations (ng/ml). The r-square value
of 0.43 indicates that the model accounts for 43% of the overall variance in plasma concentrations. (From Shader RI, Harmatz
JS, Oesterheld JR, et al. Population pharmacokinetics of methylphenidate in children with attention-deficit hyperactivity
disorder. J Clin Pharmacol 1999;39:775–785, with permission.)

Implications
Pharmacokinetically based approaches to the treatment of ADHD with MP are not clearly established (21 ,22 ,23 ,24 and 25 ). In the
present study of prescribing patterns in particular clinical practices, the mean prescribed per dose amount for the whole study
population was 0.335 mg/kg per dose (range = 0.044–0.568), and 36% of the children received between 0.25 and 0.35 mg/kg per
dose. The mean total daily dose was 0.98 mg/kg/day for the entire sample, and increased significantly in association with larger
body weight. This may reflect the clinicians’ considering body weight in their choice of total daily dosage, or it may be that the dose
was titrated according to response, which in turn was influenced by associations among concentration, clearance, and weight.

The pharmacokinetic model explained 43% of the variability in plasma MP concentrations during typical naturalistic therapy. The
model fit equally well for both genders. Assuming that clearance is proportional to body weight in the context of intercorrelated age
and weight allows age, weight, and daily dosage to be used to predict plasma concentrations of MP during clinical use in children.
These findings support the value of prescribing MP on a weight-adjusted basis.

Our typical population value of elimination half-life was 4.5 hours, with a confidence interval of 3.1 to 8.1 hours. This estimate
somewhat exceeds the usual range of half-life values reported in single-dose kinetic studies of MP (25 ,26 ). This could reflect the
relatively small number of plasma samples from the terminal phase of the plasma concentration curve, upon which reliable
estimates of beta are dependent. MP kinetics may also have a previously unrecognized dose-dependent component, in which
estimated values of half-life are larger at steady state than following a single dose.
 P.510

The single-sample approach described in this study allows relatively noninvasive assessment of pharmacokinetic parameters in a group of children and adolescents under naturalistic
circumstances of usual clinical use, when blood sampling is not otherwise clinically indicated. This approach in general can be applied to other special populations such as neonates,
the elderly, or individuals with serious medical disease.

KINETIC-DYNAMIC MODELING
Part of "38 - Pharmacokinetics, Pharmacodynamics, and Drug Disposition "

Principles
Pharmacokinetics is the discipline that applies mathematical models to describe and predict the time course of drug concentrations in body fluids, whereas pharmacodynamics refers
to the time course and intensity of drug effects on the organism, whether human or experimental animal (Fig. 38.4 ). Both have evolved as the techniques for measuring drug
concentrations, and drug effects have become more accurate and sensitive. Evolving in parallel is kinetic-dynamic modeling, in which the variable of time is incorporated into the
relationship of effect to concentration (Fig. 38.4 ) (27 ,28 ,29 ,30 ,31 and 32 ). A concentration-effect relationship is, in principle, the most clinically relevant, because it potentially
validates the clinical rationale for measuring drug concentrations in serum or plasma.

FIGURE 38.4. Schematic relation between pharmacokinetics, pharmacodynamics, and kinetic-dynamic modeling, based on the status of the variables of time (t), concentration
(C), and effect (E). Note that kinetic-dynamic modeling incorporates both pharmacokinetics and pharmacodynamics, with time subsumed into the relation of concentration and
effect.

A kinetic-dynamic study in clinical psychopharmacology typically involves medication administration (usually under placebo-controlled, double-blind laboratory conditions) followed by
quantitation of both drug concentration and clinical effect at multiple times after dosing. Measures of effect necessarily depend on the type of drug under study. For sedative-
anxiolytic drugs such as benzodiazepines, effects of interest may include subjective or observer ratings of sedation and mood; semiobjective measures of psychomotor performance,
reaction time, or memory; or objective effect measures such as the EEG or saccadic eye movement velocity. The various measures differ substantially in their relevance to the
principal therapeutic actions of the drug, the stability of the measure in terms of response to placebo or changes caused by practice or adaptation, the objective or subjective nature
of the quantitative assessment, and the comparability of results across different investigators and different laboratories (Table 38.1 ). The extent to which the various
pharmacodynamic measures provide unique information, as opposed to being overlapping or redundant, is not clearly established.

TABLE 38.1. PHARMACODYNAMIC ENDPOINTS APPLICABLE TO STUDIES OF GABA-BENZODIAZEPINE AGONISTS

Pharmacokinetic and pharmacodynamic relationships initially are evaluated separately, and the relationship of effect versus concentration at corresponding times is examined
graphically and mathematically. Effect measures are usually expressed as change scores: the net effect (E) at postdosage time t is calculated as the absolute effect at this time (Et)
minus the predose baseline value (Eo), that is, E = Et - Eo. Several mathematical relationships between effect and concentration (E versus C), often termed “link” models, are of
theoretical and practical importance (5 ,32 ). The “sigmoid Emax” model, incorporates a value of Emax, the maximum pharmacodynamic effect, and EC50 is the “50% effective
concentration,” the concentration that is associated with half of the maximum effect (Fig. 38.5 ). The exponent A reflects the “steepness” of the concentration-response relationship
in its ascending portion. The biological importance of A is not established.

FIGURE 38.5. Three mathematical relationships between concentration (C) and change in pharmacodynamic effect (E) that are commonly applied in kinetic-dynamic modeling
procedures. For the sigmoid Emax model, Emax is maximum pharmacodynamic effect, EC50 is the concentration producing a value of E equal to 50% of Emax, and A is an exponent. For
the exponential and linear models, m is a slope factor.

A concentration-effect relationship that is consistent with the sigmoid Emax model may be of mechanistic importance, because drug-receptor interactions often fit the same model. The
Emax and EC50 values allow inferences about questions such as the relative potency or efficacy of drugs producing the same clinical effect, individual differences in drug sensitivity, the
mechanism of action of pharmacologic potentiators or antagonists, and the possible clinical role of new medications.

The sigmoid Emax model does not necessarily apply to all concentration-effect data (32 ). When experimental data are not consistent with the model, the corresponding misapplication
of the sigmoid Emax relationship can lead to misleading conclusions about Emax and EC50. Some data sets are consistent with less complex models, such as exponential or linear equations
(Fig. 38.5 ); in these cases, the concepts of Emax and EC50 are not applicable. Kinetic-dynamic modeling is further complicated when drug concentrations measured in serum or plasma do
not reflect the concentration at the site of action, which is sometimes termed the “effect site.” This is illustrated by the data described below.
 P.511

Application: Kinetics And Dynamics Of Intravenous Lorazepam

In this study the benzodiazepine derivative lorazepam was administered intravenously according to a complex bolus-infusion scheme
(33 ). On the morning of the study day, a rapid intravenous dose of lorazepam, 2 mg, was administered into an antecubital vein,
coincident with the start of a zero-order infusion at a rate of 2 μg/kg/h. The infusion continued for 4 hours and then was terminated.
Venous blood samples were drawn from the arm contralateral to the site of the infusion prior to drug administration and at multiple
time points during 24 hours after the start of lorazepam infusion. Samples were centrifuged, and the plasma separated and frozen
until the time of assay. The EEG was used as the principal pharmacodynamic outcome measure (Table 38.1 ). The EEG was recorded
prior to lorazepam administration, and at times corresponding to blood samples. EEG data were digitized over the power spectrum
from 4 to 30 cycles per second (Hz), and analyzed by fast Fourier transform to determine amplitude in the total spectrum (4 to 30
Hz) and in the beta (12 to 30 Hz) frequency range (33 ,34 and 35 ). Concentrations of lorazepam in plasma samples were
determined by gas-chromatography with electron-capture detection.

Analysis of Data
The relative EEG beta amplitudes (beta divided by total, expressed as percent) in the predose recordings were used as the baseline.
All values after lorazepam administration were expressed as the increment or decrement over the mean predose baseline value,
with values averaged across eight recording sites. The EEG change values were subsequently used as pharmacodynamic effect (E)
measures in kinetic-dynamic modeling procedures described below. For pharmacokinetic modeling, the relation of plasma lorazepam
concentration (C) to time (t) was assumed to be consistent with a two-compartment model (Fig. 38.6 and Fig. 38.7 ).

FIGURE 38.7. Plasma lorazepam concentrations (solid circles) together with the pharmacokinetic function determined by
nonlinear regression (solid line), in a representative volunteer subject. Shown are the derived pharmacokinetic variables of
elimination half-life (t1/2), volume of distribution (Vd), and clearance (CL).

FIGURE 38.6. Schematic representation of the kinetic-dynamic model for the lorazepam study. Intravenous lorazepam was
assumed to have kinetic behavior consistent with a two-compartment model: reversible distribution to a peripheral
compartment, and first-order elimination (clearance) from the central compartment (rate constant: Ke). Lorazepam in plasma
was postulated to equilibrate with a hypothetical effect site, from which the exit rate constant is KEO. Finally, effect-site
concentrations were presumed to be the principal determinant of pharmacodynamic effect, via a kinetic-dynamic link model
as shown in Fig. 38.5.

Examination of plots of pharmacodynamic EEG effect versus plasma lorazepam concentration (E vs. C) indicated counterclockwise
hysteresis (see below), suggesting a delay in equilibration of lorazepam between plasma and the site of pharmacodynamic action in
brain. This equilibration effect has been described in previous clinical and experimental studies of lorazepam (34 ,36 ,37 ,38 and
39 ). Accordingly the relationship was modified to incorporate a distinct “effect site,” at which the hypothetical lorazepam
concentration is CE (Fig. 38.6 ). The apparent rate constant for drug disappearance from the effect compartment is kEO; this rate
constant determines the apparent half-life of drug equilibration between
 P.512

plasma and effect site. Under these assumptions, the relation of E to CE was postulated to be consistent with a sigmoid Emax model
(Fig. 38.5 ).

Results
Kinetic variables for lorazepam were similar to those reported in previous single-dose studies of lorazepam pharmacokinetics
(34 ,40 ,41 ,42 ,43 ,44 and 45 ). Overall mean values were volume of distribution, 1.7 L/kg; elimination half-life, 14 hours;
clearance, 1.44 mL/min/kg. The bolus-infusion scheme rapidly produced mean plasma lorazepam concentrations in the range of 18
to 19 ng/mL, values close to the mean predicted value of 24 ng/mL.

Lorazepam infusion produced significant increases in EEG beta amplitude throughout the 24-hour duration of the study. The
maximum change over baseline was measured at 0.25 to 0.75 hours after the initiation of lorazepam dosage, whereas the maximum
plasma concentration was measured immediately after the loading dose (Fig. 38.8 ). The effect-site model eliminated the hysteresis,
with a mean equilibration half-life of 8.8 minutes (Fig. 38.9 ).

FIGURE 38.9. Left: Mean values of plasma lorazepam concentration versus pharmacodynamic EEG effect at corresponding
times, with arrows indicating the direction of increasing time. As indicated in Fig. 38.8, the maximum EEG effect is delayed,
and does not correspond in time to the maximum plasma concentration. Right: The scheme shown in Fig. 38.6 was applied to
the data points, with the link model being the sigmoid Emax relationship shown in Fig. 38.5. The data points (closed triangles)
are the hypothetical effect site concentrations and pharmacodynamic effect values at corresponding times. The solid line is
the link model function determined by nonlinear regression, yielding the indicated values of Emax and EC50. The overall mean
equilibration half-life was 8.8 minutes.

FIGURE 38.8. Mean values of plasma lorazepam concentration, and of EEG beta amplitude, during the first hour of the study.
Note that pharmacodynamic EEG effects are delayed following the peak value of lorazepam in plasma.

Implications
Maximum EEG effects of lorazepam were significantly delayed following the initial intravenous bolus dose. Previous single-dose
pharmacodynamic studies of lorazepam, using the EEG or other methods for quantitation of benzodiazepine
 P.513

effect, consistently demonstrate a delay in attainment of maximum drug effect compared to attainment of peak concentrations in
plasma (34 ,36 ,37 ). After rapid intravenous dosage, for example, maximum effects may be delayed for an average of 30 minutes
after dosage. Experimental studies of the time-course of whole-brain concentrations of lorazepam, or of the degree of
benzodiazepine receptor occupancy, indicate that the delay is attributable to the slow physical entry of lorazepam into brain tissue,
probably because of the relatively low lipid solubility of lorazepam (34 ,38 ,39 ). The delay was mathematically consistent with a
kinetic-dynamic model incorporating a hypothetical “effect site” distinct from the central compartment. The half-life for
equilibration between plasma and the effect compartment was approximately 9 minutes. This matches clinical experience indicating
that intravenous lorazepam cannot easily be used in situations requiring minute-to-minute titration of sedative or amnestic effects
(40 ). Nonetheless, intravenous lorazepam can be used for the treatment of status epilepticus, although its onset of action may be
somewhat slower than that of intravenous diazepam (46 ,47 ).

CYTOCHROMES P-450 IN PSYCHOPHARMACOLOGY: THE IMPORTANCE

OF P-450-3A ISOFORMS
Part of "38 - Pharmacokinetics, Pharmacodynamics, and Drug Disposition "

The cytochrome P-450 (CYP) superfamily of drug metabolizing enzymes is now established as being of primary importance for the
metabolism and clearance of most drugs used in psychopharmacology and in other areas of clinical therapeutics (6 ,7 ,8 and
9 ,48 ,49 ,50 ,51 ,52 ,53 ,54 and 55 ) (Fig. 38.10 ). For the CYP isoforms most relevant to human drug metabolism, each has its own
distinct pattern of relative abundance, anatomic location, mechanism of regulation, substrate specificity, and susceptibility to
inhibition and induction by other drugs or foreign chemicals (Table 38.2 ). The expression and in vivo function of at least two CYP
isoforms (CYP2D6 and CYP2C19) are regulated by a genetic polymorphism, such that some members of a population fail to express
“normal” levels of enzyme or expresses poorly functional protein (56 ,57 ,58 ,59 ,60 ,61 and 62 ). Individuals identified as “CYP2D6
poor metabolizers,” as an example, have very low clearance of drugs that are major substrates for biotransformation by CYP2D6
(such as desipramine, nortriptyline, venlafaxine, tramadol, and dextromethorphan). Such individuals are at risk for developing high
and potentially toxic plasma concentrations of these
 P.514

substrate drugs despite dosages in the usual therapeutic range.

FIGURE 38.10. Nomenclature system for the cytochrome P-450 (CYP) superfamily of enzymes. Following the CYP designation,
the number-letter-number sequence indicates the family, subfamily, and specific isoform.

TABLE 38.2. OVERVIEW OF HUMAN CYTOCHROMES P-450

The CYP3A Isoforms

The overall importance of the CYP3A subfamily of drug-metabolizing enzymes, particularly in the field of psychopharmacology, has
become increasingly evident over the last decade (6 ,7 ,8 and 9 ,63 ,64 ,65 ,66 ,67 ,68 and 69 ) (Table 38.3 ). The CYP3A isoforms
are the most abundant of the CYPs, accounting on average for approximately 29% of identified cytochrome P-450 in liver (70 ) (Table
38.2 ). Within the CYP3A subfamily, CYP3A4 is the most important in the adult human, in terms of drug-metabolizing activity as well
as quantitative dominance. CYP3A5, another CYP3A isoform, is also detected in varying amounts in some human livers and in
esophagus, but quantities of CYP3A5 are less than quantities of CYP3A4. It is not established to what extent hepatic CYP3A5 is of
clinical significance for drug-metabolizing activity. CYP3A7 is principally a fetal-specific isoform. The location and sequence of the
genetic element responsible for CYP3A4 expression have been identified, as well as a regulatory segment located on the 5′ flanking
region corresponding to the CYP3A gene.

TABLE 38.3. PSYCHOTROPIC DRUG SUBSTRATES FOR HUMAN CYP3A

CYP3A4 typically functions as a high-capacity, low-affinity enzyme. Its high substrate capacity is a consequence of both the
relatively high value of maximum reaction velocity (Vmax, expressed in nanomoles of product produced per unit time per milligram of
protein) in a Michaelis-Menten relationship, as well as the high quantitative abundance of the protein in hepatic tissue. The low-
affinity characteristic is reflected in the high Km value (substrate concentration corresponding to 50% of Vmax) in a Michaelis-Menten
relationship. One consequence is that CYP3A-mediated metabolism usually is not “saturable” at substrate concentrations within the
therapeutic range, because this range is likely to be far below the reaction Km. Furthermore, in situations in which CYP3A is one of
several cytochromes contributing to metabolism [e.g., amitriptyline N-demethylation (71 ), citalopram N-demethylation (72 ), or
zolpidem hydroxylation (73 )], the relative importance of CYP3A will increase at higher substrate concentrations. However, this is
not invariably true. Nefazodone is a CYP3A substrate, but Km values for production of the various metabolites are relatively low (74 ),
and kinetics are nonlinear (75 ). Midazolam has a low Km for the principal pathway (76 ,77 ), and there is evidence of nonlinear
kinetics at higher concentrations in humans (78 ).

Significant quantities of CYP3A exist in gastrointestinal (GI) tract mucosa (65 ,69 ,79 ). The quantitative expression/
 P.515

activity of GI tract CYP3A is not correlated with its expression/activity in liver, even though the expressed protein is identical at the
two sites. For a number of moderate or high-clearance CYP3A substrates (e.g., midazolam and triazolam), GI tract metabolism
contributes importantly to presystemic extraction (first-pass metabolism) after oral dosage (79 ,80 and 81 ); incomplete oral
bioavailability therefore results from a combination of GI tract and hepatic presystemic extraction (Fig. 38.11 ). For low-clearance
CYP3A substrates having oral bioavailability in the range of 80% to 90% or greater (e.g., alprazolam), the contribution of the GI tract
is apparently small.

FIGURE 38.11. Relative contributions of CYP3A enzymes present in gastrointestinal (GI) tract mucosa, and in the liver, to net
bioavailability (F) of orally administered midazolam and triazolam. Both of these compounds have net F values of less than 50%
(29% for midazolam, 44% for triazolam). Both compounds undergo approximately 50% extraction during passage through the
G.I. tract mucosa. However midazolam undergoes another 38% extraction across the liver, compared to only 12% for triazolam.

Inhibition and induction by other drugs or chemicals may modify CYP3A activity both in vitro and in vivo (Table 38.4 ). Identification
of these compounds is of clear clinical importance, because it may allow anticipating of drug interactions that may be either
potentially hazardous or of therapeutic benefit (6 ,7 ,8 and 9 ,65 ,69 ,79 ,80 ,81 ,82 ,83 ,84 and 85 ). Inhibiting drugs may also be
used for investigating the relative contribution of CYP3A to net clearance, or for distinguishing the contribution of hepatic and GI
tract CYP3A to overall presystemic extraction (81 ). Among the most potent CYP3A inhibitors are the azole antifungal agents
(ketoconazole, itraconazole, fluconazole), the antidepressants nefazodone and fluvoxamine, and the calcium channel antagonists
verapamil and diltiazem. These compounds produce “reversible” inhibition, by a competitive, noncompetitive, or mixed mechanism.
Other potent inhibitors, such as the macrolide antimicrobials erythromycin and clarithromycin produce “mechanism-based”
inhibition via a metabolic intermediate that complexes with and inactivates the CYP3A enzyme (86 ,87 ). The HIV protease inhibitor
ritonavir and the nonnucleoside reverse transcriptase inhibitor delavirdine also are potent CYP3A inhibitors (88 ,89 ,90 and 91 ). A
component of grapefruit juice inhibits CYP3A in the GI tract (92 ). Inducers of CYP3A include carbamazepine, rifampin,
phenobarbital, nevirapine, dexamethasone, St. John’s wort, and possibly venlafaxine. Ritonavir is an inducer as well as an inhibitor,
yielding a net effect on CYP3A metabolism that is difficult to predict (88 ,89 ,90 and 91 ,93 ,94 and 95 ).

TABLE 38.4. REPRESENTATIVE DRUGS HAVING CLINICALLY IMPORTANT EFFECTS

ON THE HUMAN CYP ENZYMES
Variability among individuals in CYP3A activity is substantial, even when relatively homogeneous groups of healthy subjects are
studied. A consistent finding in population studies of CYP3A activity is that distributions are unimodal, without evidence of genetic
polymorphic regulation (96 ,97 ). However, several studies of CYP3A substrates have demonstrated a small number of individuals
with unusually low clearance (96 ,98 ). The explanation for these observations is unclear, but the genomic determinants of such
individual variations in clinical CYP3A activity have become
 P.516

an active research topic. A number of genetic variants or single nucleotide polymorphisms in either the promoter or coding regions
of the human CYP3A4 gene have recently been described (99 ,100 ,101 ,102 ,103 ,104 and 105 ). One of the promoter region
polymorphisms, designated as CYP3A4*1B, is more prevalent in the African-American as opposed to the Caucasian populations.
However, there is no evidence to indicate that any of the identified CYP3A4 variants accounts for individual variation in clearance of
CYP3A substrates.

In Vitro Models of Drug Metabolism

In vitro systems now are extensively utilized to provide presumptive answers to fundamental clinical questions regarding drug
metabolism and drug interactions, and to guide the planning of clinical pharmacokinetic studies (6 ,7 ,8 and
9 ,48 ,49 ,50 ,51 ,52 ,53 ,54 and 55 ,71 ,72 ,73 and 74 ,82 ,83 and 84 ). If drug X is biotransformed in humans to metabolite Y, two
core questions occur: (a) What CYP isoform or isoforms mediate the biotransformation of X to Y? (b) What CYP isoforms do X or Y
themselves either induce or inhibit?

Human liver microsomes generally are an important component of currently utilized in vitro systems. These preparations contain the
various human CYPs in proportion to their abundance in human liver in vivo. If biotransformation of a specific substrate to its initial
metabolite or metabolites can be replicated in microsomal preparations (Fig. 38.12 ), inhibition of that reaction by a relatively
specific chemical inhibitor can be used as evidence supporting the contribution of the corresponding cytochrome. Chemical
inhibitors can also be used in clinical studies, but the in vitro model has the advantages of lower cost, more rapid implementation,
no risk of human drug exposure, the availability of a greater number of potential chemical inhibitors for research purposes, and the
possibility of determining both the quantitative and qualitative contributions of specific cytochromes. Antibodies with relatively
specific inhibitory activity against individual human cytochromes can also be used to support or confirm data from in vitro chemical
inhibition studies (106 ,107 ). In vitro approaches have been strengthened with the availability of microsomes containing pure
human cytochromes as expressed by cDNA-transfected human lymphoblastoid cells (108 ,109 and 110 ). These heterologously
expressed pure cytochromes further support definitive identification of cytochromes mediating a specific reaction in vivo.

FIGURE 38.12. Example of an in vitro metabolism study using human liver microsomes (73). The substrate in this study was
zolpidem, of which varying concentrations were incubated with liver microsomes and appropriate reaction cofactors. At each
concentration of zolpidem, the rate of formation (V) of the principal metabolite of zolpidem (termed the M-3 metabolite) was
determined. The relation between substrate concentration (S) and reaction velocity (V) was analyzed by nonlinear regression
to determine the maximum reaction velocity (Vmax) and the substrate concentration (Km) producing a reaction velocity of 50% of
Vmax. Left: A substrate concentration range up to 2,000 μM. Right: The lower range of concentrations shown on an expanded
scale.

The quantitative inhibitory potency of a series of drugs and their metabolites against specific index reactions can also be
determined using human liver microsomes in vitro (6 ,7 ). The first of two general approaches uses fixed concentrations of the index
substrate co-incubated with varying concentrations of the inhibitor in question. The relation of decrement in metabolite formation
rate to inhibitor concentration is used to estimate a 50% inhibitory concentration (IC50). This procedure is expeditious and relatively
inexpensive, and the numbers can be used to compare the potency of a series of inhibitors (Fig. 38.13 ). IC50 values themselves are
not dependent on knowledge of the specific biochemical mechanism of inhibition. However, IC50 values depend on substrate
concentration when inhibition is competitive, and
 P.517

can be applied to in vitro–in vivo scaling only when the mechanism of inhibition is noncompetitive (111 ,112 ). Inhibitory potency
can also be estimated by determining the inhibition constant (Ki), a number that reflects inhibitory activity in reciprocal fashion.
Estimation of Ki requires the study of multiple substrate concentrations and multiple inhibitor concentrations and therefore involves
more work, time, and expense. The numerical value of Ki depends on the specific biochemical mechanism of inhibition, which may
be unknown. Nonetheless, the inhibitor Ki, is independent of substrate concentration and can be used under some defined
circumstances for quantitative in vitro–in vivo scaling of drug interactions. In general, Ki is always less than or equal to IC50; Ki will be
essentially equal to IC50 if inhibition is noncompetitive, or if inhibition is competitive and the substrate concentration is far below
the reaction Km. Both Ki and IC50 should provide similar or identical rank-order estimates of relative inhibitory potency for a series of
inhibitors of a specific reaction. When the inhibition is purely competitive, Ki values for a specific inhibitor should theoretically be
identical across different substrates metabolized by that particular CYP. However, this principle is not supported by experimental
data, probably because of in vitro experimental artifacts, and because actual biochemical mechanisms of inhibition are not purely
competitive. Therefore, absolute values of Ki or IC50 cannot be assumed to cross different substrates for the same cytochrome,
although the relative rank order of inhibitory potency should be maintained.

FIGURE 38.13. Example of an in vitro study of inhibition of CYP2D6 by two antipsychotic agents, perphenazine and clozapine.
A fixed concentration of the substrate dextromethorphan was incubated with liver microsomes, appropriate cofactors, and
varying concentrations of perphenazine or clozapine. Rates of formation of dextrorphan (mediated by CYP2D6) with inhibitor
present were expressed as a ratio versus the control velocity with no inhibitor. The relation of the velocity ratio to inhibitor
concentration can be used to calculate a 50% inhibitory concentration (IC50). The results indicate that perphenazine (IC50 = 0.9
μM) is likely to be a clinically important inhibitor of CYP2D6, whereas clozapine (IC50 = 119 μM) is a weak inhibitor.

Limitations and drawbacks of in vitro systems should be recognized. In vitro studies generally utilize substrate concentrations that
are one or more orders of magnitude higher than those encountered clinically, even if extensive partitioning of lipophilic drugs from
plasma into liver tissue is accounted for. If mathematical models and parameter estimates are valid, the outcome of studies of
higher concentrations can be extrapolated down to clinically relevant substrate concentrations. However, a clinically important
“high-affinity” metabolic reaction (i.e., one with a low Km value) could be missed if low substrate concentrations cannot be
accurately measured due to limitations of assay sensitivity. The specificity of chemical inhibitor probes is of concern for in vitro and
in vivo studies, because all inhibitors ultimately become nonspecific at higher concentrations. Finally, data from cDNA-expressed
human cytochromes can be misinterpreted unless they are considered in the correct context. Pure cytochrome studies can yield
quantitative data on the activity of one or more particular cytochromes as a mediator of a specific reaction. However, this
information cannot be extrapolated to an estimate of the relative activity of different cytochromes, either in vivo or in liver
microsomes in vitro, without a parallel estimate of the relative quantitative abundance of the cytochromes in question. That is, the
importance of a specific cytochrome depends on both activity and abundance.

DRUG INTERACTIONS IN PSYCHOPHARMACOLOGY

Part of "38 - Pharmacokinetics, Pharmacodynamics, and Drug Disposition "

During the last two decades the general problem of pharmacokinetic drug interactions has received increased attention. New classes
of medications introduced into clinical practice over this period include the selective serotonin reuptake inhibitor (SSRI) and related
mixed mechanism antidepressants, the azole antifungal agents, newer macrolide antimicrobial agents, and the highly active
antiretroviral therapies (HAARTs) used against HIV infection and AIDS (Table 38.4 ). These and other classes of medications have had
a major beneficial impact on the therapy on some serious and life-threatening illnesses, but many of the drugs have the secondary
pharmacologic property of inducing or inhibiting the human CYP enzymes, thereby raising concerns about pharmacokinetic drug
interactions during multiple drug therapy.

One major objective of the drug development process is to generate data on drug interactions, so that treating physicians have the
information necessary for safe clinical therapy involving multiple medications. However, the number of possible drug interactions is
very large, and time and resources available for implementation of controlled clinical pharmacokinetic studies are inevitably limited.
 P.518

Some needed studies will therefore be postponed until after a new drug is marketed, and some studies may be bypassed altogether.
As discussed above, in vitro data are becoming increasingly important as a resource for identifying probable, possible, or unlikely
drug interactions, and thereby encouraging rational planning and allocation of resources to more definitive clinical studies.

Pharmacokinetic Versus Pharmacodynamic Drug Interactions

A pharmacokinetic interaction implies that the drug producing the interaction (the “perpetrator”) causes a change in the metabolic
clearance of the drug being affected by the interaction (the “victim”), in turn either decreasing or increasing concentrations of the
victim drug in plasma and presumably also at the site of action. This change may or may not alter the clinical activity of the victim
drug. One pharmacokinetic interaction variant involves modification by the perpetrator of the victim drug’s access to its
pharmacologic receptor site, without changing the systemic clearance or plasma levels of the victim. A familiar example is the
antagonism of benzodiazepine agonist activity by flumazenil; a less familiar example is benzodiazepine receptor antagonism by
ketoconazole (113 ).

A pharmacodynamic interaction involves either inhibition or enhancement of the clinical effects of the victim drug as a result of
similar or identical end-organ actions. Examples are the increase or decrease of the sedative-hypnotic actions of benzodiazepines
due to coadministration of ethanol or caffeine, respectively.

Mechanisms of Inhibition Versus Induction of Metabolism

Drug interactions due to inhibition as opposed to induction of CYP-mediated metabolism involve mechanistically different processes.
Chemical inhibition is an immediate phenomenon that becomes evident as soon as the inhibitor comes in contact with the enzyme,
and is in principle reversible when the inhibitor is no longer present [an exception is “mechanism-based” inhibition (86 )]. The
magnitude of inhibition depends on the inhibitor concentration at the site of the enzyme in relation to the intrinsic potency of the
inhibitor. In vitro systems can be used to develop quantitative estimates of inhibitory potency, such as the inhibition constant (Ki) or
the 50% inhibitory concentration (IC50) (Fig. 38.13 ). However, application of Ki or IC50 values from in vitro systems to quantitative
predictions of drug interactions in vivo is not straightforward, and requires knowledge of the effective concentration of inhibitor
that is available to the enzyme. A generally applicable scheme for relating total or unbound plasma concentrations of inhibitor to
effective enzyme-available concentration has not been established. In any case, the theoretical assumption that unbound plasma
concentrations are equal to enzyme-available intrahepatic concentrations is incorrect in reality, and may yield underestimates of
observed in vivo drug interactions by as much as an order of magnitude or more (8 ,83 ,85 ).

Induction of CYP-mediated metabolism requires prior exposure to a chemical inducer, which signals the synthetic mechanisms to
upregulate the production of one or more CYP isoforms (114 ,115 ,116 ,117 and 118 ). This process takes time, and the increase in
CYP activity is of slow onset following initiation of exposure to the inducer, and slowly reverts to baseline after the inducer is
removed. Increased CYP expression/activity due to chemical induction therefore reflects prior but not necessarily current exposure
to the inducer. The extent of CYP induction probably depends on the dosage (concentration) of the inducer and on the duration of
exposure. Induction, unlike inhibition, is not easily studied in vitro, because induction requires intact cellular protein synthesis
mechanisms as are available in cell culture models.

Inducers and inhibitors of CYP3A can be expected to influence both hepatic and gastrointestinal CYP3A, although not necessarily to
the same extent. Very strong inhibitors (such as ketoconazole) or very strong inducers (such as rifampin) will produce substantial
changes in both hepatic and gastrointestinal CYP3A. A uniquely complex situation arises for ritonavir, which is both an inhibitor and
inducer of CYP3A. Interactions of ritonavir with CYP3A substrate drugs will be time dependent. Initial exposure will produce CYP3A
inhibition, but as the duration of exposure proceeds, CYP3A induction may offset the inhibitory effects of acute exposure. The net
outcome typically is unpredictable and variable across individuals (93 ,94 and 95 ).

Perspectives on the Clinical Importance of Drug Interactions

Based on the prevalence of polypharmacy in clinical practice, noninteractions of drugs are far more common than interactions.
Coadministration of two drugs usually produces no detectable pharmacokinetic or pharmacodynamic interaction, and the
pharmacokinetic disposition and clinical activity of each drug proceed independently of each other. A less common outcome of drug
coadministration is a kinetic interaction that could be detected in controlled laboratory circumstances, but that is not clinically
important in usual therapeutic circumstances because (a) the interaction, whether or not statistically significant, is not large enough
to produce a clinically important change in dynamics of the victim drug; (b) the therapeutic index of the victim drug is large enough
so that even a substantial change in plasma levels will not alter therapeutic effects or toxicity; or (c) kinetics and response to the
victim drug are so variable that changes in plasma levels due to the drug interaction are far less important than inherent variability.
Even less common are clinically important interactions that require
 P.519

modification in dosage of the perpetrator, the victim, or both. Finally, the most unusual consequence of a drug interaction is a
hazardous and contraindicated combination, as in the case of ketoconazole and terfenadine. These situations are rare, but
unfortunately receive excessive attention in the public media.

Many secondary sources and compendia are available as summary guides to the extensive literature on drug interactions, but these
sources do not necessarily assist clinicians in deciding which interactions should generate serious concern in the course of drug
therapy. A useful general guideline is that drug interactions are more likely to be important when (a) the perpetrator drug is a
powerful inducer or inhibitor, and produces a very large change in the kinetics and plasma levels of the victim drug; or (b) the
therapeutic index of the victim is narrow. Case (a) is exemplified by powerful inducers or inhibitors of CYP3A (ketoconazole,
ritonavir, rifampin) coadministered with CYP3A substrates, or powerful inhibitors of CYP2D6 (quinidine, fluoxetine, paroxetine)
coadministered with CYP2D6 substrates. Case (b) is exemplified by victim drugs such as phenytoin, warfarin, and digoxin, for which
small changes in plasma levels could have important clinical consequences.

Application of Kinetic-Dynamic Methods to Study Drug Interactions

Drug interaction study protocols often incorporate pharmacodynamic endpoints to allow estimating the clinical consequences of drug
interactions along with the usual pharmacokinetic outcome measures. The level of complexity of an integrated kinetic-dynamic
study depends on the nature of the pharmacodynamic actions of the drug under study as well as the type of pharmacodynamic
outcome measures that are required. A number of methodologic principles and dilemmas are illustrated by kinetic-dynamic design
options for drug interaction studies involving sedative-hypnotic and anxiolytic drugs acting on the γ-aminobutyric acid (GABA)-
benzodiazepine receptor system.

Biotransformation of the benzodiazepine triazolam is dependent on the activity of human CYP3A isoforms (119 ). Metabolism is
strongly inhibited in vitro and in vivo by CYP3A inhibitors such as ketoconazole, itraconazole, ritonavir, and nefazodone
(95 ,119 ,120 ,121 and 122 ). Some, but not all, of the macrolide antimicrobial agents also are CYP3A inhibitors via “mechanism-
based” inhibition, in which the parent compound binds to the metabolically active site on the CYP3A enzyme, yielding a metabolic
intermediate that inactivates the enzyme (86 ,87 ). We tested the inhibitory potency of four macrolide antimicrobial agents
[troleandomycin (TAO), erythromycin, clarithromycin, azithromycin] versus triazolam hydroxylation using human liver microsomes in
vitro (123 ). Appropriate mean IC50 values were TAO, 3.6 μM; erythromycin, 30 μM; and clarithromycin, 28 μM. These values indicate
that all three compounds produce substantial in vitro inhibition of triazolam hydroxylation and have the potential to produce a
significant interaction with triazolam in vivo. However, azithromycin was a very weak inhibitor of triazolam in vitro (IC50 >250 μM),
and is anticipated to produce no significant interaction in vivo.

In a clinical pharmacokinetic-pharmacodynamic study (123 ), a series of healthy volunteers were exposed to the following treatment
conditions:

A. Triazolam placebo plus macrolide placebo

B. Triazolam (0.125 mg) plus macrolide placebo
C. Triazolam (0.125 mg) plus azithromycin
D. Triazolam (0.125 mg) plus erythromycin
E. Triazolam (0.125 mg) plus clarithromycin

Dosage schedules of the coadministered macrolides were chosen to be consistent with usual dosage recommendations. The five trials
were randomized in sequence, and the treatment conditions were double-blind.

Following each dose of triazolam (or placebo to match triazolam), multiple venous blood samples were drawn over a period of 24
hours, and multiple pharmacodynamic testing procedures were performed. Triazolam plasma concentrations were determined by
gas chromatography with electron capture detection (Fig. 38.14 ).

FIGURE 38.14. Mean plasma triazolam concentrations following single 0.125-mg doses of triazolam during trials B, C, D, and E.
Note that coadministration of triazolam with azithromycin (AZI, trial C) produced plasma levels nearly identical to triazolam
administered with placebo (PL, trial B). However, coadministration with erythromycin (ERY, trial D) or clarithromycin (CLAR,
trial E) produced a large increase in plasma triazolam concentrations. (Adapted in part from Greenblatt DJ, von Moltke LL,
Harmatz JS, et al. Inhibition of triazolam clearance by macrolide antimicrobial agents: in vitro correlates and dynamic
consequences. Clin Pharmacol Ther 1998;64:278–285, with permission.)
 P.520

Mean clearance of triazolam during trials B and C was nearly identical (413 and 416 mL/min, respectively); that is, coadministration
of azithromycin had no effect on the pharmacokinetics of triazolam (Fig. 38.14 ). However, triazolam clearance was significantly
reduced to 146 mL/min by erythromycin (trial D), and to 95 mL/min by clarithromycin (trial E) (Fig. 38.14 ). Thus the in vivo kinetic
results are highly consistent with the in vitro data.

The pharmacodynamic data indicated that the benzodiazepine agonist effects of triazolam plus placebo (trial B), and of triazolam
plus azithromycin (trial C) were similar to each other, and greater than the effects of placebo plus placebo (trial A). However,
coadministration of triazolam with erythromycin (trial D) or with clarithromycin (trial E) augmented the pharmacodynamic effects of
triazolam when compared to trials B or C. The outcome was similar whether based on subjective measures, a semi-objective
measure (the Digit-Symbol Substitution Test, DSST), or the fully objective measure (the EEG) (Fig. 38.15 ). Kinetic-dynamic modeling
indicated that the increase in benzodiazepine agonist effects of triazolam caused by coadministration of erythromycin or
clarithromycin was fully consistent with the increase in triazolam plasma concentrations (Fig. 38.16 ).

FIGURE 38.16. Relation of mean plasma triazolam concentrations to mean changes over baseline in DSST score at the
corresponding times. The solid line represents the kinetic-dynamic model relationship based on an exponential function as
shown in Fig. 38.5. (From Greenblatt DJ, von Moltke LL, Harmatz JS, et al. Inhibition of triazolam clearance by macrolide
antimicrobial agents: in vitro correlates and dynamic consequences. Clin Pharmacol Ther 1998;64:278–285, with permission.)

FIGURE 38.15. Mean (± standard error, SE) 4-hour pharmacodynamic effect areas for the digit-symbol substitution test (DSST)
score (left), and for the EEG beta amplitude (right), during the five trials. Note that decrements in DSST score, and increases
in EEG beta amplitude, were very similar between trials B and C, whereas effects were significantly enhanced during trials D
and E.

COMMENT
Part of "38 - Pharmacokinetics, Pharmacodynamics, and Drug Disposition "

Pharmacokinetic drug interactions in clinical psychopharmacology are assuming increasing importance as polypharmacy becomes
more common, and more drugs with enzyme-inducing or -inhibiting properties are introduced into clinical practice. Contemporary
approaches to the basic and clinical investigation of drug interactions and their pharmacodynamic consequences are illustrated in
this chapter. It is evident that technologic and conceptual advances in pharmacokinetics, pharmacodynamics, and drug metabolism
may be usefully applied to the evaluation of drug interactions. An ideal approach would incorporate the collaborative participation
of individuals representing expertise in molecular pharmacology, cytochrome biochemistry, in vitro metabolism, clinical
pharmacokinetics-pharmacodynamics, and clinical therapeutics.

ACKNOWLEDGMENTS
Part of "38 - Pharmacokinetics, Pharmacodynamics, and Drug Disposition "
 P.521

The work was supported by grants MH-34223, MH-01237, DA-05258, DA-13209, MH-58435, DK-58496, and RR-00054 from the U.S. Department of Health and Human Services.

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39
The Role of Pharmaceuticals in Mental Health Care Outcomes

Ramin Mahmoud

Chris M. Kozma

C. E. Reeder

Amy Grogg

Brian Meissner

Ramin Mahmoud and Amy Grogg: Janssen Research Foundation, Titusville, New Jersey.

Chris M. Kozma: Strategic Outcomes Service of Care Sciences, Inc., Philadelphia, Pennsylvania.

C. E. Reeder and Brian Meissner: College of Pharmacy, University of South Carolina, Columbia, South Carolina.

This chapter discusses the role of pharmaceutical outcome evaluations in mental health care. The first section discusses the
importance of pharmaceutical outcome evaluations. The second section describes techniques used in economic evaluations of
pharmaceuticals (i.e., pharmacoeconomic methods). The final section discusses how mental health care outcomes data may be used
in practice.

 WHY OUTCOMES?
 PHARMACOECONOMICS
 CONDUCTING PHARMACEUTICAL OUTCOMES RESEARCH
 HUMANISTIC MEASURES
 USING OUTCOMES DATA IN PRACTICE
 CONCLUSION

WHY OUTCOMES?
Part of "39 - The Role of Pharmaceuticals in Mental Health Care Outcomes "

The idea that outcomes associated with the provision of health care are important is not new. In the 1960s Avedis Donabedian (1 )
presented health outcomes as changes in health status that were attributable to antecedent health care. For many years, however,
evaluations of health care focused on the structure or process of care. As health care moves into the new millennium, financing of
health care is evolving from individual providers being solely responsible for patient outcomes to an environment where payers,
institutions, and providers are being held accountable for quality and cost of care. As financing of health care has moved to a more
centralized locus of control, evaluation of outcomes has become more feasible and desirable. In a book titled Who Shall Live, Victor
Fuchs (2 ) discussed three factors that can be balanced in our health care system: costs, quality, and access. Over time the
pendulum swings from one to another of these dimensions. If costs containment goes too far, then quality or access may suffer.
Likewise, it is possible in today’s technologically driven environment to provide a level of quality that is affordable to only a small
segment of our society. In an environment of limited resources and high demand for health care, a quality, cost, access trade-off is
essential. The issue then becomes how do we define and measure quality so that these trade-offs can be made in pursuit of
efficiency and/or equity. Many believe that quality should be defined and measured in terms of patient outcomes.
In simple terms, outcomes are the “end results.” Improvement in outcomes is a primary reason for medical intervention, including
use of pharmacologic therapies. There is a belief that the use of pharmaceuticals will have a positive impact on the “end results” of
patient care. Historically, this belief is self-evident in the treatment of mental health disorders with the use of drugs to treat
psychoses and depression—conditions for which treatment was revolutionized by pharmaceuticals (3 ,4 ). However, with newer,
more costly pharmaceuticals, such as selective serotonin reuptake inhibitors (SSRIs) and atypical antipsychotic agents, many payers
and health professionals have questioned the value that is received for the resources expended on these agents. Consequently,
numerous studies have been directed at these issues. For example, SSRIs have been compared to tricyclic antidepressants (TCAs) for
the treatment of depression (5 ,6 ,7 and 8 ). Payers are interested not only in the most efficacious antidepressant but also in which
agent should be chosen as first- or second-line therapy, the appropriate course (length) of therapy, and whether initial therapy
should be augmented with an additional agent (9 ,10 ,11 and 12 ). Given the wide variations in care and the possibility that
alternative treatments could lead to similar outcomes, particularly under the less than ideal conditions of “usual care” practice,
health care payers are concerned about which treatments regimens lead to the most efficient outcome. In a resource-constrained
environment, it is both reasonable and responsible to ask this question:
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Would the resources expended for one alternative be more efficient if devoted to another alternative? One useful way to address
this question is with data on patient and cost outcomes.

There is an inextricable but sometimes complex relationship between quality of care and outcomes. Outcomes data are one way of
evaluating quality. It is noteworthy, however, that establishing quality “thresholds” requires a value judgment. What is acceptable
quality to one person may be unacceptable to another at any given level of cost. Outcomes data cannot provide answers to
questions that require fundamental value judgments. On a macro level, an analysis using measures of quality and cost will not
define the percentage of gross domestic product (GDP) that a nation should spend to achieve a certain level of quality in health care,
but rather will provide tools and information to assist decision makers in efficiently allocating scarce resources. On a micro level,
there is no specific quality of life score on an instrument that indicates if or when a drug product should be reimbursed. There is no
single clinical measure that indicates that a patient is in perfect health. All outcomes data require the interpretation and evaluation
of a medical decision maker. Outcomes data provide one more, albeit in many cases relevant, piece of information on which to base
decisions.
In the previous discussion, outcomes were defined in abstract terms (i.e., changes in health status, end results). While there is an
inherent belief that many pharmaceuticals improve outcomes, for outcomes to be documented and improved, this terminology must
be defined in more concrete terms. One conceptualization of health care outcomes is the economic, clinical, humanistic outcomes
model (ECHO) (13 ). This conceptualization portrays health outcomes along three dimensions. Clinical outcomes are outcomes
related to the effects of medical treatments or disease on medical events such as hospitalization or death (i.e., end results).
Economic outcomes are usually expressed as costs (e.g., dollars) associated with an intervention, and are often considered as ratios
of costs to some measure of the consequences of a disease and its treatment. Humanistic outcomes are measures of the impact of
disease or treatment on patient’s lives. In addition to these outcomes, there are many intermediate variables that are important
when measuring the effects of a disease or treatment. These variables are referred to in the published literature by many names
including process variables, surrogate outcomes, or intermediate variables. In many cases making a clear distinction between these
consequences of pharmaceutical use is probably not necessary; however, when reviewing literature, it is important to consider
whether a consequence is a “true” outcome or an intermediate variable. For example, a score on a depression inventory is probably
closer to an intermediate variable, whereas events such as rehospitalization or suicide reflect the “end results” or outcomes one
would like to prevent.
Mental health care is expensive. For example, it is estimated that $44 billion is spent annually on the treatment of depression and
$100 billion is spent annually on the treatment of Alzheimer’s disease (14 ,15 ). The cost to treat schizophrenia has been estimated
at $33 billion per year, accounting for 22% of dollars spent to treat all categories of mental illness and 2.5% of total health care
expenditures (16 ,17 ). Increasing competition for scarce resources encourages decision makers to use outcomes data to evaluate
the effectiveness and efficiency of treatment options for depression, Alzheimer’s disease, and other mental health disorders. These
issues are not new to health care providers, but the development of drug formularies as mechanisms to control costs has generated
a need for outcomes studies to evaluate the benefits obtained from new pharmacologic agents. Shortly after SSRIs were released on
the market, questions arose regarding whether health care outcomes were better for patients treated with SSRIs than for patients
treated with traditional TCA therapy (5 ,6 and 7 ). This new class of pharmaceuticals was more costly than prior standard therapy
(the TCAs), prescribed for a wide variety of patients, and had (in clinical trials) fewer side effects. Although the products were
shown to be superior in some domains in clinical trials, there was a practical question regarding whether these benefits were
realized in real-world practice and if so, at what net incremental cost (18 ). Do patients treated with SSRIs consume less acute care
services, require fewer specialist visits, or have lower suicide rates? Is the total cost per acute depressive episode (successfully
treated case) therefore lower with the newer products despite higher drug acquisition costs? Does treatment with SSRIs cost more,
but provide better humanistic outcomes such as quality of life or quality-adjusted life years? These are the types of questions that
outcomes research and pharmacoeconomic evaluations attempt to answer. This chapter does not specifically address these
questions, but rather uses these questions to illustrate issues in outcomes research. It is noteworthy to recognize that not all the
outcome questions of interest are likely to be addressed in a single study; rather, answers will come from an evaluation of a body of
literature.
Most health care professionals are familiar with the clinical aspects of the treatment of mental health diseases. Given the
substantial clinical information in the remainder of this text, this chapter focuses on evaluation of the economic and humanistic
outcomes related to pharmaceutical use. Specifically, the techniques of pharmacoeconomics will be reviewed as well as the
instruments for evaluating humanistic outcomes in mental health care populations.

PHARMACOECONOMICS
Part of "39 - The Role of Pharmaceuticals in Mental Health Care Outcomes "

In the current health care environment, many decisions are driven by costs. At a minimum, health care systems are looking for
systematic methods for reducing costs. Although
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the fraction of the health care dollar spent on pharmaceuticals is low, it is clear that as both the pressure to reduce costs and the
percentage of health care dollars spent on pharmaceuticals grow, so does interest in the costs of medications. Economists, however,
are quick to point out that the acquisition cost of the pharmaceutical is not the most appropriate unit of analysis. It is possible that
the acquisition cost of many pharmaceuticals may be offset by reductions in other more expensive forms of care. If the use of an
expensive atypical antipsychotic leads to reductions in hospitalizations, then the “value” of the pharmaceutical from a total cost
perspective is greater than the acquisition cost of the pharmaceutical. This is a key idea behind pharmacoeconomics.
Pharmacoeconomics provides a set of techniques that allow consideration of the costs and consequences of alternative
pharmaceutical therapies (19 ).

Studies are typically categorized by whether they consider costs, outcomes, or both cost and outcomes. In addition, studies can also
be categorized by whether or not they consider alternatives. For example, traditional clinical trials focus on comparing the
consequences of alternatives when one of the alternatives is typically a placebo. Although placebo comparison is highly relevant
from the perspective of a regulatory agency striving to meet its special mandate, from the perspective of many health care decision
makers a comparison with placebo is meaningful only if it is a relevant treatment alternative. Pharmacoeconomic studies best
provide a comparison of relevant alternatives.

Studies that evaluate only cost for one alternative are referred to as cost descriptions. Other studies may also consider
consequences. In these cases the study would describe both the costs and consequences of a single alternative leading to a cost-
outcome description. If two alternatives are compared but only costs are considered, then the study is a cost evaluation. However,
the primary concern of pharmacoeconomics is the comparison of both costs and consequences simultaneously for two for more
relevant alternatives. There are four specific techniques that are typically used when conducting pharmacoeconomic studies (20 ):

 Cost-minimization analysis
 Cost-effectiveness analysis
 Cost-utility analysis
 Cost-benefit analysis

In each of these cases the numerators are the costs of inputs for a given decision. For example, if the total cost of care for the
treatment of depression is considered, input costs might include cost of drug, cost of physician visits (family practitioner, internist,
and specialist), behavioral therapy, hospitalization, and emergency department use. (Cost is discussed in greater detail below.) Next,
the appropriate outcomes or consequence must be specified in the denominator.

Cost-minimization analysis assumes, not always explicitly, that the outcomes are equal. If this is a valid assumption, then the
decision is based entirely on the costs of the inputs. The classic example of a cost minimization analysis is the use of generic versus
branded products. If the chemical entities and formulations are identical, then there is no reason to suspect that the outcomes
associated with the use of either product would be different. In this case, the decision is based solely on the costs of the inputs. The
difficulty with cost-minimization analysis is establishing that outcomes are equal. Even in the case of generic pharmaceuticals there
are examples where alternative formulations have been questioned. Additionally, products may be equivalent on some outcomes
such as clinically significant improvement in depression, but not with regard to others such as side-effect profiles.

Cost-effectiveness assesses the consequences in natural units. These natural units may include outcomes such as years of life saved,
hospitalizations avoided, or scores on a symptom scale. Jonsson et al. (21 ) used the Mini-Mental State Examination as a mechanism
to assess time in a nonsevere disease state. This information was incorporated into a Markov state transition model to compare the
cost-effectiveness of newer medications for the treatment of Alzheimer’s disease to standard care. In many cases it is possible to
develop several cost-effectiveness ratios for a comparison of relevant treatment alternatives. For example, in a comparison of
atypical and conventional antipsychotics, cost-effectiveness ratios such as cost per hospitalization avoided, cost per symptom free
day, or cost per schizophrenic exacerbation might all have meaning.

A recent cost-effectiveness study for the treatment of depression provides an excellent example of how decision makers can utilize
these tools to best allocate scarce resources. Nuijten and colleagues (8 ) developed a Markov process to model the cost-
effectiveness of long-term treatment with a new antidepressant compared to standard treatment with TCAs. The outcomes were
time without depression, direct costs, and indirect costs (lost workdays). Clinical data were obtained from the published literature
and costs were measured from the perspective of the German health care system. The new antidepressant was found to be
associated with a 1.5 months longer time without depression than the TCA and with less cost to the health care system. In this case
the new drug was less costly and more effective; thus by definition it is a more cost-effective choice.

Cost-utility analysis is a special case of cost-effectiveness analysis, in which the denominator is quality-adjusted life years (or
something conceptually similar). The quality-adjusted life year may, for example, be calculated using patient utilities (from zero to
one) for being in a given health state (or series of health states) and multiply them by the number of years of life expected in each
health state. This analysis benefits from combining length of life with quality of life. For example, people using different
antipsychotic medications may have similar life expectancy. However, if patients taking some antipsychotics have fewer side effects
or greater
 P.528

efficacy, they may experience an improved quality of life. As a result, the quality of the remaining years of life may not be equal for
the two treatments. Cost-utility ratios factor this quality difference into the analysis. A typical ratio presented for cost-utility
analysis is a cost per quality-adjusted life year (QALY).

Cost-utility analyses have the ability to compare QALYs over multiple treatment regimens. Revicki et al. (22 ) completed a cost-
utility analysis in a managed care setting and compared outcomes of two SSRIs, a TCA, and a stepped approach that began with a
TCA that was replaced with an SSRI if the TCA treatment failed. The outcomes measures were lifetime medical costs, QALYs, and
cost per QALY gained. The analysis for a base case found that lifetime medical costs ranged from $15,348 to $16,669 per patient,
that QALYs gained ranged from 14.32 to 14.64, and that cost per QALY gained ranged from $2,555/QALY to $6,346/QALY. The model
allowed certain factors, such as compliance, to be varied.

The final pharmacoeconomic method is cost-benefit analysis. Cost-benefit analysis values the denominator in dollars and calculates
a return on investment. Cost-benefit analysis allows comparison of alternatives that lead to dissimilar outcomes. Should a hospital
open a gift shop or provide a vaccination program for influenza? The answer would be provided in the following terms: for every
dollar invested in a gift shop, there is a return of $1.13; for every dollar invested in a vaccination program, there is a return of $1.25.
Thus the vaccination program would be the more attractive investment. A disadvantage of cost-benefit analysis is that it requires all
consequences to be valued in dollars. For example, suppose use of a medical alternative increases survival by 1 year. How do you
put a cost on 1 year of life? In health care, valuing in dollars such consequences as life years gained and disability days avoided may
be considered difficult or unacceptable by many people.

These four methods form the cornerstone of pharmacoeconomics. There are, however, many issues that affect the conduct and
interpretation of pharmacoeconomic analyses. Some of the principal issues are discussed in the following sections.

Costs
Valuation of costs in pharmacoeconomic analyses can be difficult. There are two primary issues: first, which costs should be
included in an analysis, and second, how should those costs be valued. The costs of inputs in a pharmacoeconomic analysis typically
include direct medical costs, direct nonmedical costs, and indirect costs. Direct medical costs include costs such as physician visits,
hospitalization, emergency department use, and pharmaceuticals. Examples of direct medical costs associated with the treatment
of Alzheimer’s disease include diagnostic tests, medications, and efforts to monitor or treat side effects, acute hospital care,
physicians’ services, home health care, and nursing home care (23 ,24 ). In other words, direct medical costs include any costs that
are directly related to medical treatment. Direct nonmedical costs include items such as cost of transportation to the doctor’s office
and cost of child care while the parent is hospitalized. An example of a direct nonmedical costs for the treatment of Alzheimer’s
disease is in-home day care (25 ). Examples of indirect costs are costs that arise from lost work or lost patient or caregiver
productivity. In conducting a cost analysis, the first challenge is to decide which costs are relevant for the comparison. The issue of
perspective (who pays?) becomes critical. Once this hurdle has been cleared, then the issue arises of how costs will be assigned. For
example, if prescription costs during hospitalization are included in a pharmacoeconomic analysis, how should the basis for costs be
established? Should it be based on actual acquisition costs, charges, or cost-to-charge ratios as a percentage of the entire hospital
bill? Clearly, these types of valuation decisions need to be disclosed and discussed in pharmacoeconomic studies.

Analyzing the results of pharmacoeconomic studies requires the evaluator to assess the types of costs included in each study. Large
variations in results can be attributable to different cost components (25 ). In the medical treatment of Alzheimer’s disease, the
acquisition cost of the medication is only a small percentage of the total cost. One major component of treating Alzheimer’s disease
is the indirect costs absorbed by family members. Researchers encounter difficulties in estimating the cost of such informal care. As
a result, investigators may account for those costs absorbed by family members in different ways, which may contribute to varying
conclusions (26 ).

There may also be intangible costs. These costs include things such as pain and suffering. Intangible costs are even more difficult to
value in monetary terms. In diseases where intangible costs are significant, it is important to recognize whether any effort has been
made to account for these costs. In many cases they are not included. Additional analysis may need to be considered to make fair
decisions.

One mechanism to quantify intangible costs is a willingness-to-pay approach. O'Brien et al. (27 ) performed a willingness-to-pay
evaluation in a group of patients with mild to moderate depression. The study was designed to compare a new antidepressant with
TCAs. The drugs had similar efficacy but different adverse event profiles. Participants were asked to rank a series of adverse effects
and then to quantify the maximum amount they would pay for a new drug that reduced each adverse event. On average,
participants were willing to pay an additional $14 per month to reduce the risk of blurred vision from 10% to 5%. When asked their
willingness to pay to avoid multiple simultaneous side effects, the range was $23 to $77 per month.

If costs, or benefits, are analyzed over time periods that exceed 1 year it is necessary to apply discounting. Discounting costs is a
concept that reflects the “time value of money.”
 P.529

A dollar today is worth more than a dollar received in the future. Discounting reduces the value of dollars that will be realized more than a year in the future to reflect a present value.
A similar concept applies to health benefits.

Perspective
One of the major factors that influences pharmacoeconomic analyses is the perspective taken when conducting the analysis. Using the earlier cost example, if a study is conducted
from the perspective of a hospital, the use of actual costs may be appropriate. However, if the same study were conducted from the perspective of a managed care organization,
charge data may be more relevant. The perspective of a pharmacoeconomic analysis should always be disclosed in a publication. Given that there are many possible perspectives, it is
insightful to evaluate studies from a broad perspective. The societal perspective is the broadest and takes into consideration all costs and consequences relevant to society. For various
reasons, this perspective is often used as a “reference case,” to permit comparisons across studies that may otherwise use differing perspectives. When measuring the impact of
pharmaceuticals on mental health disorders, the perspectives of both society and providers are important (14 ,28 ,29 ). Because of the desire to serve the needs of health system
decision makers, the perspective taken in many studies is that of the payer. A substantial number of patients prescribed antipsychotic medications have their health care paid by
Medicaid (30 ). Therefore, the perspective of Medicaid is important when evaluating the cost of schizophrenia treatment. The payer perspective, however, may not include all costs
relevant to society. For example, lost productivity may not be relevant from the Medicaid perspective. It is critical that the study perspective is disclosed when a pharmacoeconomic
analysis is published or evaluated. Generalizing results from a specific setting to a different setting is unwise because the relevant costs and outcomes vary between settings.

CONDUCTING PHARMACEUTICAL OUTCOMES RESEARCH

Part of "39 - The Role of Pharmaceuticals in Mental Health Care Outcomes "

There have been several texts and journal articles describing the steps for conducting pharmaceutical outcomes research and pharmacoeconomics (19 ,20 ). This section does not
reproduce these lists of steps, but rather presents some of the issues from these materials that are pertinent to the evaluation of mental health applications.

Decision Analysis
Decision analysis is a systematic approach to structuring decisions over time. Decision trees are developed with branches representing alternative decisions or probabilistic
relationships. For example, a decision may involve a choice between two drugs. These drugs may either have side effects or not, and treatment may be either successful or not
successful (Fig. 39.1 ).

FIGURE 39.1. Example of a decision tree.

For any of the nodes where a chance relationship exists (represented by circles), the probabilities associated with that event are shown (e.g., the probability of a side effect
associated with drug A is 0.7). When the outcomes are valued in dollars (i.e., the cost of following a particular path), the expected values that are calculated from a decision tree
analysis can provide cost estimates that are used in the numerator of a pharmacoeconomic ratio. Expected values are calculated by summing the product of the probabilities and the
costs. The expected values are shown in Fig. 39.2 .

FIGURE 39.2. A solved decision tree.

In this case, assuming that the cost at the end of each branch represents the total cost of care for the selected drug, the expected costs associated with the use of drug A is $144.25
and $91.50 for drug B. If outcomes were assumed to be equal (i.e., cost-minimization) then the least expensive alternative would be drug B. However, outcomes are not equal. As can
be seen from the decision tree, the probability of success while using drug A is greater than for drug B. If the path probabilities for the successful branches are summed for each
alternative, it can be seen that the probability of successful treatment with drug A is 0.815, and the probability of successful treatment with drug B is 0.720. Therefore, the total cost
of treatment while on drug A is higher, but so is the effectiveness (i.e., chance for successful treatment). The real question is one of cost-effectiveness. Is the additional cost of drug A
worth the additional benefits? This would be assessed with an incremental cost effectiveness ratio:
 P.530

Is an additional successful case worth paying an additional $555.26? This is a value judgment that depends on the situation
surrounding the decision. Again, outcomes research does not answer the question regarding which product should be used; it simply
provides information regarding the efficiency with which these two products produce a desired outcome.

This method, while powerful, is often limited by the availability of data to drive the model and the forced simplification of models,
which often results from limited driving data. It is important when reading published modeling exercises such as decision trees or
Markov models to evaluate them carefully, as results are highly dependent on the specifics of the structure selected (and how
closely it reflects clinical reality) and on the data selected for input. The latter problem can and should properly be addressed by
sensitivity analysis, for which there are several techniques. The former problem can be tested by the careful evaluation of experts
or the demonstrated ability of a model to predict measurable outcomes, a relatively uncommon exercise in pharmacoeconomics.

In general, the reader should look for some effort to discuss or examine “parameter” uncertainty, “model structure” uncertainty,
and “model process” uncertainty. With regard to parameter uncertainty (the term parameter refers, for example, to estimates of
probabilities or cost or health outcome), univariate analysis alone is often inadequate, and some attempt at multivariate evaluation
is desirable. There are different formal approaches to evaluation of cost-effectiveness uncertainty using either frequentist or
Bayesian approaches to generation of confidence (or “credible”) regions, including simulation and the delta method. Model
structure uncertainty refers to the separate uncertainty about the manner in which parameters should properly be combined (e.g.,
Are effects linear or nonlinear fashion? Are effects additive or multiplicative?). An approach to evaluation of this kind of uncertainty
is to simply examine results for different plausible alternatives. Model process uncertainty refers to the fact that different analysts
may come to different conclusions due to a spectrum of differences in approach. There are other key methodologic pitfalls in proper
conduct of studies of this type (e.g., adhering to use of incremental cost-effectiveness ratios), a fact that highlights the need for
readers to evaluate each study carefully.

The purpose of this discussion is not to fully explain the sometimes complex process of conducting a decision analysis, but rather to
suggest the usefulness of this tool in outcomes research. It is also to encourage the reader to develop the critical skills necessary to
evaluate studies of this type, much as similar skills have been developed for evaluation of ordinary controlled clinical trials. The
reader is referred to Clinical Decision Analysis by Weinstein and Fineberg (31 ) for a more complete description of the process of
conducting decision analyses, and to Cost-Effectiveness in Health and Medicine, edited by Gold (32 ) for in-depth discussion of many
important issues in cost-effectiveness analysis.

In a final example, we refer to a study using decision analysis to permit evaluation of a systematically developed time-ordered series
of events. It incorporates previously published clinical trial data into a model to estimate long-term effects. In this study, Dardennes
et al. (33 ) developed a decision analysis model to compare outcomes and costs of treating major depression with an SSRI, a TCA, or
serotonin norepinephrine reuptake inhibitor (SNRI). The perspective of the study was that of a national health care system, and the
clinical outcomes data used in the model were derived from published meta-analyses. The analysis found that the SSRI and TCA had
comparable efficacy but dissimilar tolerance profiles and that the SNRI had both efficacy and tolerance advantages compared to the
SSRI. Direct cost data (hospitalization, medication, physician visits, and laboratory tests) and the efficacy data from the model were
entered into a decision tree. The decision tree analysis provided estimates of the expected cost of treatment per
 P.531

depressive episode that could be used by the health service in its treatment approval process.

Data Sources
Data for pharmaceutical outcomes studies can come from several sources. Many pharmaceutical companies routinely include
pharmaceutical outcome (other than just clinical) measurements in their development trials. In addition, postmarketing studies with
direct comparisons to relevant alternatives (i.e., intended to serve the needs of pharmaceutical users rather than regulators) are
becoming more common. However, conducting single studies that contain all pharmaceutical outcomes of interest for a given
product are expensive, and data collection of all relevant information is difficult. Therefore, many pharmaceutical outcome studies
contribute to the body of knowledge by evaluating components of the overall picture. Additionally, many pharmacoeconomic
analyses are based on models. These models typically use published literature, expert opinion, or data from administrative or
encounter databases to get information on probabilities and costs.

The impact of this component approach to building an understanding of pharmaceutical outcomes is that data come from many
sources ranging from experimental and nonexperimental research designs to expert opinion and models based on data from multiple
and frequently diverse sources. Therefore, when reviewing pharmaceutical outcomes research, it is critical to understand the
potential impact of the source of information on the results.

A frequent source of outcomes data in mental health research is randomized clinical trials conducted by the pharmaceutical industry.
These trials, however, are often placebo controlled and typically contain (as expected) mostly clinical information. In mental health,
however, patient self-reported items (i.e., humanistic measures) are frequently included. There are also many studies that rely on
chart review and quasi-experimentation to document differences in resource use for patients using various pharmaceutical agents.
Examples include recent comparisons of tricyclic antidepressants and SSRIs, and atypical versus conventional antipsychotic agents.
Many of these studies were retrospective and were conducted through chart reviews or administrative data using quasi-experimental
techniques. Finally, economic models have been built using published data or expert opinion, where data were not available.

Historically, randomized controlled trials have been the “gold standard” (5 ). Studies of this type allow the efficacy and safety of a
drug to be established. Unfortunately, some of the strengths of such studies can also be a source of less commonly recognized
weaknesses. This is a result of the artificial treatment environment purposefully created in efficacy trials, and may be particularly
an issue in mental health because of the wide gap recognized to exist between the “optimal” care provided in such trials and the
realistic patterns of care experienced by most patients. The primary care provider deals with other issues that influence the effects
of a medication such as side effects, dose titration, and out-of-pocket expenses. As a result, real-world effects can be difficult to
extrapolate from ordinary clinical trials. This issue is discussed further below. Randomized trials failed to differentiate the SSRIs and
TCAs, except for their side-effect profiles. However, SSRIs may have some advantage over TCAs in the primary care practice setting
(6 ,28 ,29 ). In summary, data for building evidence about the value of pharmaceutical outcomes in mental health has been drawn
from a number of sources using a variety of experimental and nonexperimental designs. Each of these sources of data and type of
experimentation affect the degree of evidence obtained. Review of pharmaceutical outcomes research in mental health care
requires careful consideration of the source and strength of the evidence presented.

HUMANISTIC MEASURES
Part of "39 - The Role of Pharmaceuticals in Mental Health Care Outcomes "

Humanistic measures assess how disease or treatment affects patients. Humanistic measures are most important from the
perspective of the patient. A primary goal for treatment of any disease should be for patients to function normally, have an
acceptable quality of life, and be satisfied with their treatment. This is especially true for mental health disorders where impacts on
both physical and social functioning may be significant. In many cases, patients and their friends and families might best judge the
success of treatment. Until recently, humanistic measures have taken a back seat to traditional clinical measures and to some
extent economic measures. This is in part due to greater variability from patient self-reported measures compared to standard
clinical measures (34 ). The development of valid and reliable instruments is a relatively recent phenomenon. The most common
conceptualization of humanistic outcomes used in the evaluation of pharmaceuticals is health-related quality of life.

Health-related quality of life encompasses factors such as functional status, physiologic status, social and emotional well-being, and
life satisfaction (35 ). Health-related quality of life information allows health care providers and payers to make decisions based not
only on clinical effectiveness, or costs but also on effects that are important to patients. Measurement of health-related quality of
life may be especially important in chronic diseases for which we have no cure. There are many humanistic measures available for
assessing mental health disorders. Generic and disease-specific instruments are available for a variety of disorders. Discussion of
every instrument is not feasible; however, a few examples are provided.

Generic instruments are global in content and cover a number of dimensions relevant to overall health-related quality of life. One of
the most widely used generic health-related
 P.532

quality of life instruments is the Medical Outcomes Study Short Form 36 (MOS SF-36). The MOS SF-36 captures eight dimensions of
health-related quality of life: physical functioning, role limitations due to physical functioning, bodily pain, general health, vitality,
social functioning, role limitations due to emotional problems, and mental health (36 ).

A modified version of the Sickness Impact Profile has also been developed for use in patients with mental illnesses.

Disease-specific health-related quality of life instruments focus on dimensions that are most relevant to the particular disease and
are therefore more sensitive to subtle changes in the disease or its treatment. Examples of disease specific instruments used in
schizophrenia include the Quality of life Scale (37 ), the Social Performance Schedule (38 ), and the Quality of life Interview (39 ).
Several review papers have been published on the use of quality of life instruments in mental health conditions (40 ,41 and 42 ).
These articles highlight that quality of life measurement in mental health conditions, and in particular specific drug comparison, is a
developing science. Many of the available studies are observational or cross-sectional. However, quality of life measurement is
increasingly being built into clinical trials. There continues to be debate regarding mental health patients’ ability to complete
quality of life questionnaires, highlighting the importance of population-specific assessment of instrument validity and reliability.
However, several articles have shown that it is possible for patients with severe mental health problems to successfully complete
these forms (43 ,44 ). Lenert’s group (45 ) has shown that even when posed the conceptually challenging task of the standard
gamble, patients with mental illness have been able to perform adequately. It is important to note that although agreement is not
universal, there are many researchers who believe that in principle the best source for measuring patients’ quality of life or
preferences is the patients themselves whenever such measurement is possible. Much has been published on this subject and in
particular on the issue of whose values to use in creation of reference case analyses (to be used for comparisons across studies). But
rarely is the view of the health care provider or other proxy considered superior to that of either the patient or of society in general.

Health-related quality of life has many applications in the treatment of mental health disorders. For example, Simon et al. (46 )
completed an analysis that evaluated clinical effectiveness, health-related quality of life, and economics of treating depression. The
study took place within a staff model HMO and utilized net costs. Patients starting new antidepressant therapy were randomized to
an SSRI or TCA for 24 months. The primary care providers were allowed to adjust doses and medications or discontinue medications
as they deemed appropriate. The quality of life outcome was measured using the Medical Outcomes Study SF-36 Health Survey at 6,
9, 12, 18, and 24 months. The results indicated no significant difference in quality of life or severity of depression when comparing
treatment groups (46 ). Other studies have evaluated health-related quality of life in the treatment of depression and utilized
similar generic rating scales (47 ).

Numerous Alzheimer’s disease–specific quality of life tools are available. However, there is a lack of understanding of how to
quantify changes in scores. It is important to note that the cognitive impairment of Alzheimer’s disease at times requires the
administration of the tool to a care provider. The tools assess functions such as daily activities, memory, emotional well-being, and
other aspects such as finances (48 ).

One other area of humanistic measurement concerns the relationship between humanistic and economic outcomes. Cost utility
analysis uses patient preferences in the form of utilities to combine cost information with patient preferences. Utilities are usually
measured by three techniques: rating scales, the standard gamble, or time trade-off technique (49 ). Utility scores differ from
quality of life measurements. While some quality of life instruments can be used to capture utilities, most cannot. Utilities are a
measure of overall patient well-being that lie on a scale between 0 and 1. Utilities typically measure the difference in patient’s
preferences between perfect health and impaired health states. At present, utilities have been measured for only a few health
states. Unfortunately, utility values are difficult and expensive to measure. They require detailed patient interviews with large
numbers of subjects with and without the disease. Additionally, there are many questions about patient’s ability to give reliable and
valid responses. While these techniques have been used in mental health care, more widespread use is dependent on the
development of reliable and valid measures of utility or preference for alternative health states in mental health diseases.

USING OUTCOMES DATA IN PRACTICE

Part of "39 - The Role of Pharmaceuticals in Mental Health Care Outcomes "

The use of outcomes data in practice is not about applying the results of a single study. Instead, using outcomes data typically
requires synthesis across a body of literature. Outcomes data, and in particular economic and humanistic data, offer additional
pieces of information that should be incorporated into decisions. Economic, clinical, and humanistic data are all needed to make
fair evaluations of pharmaceutical products and services. In reality, however, decisions will be made even if all these data are not
available.

Uses of outcomes data in practice include reimbursement decisions, internal practice decisions, external or regulatory decisions,
and marketing of pharmaceutical products. Pharmacy and therapeutics committees are using outcomes data as a component of the
formulary decision. Where these decisions were once made almost entirely on clinical parameters, the use of economic and
humanistic data is becoming more common.
 P.533

In practice, the use of terminology such as evidence-based medicine or treatment guidelines has its roots in outcomes evaluations.
The evaluation of a body of literature to make decisions about best practice is the goal of evidence-based medicine. Evidence-based
medicine involves explicit use of what can be identified as the best evidence in making decisions about the care of both individual
patients and populations of patients (Fig. 39.1 ) (50 ). This philosophy extends into treatment guidelines that are often established
by expert panels that have reviewed the available evidence in the literature regarding effectiveness of alternative treatments.
While these efforts rely most heavily on clinical information, economic and humanistic data are being included in these
considerations.

Outcomes data is beginning to be considered in the accreditation of health care organizations. Although the measures currently used
are more process than outcomes oriented, the evolution toward outcomes can be seen. The National Committee for Quality
Assurance (NCQA) conducts accreditation of managed care organizations and has a specific program for behavioral health
accreditation. NCQA also sponsors the Health Plan Employer Data and Information Set (HEDIS) report, which is a set of standardized
performance measures designed to assist consumers with decisions about purchasing health care coverage. HEDIS 2000 includes
several measures relative to mental health care. These measures are organized into several categories. Under the effectiveness of
care category, two measures are included: follow-up after hospitalization for mental illness, and antidepressant medication
management. In the use of services category, mental health care related measures include mental health utilization, inpatient
discharges and average length of stay, and mental health utilization–percentage of subjects receiving services. These measures are
evolving to require managed care organizations to consider the outcomes of care they provide. As HEDIS measures continue to
evolve, they are expected to raise the quality of health care.

Some of the more sophisticated users of outcomes data may be pharmaceutical companies. Most major pharmaceutical
manufacturers are investing resources in departments that focus on the collection and analysis of outcomes data for their products.
Although these data are frequently used in the marketing of pharmaceutical products, they are also providing information about the
developing science of outcomes measurement.

Efficacy and Effectiveness

The evolution of the use of data for decision making is interesting. Health care organizations have evolved from requiring evidence
of efficacy to effectiveness to efficiency. Efficacy is defined as how well an intervention can ideally work. This is characterized by
the types of ordinary clinical trials that are commonly performed for regulatory purposes. This type of trial generally creates what is
believed to be ideal conditions of treatment and limits treatment to optimal patients, conditions that can be difficult or impossible
to duplicate in regular practice. Blind, prospective randomization of an adequate number of patients to a study in which outcomes
are assessed by raters blind to treatment is intended to minimize observer bias and confounding, and maximize internal validity.
Accordingly, clinical trials are excellent for providing confidence that there is a causal relationship between drug use and the
measured endpoint. Once confidence in this relationship is established, however, questions of use in the real world arise, which beg
the question of effectiveness. Efficacy results, generally using highly select, often healthier, patient populations (not least because
informed consent is required) under different practice conditions (tertiary vs. primary care, frequency of follow-up, compliance
issues, insurance issues, dosing/titration regimens, etc.), may simply not predict patient outcomes in usual practice. This leads to
the question of effectiveness, defined as the extent to which health improvements are achieved in real practice settings. Does a
pharmaceutical product work under real-world conditions? Is it really influencing outcomes that are important to patients, payers,
and clinicians? An experimental research approach to the question of effectiveness is a type of trial referred to variously as
“pragmatic trials” or “effectiveness trials.” Such trials employ random treatment assignment to address confounding, but sacrifice a
degree of internal validity in an effort to increase generalizability to the real world. This necessitates, for example, inclusion of as
many patients as possible (minimizing exclusions), using ordinary practice settings, avoiding protocol-mandated interference in
patient care, and permitting the effects of cost and payment mechanisms. The application of this useful type of trial in mental
health has been described, but rarely implemented (51 ). Alternatively, nonexperimental research designs (e.g., cohort studies,
retrospective database analyses, etc.) can often shed light on real-world outcomes when a clinical trial is impractical for reasons
such as sample size, informed consent, duration of follow-up, etc. Usually, such studies must take special care to address issues of
bias and confounding. When real-world data from either effectiveness trials or nonexperimental research are not available, as is
often the case, the evidence basis for mental health decision makers is limited to either attempting to generalize from efficacy data
or relying on some form of expert opinion.

The increasing expenditures associated with mental health disease states require decision makers to evaluate the full impact of
treatment alternatives. The evaluation should include the appropriate variables to fully evaluate patient outcomes (including quality
of life); an adequate evaluation of all relevant costs, which permits capture of potential offsets of simple drug acquisition costs; and
consideration of issues of efficacy vs. effectiveness. The tools of pharmacoeconomics
 P.534

and outcomes research provide decision makers with a mechanism for attempting to quantify and balance these factors to assist in the allocation of scarce mental health resources.

CONCLUSION
Part of "39 - The Role of Pharmaceuticals in Mental Health Care Outcomes "

Measurement of economic, clinical, and humanistic outcomes is an important tool for establishing the value of competing mental health care programs and treatments. Ultimately,
measures of quality of care in relation to commensurate costs should aid decisions about which programs to implement and which treatments to reimburse. Although no single study is
likely to provide an answer, careful evaluation of the economic, clinical, and humanistic outcomes literature may assist decision makers in making more informed decisions. Many of
the economic and clinical studies conducted to date use descriptive designs or apply modeling techniques based on the best source of available data. As the science behind outcomes
measurement evolves, the level of sophistication of the information provided will improve. Information on treatment outcomes can contribute significantly to decisions that affect the
quality of care received by mental health patients.

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40
Issues in Clinical Trial Designs

John M. Kane

J. M. Kane: Department of Psychiatry, Hillside Hospital, Glen Oaks, New York; Department of Psychiatry and Neuroscience, Albert
Einstein College of Medicine, Bronx, New York.

The introduction of the randomized, double-blind, clinical trial was one of the major advances in the development of medical
science. In the arena of psychotropic drug development this approach has proven to be of enormous value in advancing a field in
which laboratory tests and strictly objective methods for diagnosis and outcome assessment are not currently available.

Designing trials in the treatment of schizophrenia highlights some of these challenges. Schizophrenia is a complex illness affecting to
varying degrees a range of functions, including cognition, affect, behavior, mood, and motivation. The fact that this disorder affects
so many different domains, varying from individual to individual, and to some extent within individuals over time, makes
development of pharmacologic treatments even more challenging. Although there are core features of schizophrenia that involve
perception (hallucinations), cognition (attention, working memory, etc.), motivation (avolition), inferential reasoning (delusions),
and affect (blunted or inappropriate), there is no pathognomonic sign or symptom of the disease. This has important implications for
the diagnostic process, which is also complicated by the fact that the evaluation of some core features (e.g., hallucinations and
delusions) relies solely on subjective reporting, the accuracy of which is potentially influenced by the very symptoms themselves as
well as by other social situational and personality variables.

In addition, the fact that such an array of domains and functions is disturbed in this illness creates a challenge for drug development.
The tendency has been to conduct an array of assessments to evaluate drug effects in a number of domains concurrently, when in
fact different domains may require different study designs, patient selection criteria, durations of treatment, etc. In the future,
more attention will be given to those issues, and it is possible that multiple treatments will be studied rather with the goal of
finding combinations able to improve outcome across a variety of domains. It is hoped that new treatments will be developed with a
focus on specific domains such as negative symptoms and cognitive dysfunction. Although a better understanding of basic
mechanisms should facilitate further treatment advances, our current knowledge of pathophysiology remains limited. Advances in
imaging techniques and pharmacogenomics are also important potential developments on the horizon that could have enormous
impact on drug development and clinical evaluation.

Each area of psychotropic drug development has its own challenges in terms of rates of spontaneous remission, placebo response,
patterns of relapse, domains of assessment, etc., but, in general, challenges of design and methodology involve issues that cut
across the diagnostic domains.

 DESIGN ISSUES
 SELECTION OF PARTICIPANTS IN CLINICAL TRIALS
 PHARMACOKINETIC ISSUES
 ASSESSMENT OF THERAPEUTIC EFFECTS AND CLINICAL CHANGE
 PROBLEMS IN ASSESSMENT
 ASSESSMENT OF ADVERSE EXPERIENCES
 CONCLUSION

DESIGN ISSUES
Part of "40 - Issues in Clinical Trial Designs "

There are a number of critical issues in general design that need to be addressed in both the individual study as well as the
particular program of drug development. A drug development program needs to be comprehensive as well as adaptive so that early
results can inform subsequent evaluation. Although even when a drug is marketed there are still limitations in the amount of
knowledge available to clinicians, several fundamental questions should have been at least partially addressed: (a) What benefits
are likely to result from the drug? (b) What are its risks? (c) What dosage is indicated? (d) How does the new drug compare to
alternative treatments? (e) Are there specific patients most likely to benefit from the drug?

There are a number of specific concerns that should be addressed when designing clinical trials of psychotropic drugs. Some of the
most salient issues include dose finding; efficacy vs. placebo; efficacy vs. a standard reference compound; acute and long-term
adverse effects; continuation and maintenance treatment efficacy; and relative efficacy or adverse effects in specific subgroups
(e.g., early-phase illness, late-phase illness, refractory patients).

Dose-finding tolerability studies involving antipsychotic medications generally call for involvement of target patient populations
earlier in the process than with other classes of drugs because it is difficult to ethically justify administering
 P.538

these drugs to healthy volunteers for more than a week or two, and patterns of tolerance may be quite different in patients versus
healthy volunteers.

It is not always possible to accurately predict clinical dosage requirements from preclinical studies; therefore, it is important to
establish a full range of tolerable dosages in order to provide an appropriate range for efficacy studies. Drug development programs
have been delayed and at times abandoned because of inadequate dose-finding efforts in the early stages of development (1 ). In
addition, it is not unusual for dosage recommendations to change after a drug is marketed.

It is also important to have sufficient data on absorption, elimination, metabolism, and drug–drug interactions, to inform trial design.

Treatment trials generally fall into three broad categories: acute, continuation, and maintenance (or relapse prevention).
Sometimes attempts are made to study two or even three phases in the same trial, but controversy surrounds the need to
rerandomize patients before drawing conclusions about relative efficacy in maintenance-phase treatment. Patient characteristics
may vary somewhat in terms of desirability within specific trials, but overall the following issues should be considered.

Patient Characteristics
It is important to be clear on whether or not patients are in a state of acute relapse or exacerbation as opposed to partial remission
or a “stable plateau” of chronic symptomatology. At times investigators will withdraw patients from ongoing treatment in order to
transition them to a clinical trial, resulting in some symptom exacerbation. The importance of these different approaches is that
they may result in patients with very different degrees of drug responsiveness, different patterns of baseline symptomatology, and
varying degrees of “stability” in baseline symptomatology.

The ideal sample of patients is probably those who have not already been partially treated so that the full degree and time course
of response can be determined. However, given the way that subjects must be ascertained and recruited for trials, it is likely that
some treatment will have already been administered. The fact that participants have been partially treated or are in a chronic
symptomatic state does not necessarily preclude the detection of a subsequent, clinically significant drug effect, but it is likely that
the nature and magnitude of the effect will be altered.

The subjectivity of many components of symptomatology in psychiatric disorders creates special challenges. Given the fact that
many symptoms are subjective and cannot be confirmed or quantified using objective measures, the assessment of baseline status
can be difficult. Clinicians are particularly familiar with patients suffering from psychoses who are more open and explicit about
pretreatment psychopathology once they begin to improve. Some patients may not appear eligible for a trial or be willing or able to
give informed consent until they are partially treated.

A variety of subject characteristics should be considered in terms of inclusion and exclusion criteria. Specific decisions will be
influenced by the nature and goals of the particular trial.

Age is often a basis for exclusion (either too young or too old). Age can certainly affect pharmacokinetics of particular drugs. The
elderly are more likely to have comorbid medical conditions and be more sensitive to some adverse effects, and there are a variety
of issues when young patients are included in trials. These and other factors have led to a paucity of subjects at the extreme age
ranges in clinical trials. However, there has recently been increased recognition of the need for more early data on diverse age
groups, and mechanisms are being implemented to encourage their inclusion in clinical trials.

Gender can be an important variable, and women are often underrepresented in clinical trials.

Ethnicity may have implications for drug metabolism and tolerance. In addition, as pharmacogenomic strategies are developed to
extend clinical trial data, more accurate documentation of race will be critical.

Marital status can be a proxy for psychosocial adjustment and illness course, and may therefore be of prognostic significance.

Weight and body mass index have become an increasing concern from a public health standpoint and because of the considerable
weight gain observed with some psychotropic drugs and in particular several new-generation antipsychotic medications (2 ).

Diagnostic subtype can be important in helping to characterize those patients most likely to benefit from specific treatments.
Duration of illness and the duration of the current episode can be important in helping to define populations in terms of drug
responsivity as well as long-term course and outcome. A particular problem in many trials is categorizing patients’ histories in terms
of drug responsiveness. A current episode duration of more than 2 or 3 weeks could suggest that the patient is poorly or only
partially responsive to the treatments that have already been administered, or, alternatively that some other factor is complicating
treatment response (e.g., noncompliance, comorbid conditions, etc.). It can often be difficult to time the onset of illness or of a
specific episode. As putative novel compounds are developed, it may become increasingly important to test these agents in patients
who have not already been chronically exposed to other medications.

The specific type and severity of signs and symptoms required for entry into a trial will vary depending on the overall goals. Usually
a minimal threshold of severity is established for core symptoms of interest. It is hoped that studies will also focus on patients
selected on the basis of significant residual or secondary symptoms if they are associated with subjective distress and/or functional
impairment.
 P.539

If trials are designed to focus specifically on patients who were nonresponders or intolerant to other treatments, explicit criteria
should be developed to identify such groups. There is debate as to whether or not a prospective trial is necessary to confirm
treatment refractoriness, but this is certainly the most conservative approach because it also addresses to some extent the potential
change in treatment milieu and attention resulting from participation in a research trial. In addition, there is enormous variability in
the quality of retrospective assessment of treatment response.

Drug washout is a challenge in acutely ill patients. If some exacerbation in symptoms occurs, this complicates establishment of a
baseline as well as adding to ethical concerns and management issues. On the other hand, absence of a washout means a true
“baseline” is not achieved, assuming that there has been some degree of response and or adverse effects from the prior treatment.
The use of a concurrent placebo group in the treatment trial mitigates these concerns to some extent, but does not eliminate them
entirely. The type, dosage, and half-life of prior treatments will influence how long a washout is necessary to prevent potential
withdrawal effects from influencing baseline ratings. Whether or not a washout takes place (and how long it is) can have
implications for assessing the effects of subsequent treatment. The effects of withdrawal are neither consistent nor predictable,
which complicates establishment of an appropriate baseline.

Premorbid social adjustment is a variable that does have prognostic significance, particularly in schizophrenia. Poor adjustment is
associated with poorer outcome, and may be an indicator of those patients in whom early neurodevelopmental abnormalities or
prodromal symptoms were more severe.

Comorbid psychiatric disorders should be evaluated and documented. Though there are insufficient data to determine what
influence comorbid conditions are likely to have on overall response to psychotropic medications, common comorbid conditions
should be studied at some point to help assure generalizability and to inform clinical practice. In addition, some studies tentatively
suggest that different medications may have more or less impact on measures of, for example, substance abuse, suggesting that this
could also be an important outcome measure in appropriate populations.

In studying antipsychotic medications it is important to document the presence and severity of any preexisting movement disorders
in order to have an adequate baseline assessment and to ensure that a preexisting condition (or withdrawal effect) is not attributed
to subsequent treatment.

It is essential that patients be assessed for their capacity to give informed consent. It is beyond the scope of this chapter to discuss
this in great detail, but patients should be able to describe and explain in their own words the research in which they are agreeing
to participate, its goals, its experimental aspects, and its potential risks and benefits. They must understand that they have the
right to withdraw at any time and that they will not be penalized in any way if they choose to do so.

Trial Design
One of the most critical and difficult aspects of trial design is weighing and balancing what is ideal and what is feasible. An ideal
trial for which patients cannot be recruited or in which they cannot be retained will not achieve its goals. In addition, though many
questions ultimately need to be addressed, it is usually impossible to adequately address multiple questions in a single trial.

The duration of a trial will be influenced by whether or not a placebo group is included. The longer the duration, the more difficult
to justify the retention of patients on placebo, and the higher the dropout rate, the less useful are the data.

The time course of response to psychotropic medication is generally variable. The modal time frame of response has to be factored
into trial design in order to allow estimates of statistical power. In the acute treatment of schizophrenia, for example, most patients
will experience at least half of the ultimate degree of improvement within the first 4 to 6 weeks (assuming that there was not an
inordinately long titration phase). In many studies a significant drug effect is seen after only 1 to 2 weeks; however, different signs
and symptoms are likely to have a different time course of response. For example, agitation is likely to respond more rapidly than
delusions or thought disorder. In addition, there may be a subgroup of patients who are slower to respond, and for such patients
longer trials may be needed. If a between-drug comparison of the full extent of response is ultimately important, then much longer
trials are needed (e.g., 6 months or longer), and this begins to encompass the continuation phase of treatment. As more and more
domains of outcome are of interest in clinical trials (such as primary negative symptoms or cognitive dysfunction in schizophrenia),
it will be important to better characterize the time course of response for these variables in order to establish minimum and
optimum durations of trials for these purposes. Estimates of expected degrees of improvement in various domains will be critical for
statistical power calculations.

The Role Of Placebos

The decision as to whether or not to use a placebo in short-term, acute trials remains a topic of considerable controversy, and some
dynamic tension continues to exist between “regulatory” requirements, investigators, institutional review boards, patients and
families, and other interested parties. There are a number of important arguments that can be made against the routine use of a
placebo in clinical trials. Rothman and Michels (3 ) argue that when an effective
 P.540

treatment exists for a particular disease, the use of a placebo is inappropriate on both logical and ethical bases. However, the
argument suggests that the use of a placebo is appropriate in cases when an effective treatment is not available. A problem remains
in how to define effectiveness. The use of the term effective in this context is not necessarily identical to the current use of
effectiveness as differentiated from efficacy.

In a complex disease such as schizophrenia, we continue to struggle with establishing the most meaningful definitions of efficacy
and effectiveness. If we define response narrowly in terms of positive symptoms, then certainly some response to conventional
agents is expected. In the case of severe deficit symptoms or in patients who have proven refractory to other drugs, the issue is less
clear.

A particular problem arises when response to a proven effective treatment (or so-called gold standard) can vary enormously from
trial to trial and in some cases be rather low, or when response to a placebo is generally high (4 ).

The argument is often made that in developing new drugs to treat a condition for which effective treatments are already available,
the question should not be is the new drug superior to placebo but rather is the new drug superior to an already available agent.
Unfortunately, given the nature of the diseases and the adverse effects associated with some psychotropic drugs, a new drug could
be superior in one domain and inferior in another, while being a very valuable addition to the therapeutic armamentarium. The use
of placebo controls can still be important to determine whether or not in some domains a drug is inferior, but still better than a
placebo, or whether its inferiority in one domain is such that it would change the overall effectiveness equation.

To provide an example, suppose drug A were somewhat less effective than drug B in controlling acute symptoms, but some patients
did quite well on drug A. At the same time, drug B was associated with serious side effects that might result in a substantial number
of patients discontinuing the medication within a short period of time. Would we prefer to have drug A available to treat those
patients who benefited from it, while then giving drug B to those who don’t. Before approving drug A, we would want to be certain
that it was superior to a placebo, though inferior to drug B in the particular domain of acute response.

There are a host of issues relating to the use of placebos that have been discussed in more detail elsewhere. As Lavori (5 ) has
emphasized, the data sets available from current placebo-controlled trials are usually “heavily truncated, differentially by
treatment groups, and certainly nonrandomly.” He argues that most investigators “use ad hoc statistically unjustifiable maneuvers
such as last observation carried forward (LOCF)” and that “the interpretation of positive results in the context of badly truncated
data requires unverifiable assumptions, external to the observed data of the study.”

Another important consideration in the use of a placebo is the potential harm resulting from a delay in instituting active treatment.
This is a difficult question to adequately address; however, there have been some attempts to examine the consequences, both
short- and long-term, of receiving a placebo in the context of short-term trials. Overall, there do not appear to be demonstratable
deleterious effects of participating in short-term trials (6 ,7 ). The issue of lengthy delays (i.e., 6 months or longer) in implementing
treatment has been a topic of discussion in first-episode schizophrenia patients, with some authors suggesting that the longer
duration of untreated psychosis is associated with poor outcome. In one patient cohort, this effect was reported in short-term
outcome (8 ), but the effect was no longer evident in long-term follow-up (9 ). Short-term clinical trials usually involve durations of
4 to 8 weeks. Therefore, it is important to recognize potential differences in consequences between brief delays and relatively long
delays in treatment. Lavori (5 ) argues that because assessments in placebo-treated patients are usually truncated because of high
dropout rates, we do not know the full consequences of exposure to a placebo. The field would certainly benefit from more intent-
to-treat analyses as well as long-term follow-up of patients who were involved in placebo-controlled trials.

Designs involving the treatment of patients who have failed on other treatments are another challenge. One could argue that
placebo controls are more acceptable in this context because there is no effective treatment. However, it is usually the case that
these patients have demonstrated some benefit from standard, albeit inadequate, treatment. Therefore, the appropriate
comparison would be the new treatment versus standard treatment, with the only outcome of interest being the superiority of the
former.

The decision as to whether or not to use placebo or active controls or both in a particular trial is not an easy one. There are complex
issues that need to be considered, and it is hoped that further knowledge involving the determinants of heterogeneity in response
will facilitate more rational and acceptable trial designs (10 ).

A related problem is the use of rescue medication. Balancing the desire to retain subjects and the desire to prevent harm and not
withhold effective treatment is a critical issue. To what extent should other medications be available for those participants who
would otherwise be dropped from a trial due to lack of efficacy and need for alternative treatment? Extensive use of rescue
medication can make it difficult to accurately assess the drug effect (even though use of rescue medication can be a telling outcome
in and of itself). The use of adjunctive medication to treat adverse effects that occur in the course of a trial can also be a concern
(e.g., the use of antiparkinsonian medication). Here, too, rates of utilization can be an important outcome measure, yet at the
same time the additional medication might have other undesirable effects (e.g., cognitive impairment).

A number of novel designs have not been widely used, and to some extent there is a disincentive to utilize them, particularly in a
regulatory context.
 P.541

Crossover designs have been suggested as one alternative, although some exposure to a placebo is still involved. A patient receives a
potentially active compound and if response occurs, crossover to a placebo takes place. If response does not occur, the placebo
phase is not required. The placebo phase in this context helps to determine whether or not the response to medication was a true
drug effect or not. It is argued that this design has the advantage of each patient serving as his or her own control, allowing all
patients to eventually receive active medication and increasing statistical power.

The applicability of this design varies depending on the nature of the disorder being studied, the time course of response, and the
vulnerability to relapse or symptom exacerbation once active treatment is replaced by a placebo. For example, this design may be
more informative in rapid cycling bipolar patients (11 ) than in the context of an acute treatment trial in other disorders. Also, this
trial does not eliminate exposure to a placebo. From an ethical standpoint, how do we weigh the delay in providing active treatment
against the withdrawal of effective treatment once a response occurs, with the outcome of interest being an exacerbation of
symptoms?

Other alternative designs include adaptive allocation strategies. The intent of this approach is to reduce the number of subjects
exposed to placebo, ineffective, or toxic treatments. This is achieved by altering the probability of a participant’s receiving one
treatment or another based on the probability established to that point in the trial of which treatment is associated with the best
outcome. These designs are difficult to conduct, and they require knowledge of the results of completed subjects in order to
allocate treatment for the next subject. In addition, the response criteria have to be clearly established a priori. The design
becomes more complicated when three or more arms are included in a trial. Some studies have utilized such designs with success
(12 ). The ultimate goal of reducing the number of subjects exposed to inferior treatments can be achieved; ultimately, however,
the number of subjects required will depend on the effect size of interest. (For further discussion see ref. 13 .)

Active Controls
Comparisons between experimental treatments and active controls require careful consideration in terms of specific drugs, dosage,
adverse effects profiles, titration requirements, etc. If a dose of the comparator is too low, efficacy could be less than possible, and
if the dosage is too high, then adverse effects may occur more often. This issue is often a particular concern in industry-sponsored
studies, where marketing issues often influence the choice of comparator and even its dose. This highlights the potential value of
studying a range of doses of both the comparator and the experimental drug. Though this is costly, the information can be
particularly valuable in informing clinical practice. Unfortunately, this is rarely done (14 ). To some extent, this results from
unfounded assumptions that we have good data on dose-response relationships with drugs that have been in widespread use. Often
that is not the case. In addition, dosage requirements will vary depending on the population. For example, in schizophrenia, first-
episode patients in general respond to lower doses than multiepisode patients, and acute treatment usually requires higher doses
than maintenance treatment.

Another design that is being increasingly utilized is the adjunctive or add-on strategy. This is particularly useful when subjects with
partial or inadequate response are the focus of interest. Rather than switching participants from the unsatisfactory treatment to a
new treatment, participants are randomized to an added placebo or added experimental treatment. In this approach, no drug
withdrawal is necessary and the question of interest is whether or not the new treatment provides additional benefit.

The potential disadvantages of such a design include drug–drug interactions, particularly if a novel effect is anticipated from the
adjunctive treatment. Will this be influenced by the original treatment (e.g., different receptor binding profiles)? This approach is
particularly relevant when monotherapy is the exception rather than the rule. This type of design has been employed in the
development of anticonvulsant medications (15 ).

Continuation Treatment
After improvement in acute symptomatology, there is a period of consolidation and stabilization often referred to as the
continuation phase. It is assumed that discontinuation of medication during this period would be associated with a higher risk of
relapse than subsequent discontinuation. It is difficult to specify when the transition from continuation treatment to maintenance
(or prophylactic) treatment occurs, but at least 6 months is a reasonably conservative threshold. The question arises as to how to
characterize those patients who have experienced clinically significant improvement, but continue to have more than mild
symptoms. In such patients, the continuation phase could become indefinite rather than transitioning to maintenance treatment.
This is a semantic distinction because the goal of maintenance treatment is to prevent a relapse or reexacerbation of psychotic signs
and symptoms.

A continuation versus discontinuation design can be a sensitive test for drug effect. However, ethically, consent and protection
issues are a major concern when any degree of worsening becomes an outcome measure. If such designs are considered, strategies
such as sequential analyses or planned interim analysis would be important in terminating the study at the earliest appropriate time.
 P.542

Maintenance Treatment
In any potentially recurring or chronic illness, the issue of long-term treatment is critical (15 ). Clearly, the more information on
natural history and untreated course that is available from whatever source, the better in helping to define the goals and objectives
of maintenance treatment. However, as is often the case, long-term outcome data in such a context are likely to be unavailable,
and when comparisons are made with historical data there have often been changes in diagnostic criteria, ascertainment techniques,
or other factors that would limit generalizability.

In considering the role of maintenance treatment, frequency, severity, and potential consequences of relapse are critical. Is
maintenance treatment justified if a relapse is unlikely to occur for several years? This will be influenced not only by the
consequences of a potential relapse, but also by the potential consequences of the prophylactic treatment itself.

In this context, the appropriateness both from a scientific and ethical standpoint of including a placebo control is an enormous
concern. The fact that relapse rates on active medication and placebo can vary enormously from one study, one site, or one
population to another is an important consideration. Some would argue that an active comparison involving an experimental
medication could result in as many or more relapses than could occur in a placebo-controlled trial given the sample size needed to
avoid a type II error. Concerns similar to those raised previously apply here as well in terms of multiple domains of outcome and
benefit-to-risk ratio. If drug A had a significantly higher relapse rate than drug B but was much safer and more likely to be taken on
an ongoing basis, would this drug be utilized if it were shown to be superior to a placebo? How much worse than standard treatment
and how much better than placebo would a drug have to be in order to decide one way or the other? This is an unresolved issue in
terms of regulatory, scientific, and ethical concerns.

Many of the issues raised previously in the discussion of acute treatment apply here as well. Patient characteristics, age, sex,
ethnicity, age at onset of illness, duration of current or most recent episode, baseline psychopathology, comorbid conditions, etc.
are all important issues. Even premorbid psychosocial adjustment has been shown to have some predictive power in relapse
prevention studies in schizophrenia (8 ,17 ).

Issues such as reference comparator, dosage, route of administration, concomitant treatments (both pharmacologic and
nonpharmacologic), a priori relapse or exacerbation criteria, duration, and strategies to enhance and measure compliance are all
important in designing such studies.

The duration of such trials is critical in achieving overall goals. Results can be quite different during the first year of maintenance
treatment as compared to the second, with relapse rates often being higher in the first year following recovery from an acute
episode as compared to the second year (18 ). At the same time, in some studies involving dosage reduction, relapse rates were
higher in the second year than in the first (19 ).

This discussion also relates to the issue of time course of relapse in establishing appropriate durations for maintenance trials. In
schizophrenia, for example, based on historical data most relapses do not occur for several months after complete drug
discontinuation in stable outpatients. One context where time course of potential noncompliance and time course of relapse was
such that trial designs were probably inadequate to find meaningful differences was in the comparison of oral and depot medications.
A number of double-blind controlled trials were conducted in which patients were randomly assigned to depot or oral medications
and therefore had to receive both injections and tablets, one of which was a placebo. The duration of all but one of these trials was
1 year. In general, they failed to find the significant differences that had been expected given high rates of noncompliance in
schizophrenia and high rates of relapse following drug discontinuation. However, meta-analysis of these studies supports the value
of long-acting injectable preparations (20 ).

It is likely that the less than expected effects were due to an inadequate duration. Given the fact that subjects agreeing to receive
both injections and tablets in a double-blind design are on the more compliant end of the spectrum, one would not expect
noncompliance to occur rapidly. In fact, it could take many weeks or months, particularly given the frequent assessments and the
psychosocial support involved in being part of a research project. Because the relapse that ensues after complete discontinuation of
medications is not likely to occur for several months, it would be unrealistic to expect to observe a difference between depot and
oral medication in such a study if the duration was only 1 year (21 ). The only such study that lasted 2 years found no difference
between treatments in the first year, but evidence of clear separation in the second (22 ). However, the sample size was inadequate
to have sufficient statistical power to establish a significant difference, even in the second year.

The role of nonpharmacologic treatments and environmental factors in long-term studies is also important. There is clear evidence
that application of nonsomatic interventions can have significant impact on relapse rates among individuals receiving
pharmacotherapy. Although ideally nonpharmacologic treatment should be controlled, if it is not there should be documentation of
availability and utilization so that potential confounds can be identified.

Another important issue in the design of maintenance trials is whether or not rerandomization following recovery from an acute
episode is necessary to demonstrate efficacy in the maintenance phase. In some drug development programs, those patients who
respond in the context of an acute trial will be followed and relapse rates reported in comparison to a reference drug. This design
provides data
 P.543

from only those patients who responded to each drug acutely. The argument is made that to demonstrate efficacy in relapse
prevention, patients should be rerandomized or the study should be started after patients have been stabilized on any drug. This
then allows conclusions to be drawn regarding prophylactic efficacy among patients in general, not just those who responded to an
acute trial of a particular drug. (In addition, it is important to recognize high rates of attrition for other causes in acute treatment
trials.) This is not to say that there is no value in collecting long-term continuation data on a particular medication, because these
data are important in setting the stage for subsequent evaluation and comparisons.

As more domains of interest are examined in schizophrenia, it is necessary to consider the specific designs required to establish
efficacy and particular outcome measures. In recent clinical trials, attempts have been made to collect data on an array of
measures when at times important confounds can compromise interpretation. For example, in schizophrenia, primary negative
symptoms are difficult to study in the context of an acute treatment trial that has selected patients on the basis of having clinically
significant positive symptoms. Trials need to be conducted in patients selected on the basis of having residual negative symptoms
not complicated by acute positive symptoms or significant extrapyramidal side effects. Remarkably few such studies have been done.

Similar concerns surround the issue of cognitive dysfunction. Newer antipsychotics show some promise in improving measures of
cognitive function (23 ). However, studying these measures requires designs specific to their optimum assessment. In addition, the
ultimate question in measuring cognitive performance will be what impact these changes have on functioning, either psychosocial or
vocational, level of care, family burden, etc. To date, such studies have not been conducted, and it is premature to conclude that
measurable differences on specific cognitive tests will translate into meaningful differences in functioning.

SELECTION OF PARTICIPANTS IN CLINICAL TRIALS

Part of "40 - Issues in Clinical Trial Designs "

The issues discussed in the previous paragraph serve as examples of how patient selection becomes a critical focus in expanding our
knowledge of specific drug effects.

Effectiveness Research
Increasing attention has been focused on the fact that traditional randomized clinical trials often include highly selected patients
who may not be representative of the population at large. As new medications are used in routine clinical practice, there is often a
considerable gap in the knowledge base needed to inform decision making. For example, many patients with schizophrenia have
comorbid conditions (e.g., substance abuse) that could influence dosing patterns, adverse effects, overall response rates,
compliance, drug interactions, etc. The pharmaceutical industry does not necessarily have an incentive to conduct effectiveness
research, as the narrowly defined clinical trial is the most useful and probably cost-effective approach to the drug approval process.
In addition, including patients with comorbid psychiatric and medical conditions can potentially increase rates of apparent adverse
effects where attribution can be difficult.

At the same time, mechanisms should be sought for conducting effectiveness trials, which are extremely important in informing
clinical practice and public policy decisions.

Approaches to Subject Selection

Diagnosis and Phenomenologic Characterization

At present, diagnostic classification is an important element in patient selection. Although nosology shifts over time, it is important
to incorporate into the selection criteria the use of an established diagnostic system with proven validity and established reliability.
Ideally, research should involve a more systematic and formal diagnostic process than simply relying on a hospital chart diagnosis.
Formal evaluation instruments are available for specific diagnostic systems. Although the use of the complete interviews may be
overly time-consuming and not cost-effective for some types of research, at minimum a checklist indicating how patients met
specific diagnostic criteria should be completed.

As discussed previously, diagnostic subtype has not been a consistent predictor of drug response; however, as classification systems
improve and, it is hoped, subtypes become more meaningful, this element will have increasing importance in clinical trial design.

Because many psychotropic drugs are effective across a range of illnesses, a phenomenologic approach to characterizing
pharmacologic effect could be reasonable. Although issues of reliability and generalizability would have to be carefully addressed, it
is hoped that further research will lead to advances in this perspective.

Biological Classification
Although diseases such as schizophrenia have been characterized by a broad array of biologic abnormalities, there are as yet no
well-validated biological classification systems that have proven to be useful in clinical trials or in drug development. This may be
largely due to lack of systematic effects in this direction rather than an absence of potentially informative relationships. As further
advances take place in diverse perspectives ranging from neuroimaging to pharmacogenomics, it is just a matter of time before
biological classification becomes a critical ingredient in this context.
 P.544

At present, many of the findings are based on group differences and are not necessarily appropriate as selection criteria for clinical
trials. In addition, a variety of concerns including sensitivity and specificity will need to be addressed in further developing this
perspective.

PHARMACOKINETIC ISSUES
Part of "40 - Issues in Clinical Trial Designs "

The more knowledge available about pharmacokinetics and metabolism (including activity of metabolites) before large-scale clinical
trials are designed, the better. Understanding potential relationships between blood levels and therapeutic response as well as
adverse effects can be very helpful in optimizing treatment outcome. However, relevant data are often inadequate before critical
decisions about dose and dosing schedules are made. If more attention were given to these issues earlier, clinicians would have to
struggle less with establishing appropriate treatment strategies. Advances in brain imaging have set the stage for useful
investigation during early stages of drug development; however, here, too, few systematic efforts have been made to take
advantage of the potential of such studies to help establish optimum strategies for clinical trials.

Clinicians value the availability of different delivery methods for psychotropic medications, given the challenges of both acute and
long-term treatment. Oral, liquid, intramuscular, and long-acting forms should be developed and tested in clinical trials as early as
possible. Different clinical trial designs may be necessary with different preparations intended for different levels of acuity or
phases of treatment. Here, too, the more information available about pharmacokinetic and pharmacodynamic issues, the better.

Given the heterogeneity of clinical response and the enormous variability in drug absorption and metabolism, randomly assigning
patients to different plasma levels of interest can be a powerful tool in establishing dose-response relationships and optimum dosing
guidelines. Though more difficult than the standard trial, such studies are feasible, but rarely done (24 ).

ASSESSMENT OF THERAPEUTIC EFFECTS AND CLINICAL CHANGE

Part of "40 - Issues in Clinical Trial Designs "

There are many established instruments for the assessment of psychopathology in clinical trials. In some cases, these instruments
have been utilized for many years. However, there continues to be a dearth of new scale development. This is partially due to the
tedious nature of the development process and the reluctance of many sponsors of clinical trials to utilize a new instrument. As new
drugs are developed with potentially different spectrums of activity, it would be useful to have new scales designed to be sensitive
to specific therapeutic effects. This is particularly appropriate since many of the original assessment scales were validated by
proving sensitive to the effects of specific classes of psychotropic medications. (For detailed discussions of specific instruments for
clinical assessment see ref. 25 and ref. 26 .)

As outcome measures of interest become more broad, an array of separate supplemental instruments are being employed to
measure quality of life, social and vocational adjustment, cognitive functioning, and substance abuse. In designing assessment
batteries, it is important to choose instruments with proven reliability and validity as well as instruments that are likely to be
sensitive to the kind of treatment effect being measured. Meaningful clinical effects should be identified with specific measures of
change in order to ensure that the sample size provides adequate statistical power.

As increasing numbers of assessments are employed, it is also important to recognize the burden created for patients and raters.
Careful thought should go into selecting the most informative measures and planning a data analysis program with a priori primary
and secondary hypotheses.

PROBLEMS IN ASSESSMENT
Part of "40 - Issues in Clinical Trial Designs "

Because psychiatric disorders are often complex, multifaceted diseases and some key symptoms are purely subjective, the
techniques used for assessment can be critical. Information regarding psychopathology is most frequently obtained from direct
patient interview and observation, though information from other sources (e.g., family, nurses) is sometimes used. Patient report
can be impeded by intentional concealment, lack of insight, paranoid ideation, and the overall acuity and severity of the illness. It is
not uncommon for psychiatric patients to reveal more psychopathology as they begin to respond to treatment than they did prior to
its initiation. The reliability and validity of different sources of information in assessing specific domains have not been adequately
studied. In many trials assessors who are not familiar with the patient on an ongoing basis are asked to rate psychopathology.
Although these ratings can be sensitive to treatment effects, it is likely that a person who has ongoing contact with the patient in a
treatment context will provide a more accurate assessment. Here, too, research comparing different rater allocation strategies
would be helpful in determining which is most valid and cost-effective. It is critical to have the same rater evaluating the patient
throughout the trial whenever possible. Despite establishing high degrees of interrater reliability, this kind of continuity is important.

The timing of assessments and the time frame chosen for a given assessment should be determined by the goals in the study. In
general, when rating psychopathology, the previous week is a reasonable time frame. Patients are less likely to accurately recall
specific symptoms that are more
 P.545

remote in time. The time frame used for a particular assessment does not need to coincide with the interval between assessments. In a long-term trial it is not necessary to rate
patients weekly. But when they are assessed, the previous week can be the focus of the assessment.

ASSESSMENT OF ADVERSE EXPERIENCES

Part of "40 - Issues in Clinical Trial Designs "

The two major goals of drug development—to enhance therapeutic efficacy and to improve tolerability—go hand in hand, particularly in the case of psychotropic medications, where
many side effects of psychotropic drugs overlap clinical signs and symptoms of psychiatric illnesses. Given the frequent long-term nature of psychotropic drug treatment, adverse
effects become critical in influencing compliance and determining the overall benefit-to-risk ratio.

In general, the methods for detecting adverse events have been given far less attention than the methods for evaluating efficacy. Controversy exists as to the most valid means of
accurately estimating the incidence of adverse effects. Many clinical trials rely on patient self-report, with some specific queries or rating scales used to assess known adverse effects
(e.g., extrapyramidal side effects or tardive dyskinesia) that are outcomes of interest. Given the subjective nature of many adverse events, there is a concern that detailed, specific
queries across a broad range of possible symptoms will result in the elicitation of far more symptoms than an unstructured approach.

A methodologic comparison study (27 ) suggested that the general elicitation of adverse events is more practical and appropriate for routine clinical trials than a comprehensive and
lengthy interview. At the same time the field needs to acknowledge the possibility of inordinate delays in recognizing the frequency of specific adverse events such as the sexual
dysfunction associated with selective serotonin reuptake inhibitor (SSRI) antidepressants.

There is a strong argument for the use of data and safety monitoring boards when large and/or long-term studies are involved or high-risk treatments are being studied.

CONCLUSION
Part of "40 - Issues in Clinical Trial Designs "

The clinical trial remains the mainstay of treatment development. It is always hoped that further advances will evolve more rapidly than they do, but there is reason for considerable
optimism that over the next decade there will be important advances in predicting and understanding psychotropic drug response whether via functional neuroimaging,
pharmacogenomics, or other potential developments. In addition, it is hoped that increasing emphasis on studying a broader array of functionally meaningful outcome measures in the
context of better informed benefit-to-risk assessment and documentation of cost-effectiveness will lead to clinical trial designs to better address the full range of public health issues.

REFERENCES
1. Klein DF. Improvement of phase III psychotropic drug trials by intensive phase II work. Neuropsychopharmacology 1991;4:251–257.

2. Allison DB, Mentore JL, Heo M, et al. Antipsychotic-induced weight gain: a comprehensive research synthesis. Am J Psychiatry 1999;156(11):1686–1696.

3. Rothman KJ, Michels KB. The continuing unethical use of placebo controls. N Engl J Med 1994;331:394–398.

4. Leber P. The placebo control in clinical trials (a view from the FDA). Psychopharmacol Bull 1986;22:30–32.

5. Lavori PW. Placebo control groups in randomized treatment trials: a statistician’s perspective. Biol Psychiatry 2000;47:717–723.

6. Curson DA, Hirsch SR, Platt SD, et al. Does short term placebo treatment of chronic schizophrenia produce long term harm? Br Med J 1986;293:726–728.

7. Wyatt RJ, Hinter ID, Bartko JJ. The long-term effects of placebo in patients with chronic schizophrenia. Biol Psychiatry 1999;46:1092–1105.

8. Loebel AD, Lieberman JA, Alvir JMJ, et al. Duration of psychosis and outcome in first episode schizophrenia. Am J Psychiatry 1992;149:1183–1188.

9. Robinson D, Woerner MG, Alvir JMJ, et al. Predictors of relapse following response to first episode schizophrenia or schizoaffective disorder. Arch Gen Psychiatry
1999;56:241–247.

10. Mattocks KM, Horwitz RI. Placebos, active control groups, and the unpredictability paradox. Biol Psychiatry 2000;47:693–698.

11. Post RM, L’Herrou T, Luckenbaugh DA, et al. Statistical approaches to trial durations in episodic affective illness. Psychiatry Res 1998;78:71–87.

12. Tamura RN, Farris DE, Andersen JS, et al. A case study of an adaptive clinical trial in the treatment of outpatients with depressive disorder. J Am Stat Assoc 1994;89:768–
776.

13. Rosenberger WF, Hu F. Bootstrap methods for adaptive designs. Stat Med 1999;18:1757–1767.

14. Zimbroff DL, Kane JM, Tamminga CA, et al. Control, dose-response study of sertindole and haloperidol in the treatment of schizophrenia. Am J Psychiatry 1997;154:782–791.

15. Brodie MJ. Antiepileptic drugs, clinical trials, and the marketplace. Lancet 1996;347:777–779.

16. Greenhouse JP, Stangl D, Kupfer DJ, et al. Methodological issues in maintenance therapy clinical trials. Arch Gen Psychiatry 1991;48:313–318.

17. Kane JM, Rifkin A, Quitkin F, et al. Fluphenazine versus placebo in patients with remitted, acute first episode schizophrenia. Arch Gen Psychiatry 1982;39:70–73.

18. Hogarty GE. Prevention of relapse in chronic schizophrenic patients. J Clin Psychiatry 1993;54(suppl 3):18–23.

19. Marder SR, Van Putten T, Mintz J, et al. Low and conventional dose maintenance therapy with fluphenazine decanoate. Two-year outcome. Arch Gen Psychiatry
1987;44:518–521.

20. Davis JM, Barter JT, Kane JM. Antipsychotic drugs. In: Kaplan HI, Sadock BJ, eds. Comprehensive textbook of psychiatry. Baltimore: Williams & Wilkins, 1989:1591–1626.

21. Kane JM, Borenstein M. Compliance in the long-term treatment of schizophrenia. Psychopharmacol Bull 1985;21:23–27.

22. Hogarty JE, Schooler NR, Ulrich R, et al. Fluphenazine and social therapy in the after care of schizophrenic patients: relapse analysis of a two-year controlled study of
fluphenazine decanoate and fluphenazine hydrochloride. Arch Gen Psychiatry 1979;36:1283–1294.
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23. Meltzer HY, McGurk S. The effects of clozapine, risperidone, and olanzapine on cognitive function in schizophrenia.
Schizophr Bull 1999;25(2):233–255.

24. Peck CC. The randomized concentration-controlled clinical trial: an information rich alternative to the randomized
placebo-controlled trial. Clin Pharmacol Ther 1990;47:126.

25. Barnes RE, Nelson HE, eds. The assessment of psychosis: a practical handbook. London: Chapman and Hall Medical, 1994.

26. American Psychiatric Association. Handbook of psychiatric measures. Washington, DC: American Psychiatric Association,
2000.

27. Rabkin JG, Markowitz JS, Ocepak-Welikson K, et al. General versus systematic inquiry about emergent clinical events with
SAFTEE: implication for clinical research. J Clin Psychiatry 1992;12:3–10.
 P.547

Section V
Disorders of Development
Joseph T. Coyle

Disorders of Development - Introduction

Until recently, the developmental aspects of psychiatry were at best tenuously connected to the rapidly advancing neuroscience
research informing adult psychiatry. To a significant extent, this reflected the historical accident that child psychiatry evolved out
of the child guidance movement, which had a psychosocial orientation and was not associated with medical schools. Nevertheless,
the last decade has witnessed a remarkable advancement in the appreciation of the neurobiologic underpinnings of behavioral
disorders affecting children.

With increasing evidence of genetic risk factors for psychiatric disorders, the developmental features that transform genetic risk to
phenotype have become of particular interest to psychiatric research, especially with regard to prevention. Thus, the seeds of
Alzheimer's disease are sown early in the formation of the nervous system, not in the seventh decade of life. Furthermore, family
studies are disclosing the early manifestations of serious psychiatric illness including affective disorders, anxiety disorders, and
schizophrenia in children, raising the question of appropriate pharmacologic treatments. To this end, the Food and Drug
Administration (FDA) is now requiring the pharmaceutical industry to carry out controlled studies of the efficacy of all drugs that
might be used in the treatment of children, and the National Institute of Mental Health (NIMH) has funded the Research Units on
Pediatric Psychopharmacology (RUPP) to provide the infrastructure to support clinical trials of psychotropic medications in children.

The chapters in this section demonstrate the scientific vigor and rigor that are transforming pediatric neuropsychopharmacology.
This is especially so for those disorders that have traditionally been at the borderlands between psychiatry and developmental
pediatrics that now provide fertile grounds for linking behavioral pathology to specific developmental processes.

Wassink and his colleagues review the very promising and rapidly advancing area of the genetics of autism and related pervasive
development disorders. From being misperceived as being caused by poor maternal care (“refrigerator mother”), autism is now
known to be highly heritable, resulting from the interaction of several genes. McDougle reviews the evidence that psychotropic
medications can attenuate specific subsets of symptoms and pathologic behaviors that occur in the pervasive development disorders.
For too long, this area of the psychopharmacologic management of developmental disorders has rested on anecdotes and hunches;
but, increasingly now, these issues are being addressed in well-controlled clinical trials.

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common psychiatric diagnosis in children. Long the subject of
criticism by those who oppose psychopharmacology, recent research has elucidated the pathophysiology of the disorder, thereby
establishing face validity for the diagnosis. Faraone and Biederman provide a thorough review of the area with special emphasis on
the genetics of ADHD. In a related and often co-morbid clinical condition—learning disorders—much progress has been made in
understanding the neurobiologic mechanisms as well as characterizing effective interventions, as reviewed by Conners and Schulte.

Psychosis is the most extreme manifestation of psychiatric illness and in children can lead to diagnostic confusion. Joshi and Towbin
provide a lucid analysis of the causes of psychosis and how to treat them. Finally, Harris provides an overview of the emerging area
of behavioral phenotypes of neurodevelopmental disorders. Careful analysis has differentiated subtypes of developmental disorders
in which
 P.548

specific behaviors can be linked to specific genes or groups of genes in the case of deletions or reduplications. These advances have
important implication for the field of behavioral genetics.

The results from clinical trials with psychotropic medications in children disabuse us of the simplistic notion that children are simply
small adults, who should exhibit comparable responses to treatment. Nevertheless, the advances in pediatric
neuropsychopharmacology raise important questions about the interaction of family environment and social risk factors that must be
considered and addressed along with or in place of psychotropic medications.
 P.549

41
The Molecular and Cellular Genetics of Autism

Thomas H. Wassink

James S. Sutcliffe

Veronica J. Vieland

Joseph Piven

Thomas H. Wassink: Department of Psychiatry, University of Iowa College of Medicine, Iowa City, Iowa.

James S. Sutcliffe: Program in Human Genetics, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical
Center, Nashville, Tennessee.

Veronica J. Vieland: Department of Biostatistics, University of Iowa College of Public Health, Iowa City, Iowa

Joseph Piven: North Carolina Mental Retardation Research Center, University of North Carolina, Chapel Hill, North Carolina.

Autism is a behavioral syndrome that is generally associated with lifelong impairment and often confers a substantial burden on the
families of affected individuals. Epidemiologic research over the past two decades has demonstrated a significant role for hereditary
factors in the etiology of autism, stimulating an aggressive search for susceptibility genes. This chapter summarizes these efforts to
elucidate the genetic basis of this severe neurodevelopmental disorder.

 THE PHENOTYPE
 EPIDEMIOLOGY AND GENETIC MECHANISMS
 GENETIC INVESTIGATIONS OF AUTISM
 CHROMOSOMAL ABNORMALITIES
 BROADER AUTISM PHENOTYPE
 RELATED DISORDERS
 FUTURE DIRECTIONS
 CONCLUSION

THE PHENOTYPE
Part of "41 - The Molecular and Cellular Genetics of Autism "

The three core symptom domains of autism are excessive ritualistic and repetitive behaviors, deficits in communication, and
abnormal social interaction. These domains encompass a broad spectrum of behavioral and cognitive abnormalities such as speech
delay, echolalia, decreased spontaneous affection, reduced eye-to-eye gaze, motor stereotypies, rigid adherence to routine and
environment, and others. The onset of autism is variable, typically manifesting at age when the normal complements, such as
speech and prosocial activity, to the disturbed behaviors are expected to develop (usually by the age of 2). Symptoms change with
development and generally continue throughout life. When first described in 1943, infantile autism was narrowly defined, referring
to a population of children with severe manifestations of all core features and generally higher IQ levels. Kanner, for example, in his
original descriptions of autism, included elaborate stereotyped behaviors (e.g., complex rituals and marked distress in response to
change in routine or environment) as an essential, pathognomonic component. The severity of this component, however, has
gradually been relaxed so that current criteria require the presence of only milder behaviors in this domain.

Similarly, the concept of autism itself has been broadened and now includes the group of syndromes referred to as pervasive
developmental disorders (PDDs). Specific PDDs, in addition to autistic disorder, include Asperger’s syndrome, pervasive
developmental disorder not otherwise specified (NOS), disintegrative disorder, and Rett syndrome. The validity of these diagnostic
distinctions, however, is open to question. While the gene that causes Rett syndrome has recently been identified (1 ), and
disintegrative disorder involves a clear loss of function that is likely to arise from a distinct mechanism, there is little evidence to
support the remaining PDDs being etiologically distinct.

The existence of multiple symptom domains and the spectrum of related disorders demonstrate the complexity of the autism
phenotype. The core symptom domains exist in varying combinations of severity across affected individuals, and it is unclear
whether these clinically defined domains represent distinct genetic entities. Less severe symptomatology is represented as a distinct
diagnostic entity, but again whether this is genetically justified is not known.

Mental Retardation
Adding to the phenotypic complexity is the wide range of IQs associated with autism. Although some individuals with autism have
normal or even exceptional IQs, 70% to 80% are mentally retarded (2 ). Approximately one-third of those with mental retardation
(MR) are in the mildly affected range, the remainder have moderate to severe deficits, and gender proportionality differs across IQ
groups (see below).
 P.550

Biological Correlates
Investigators have searched for biological correlates of autism hoping to better define and categorize the phenotype.
Hyperserotonemia was the first biological abnormality to be reported, found by some in up to one-fourth of autistic individuals (3 ).
There is evidence to suggest that hyperserotonemia in autism may be familial (4 ,5 ), and that elevated platelet serotonin levels
may index genetic liability for autism (6 ). Dysmorphic facial features have also been investigated, with one positive finding coming
from a report linking autism to an early developmental abnormality in the branchial arches (7 ). Epilepsy occurs in 15% to 20% of
individuals with autism (8 ), much more frequently than expected by chance, though whether the presence of epilepsy defines an
etiologically meaningful autistic subgroup is unclear.

Two other biological traits that have been investigated are head size and brain morphology. Enlarged head size was noted by Kanner
in seven of the first 11 children he described in 1943. Subsequent studies have revealed that approximately 20% of autistic
individuals have macrocephaly (> 98th percentile for head circumference) (9 ,10 ), and the few published postmortem studies report
that the brains of autistic individuals are larger and heavier (megalencephalic) than those of normal controls (11 ,12 ). Retrospective
longitudinal studies of head circumference also suggest that while some enlargement may take place before birth, an increased rate
of growth appears to occur during the early postnatal period (10 ,13 ). Magnetic resonance imaging (MRI) studies confirm that brain
volume in autism is increased (14 ,15 ). This enlargement, rather than being generalized, may be confined to discrete structures
(16 ). One recent report, for example, found evidence linking abnormalities in caudate volume to ritualistic-repetitive behaviors in
subjects with autism, a finding that is similar to reported relationships in Tourette’s syndrome and obsessive-compulsive disorder
(17 ).

As with the core autistic symptomatology, however, these biological correlates are highly variable across individuals, and none is yet
able to independently identify cases or define meaningful subgroups. As our ability to measure these correlates becomes more
precise, however, their value is likely to increase. They may then serve the purpose of adding power to genetic studies by increasing
phenotypic information.

EPIDEMIOLOGY AND GENETIC MECHANISMS

Part of "41 - The Molecular and Cellular Genetics of Autism "

Prevalence
The estimated prevalence of autism has increased since the mid-1980s from 3 to 5 cases per 10,000 to a current estimate of 6 to 10
per 10,000 (2 ,18 ). This increase is most likely attributable to changing diagnostic practices and increased ascertainment.
Epidemiologic studies in the 1970s and early 1980s were based primarily on Kanner’s strict diagnostic criteria. With the
incorporation of less stringent criteria into the Diagnostic and Statistical Manual of Mental Disorders (DSM) and the International
Classification of Diseases (ICD), some individuals are now diagnosed with autism who would not have been previously (18 ). The
prevalence of the other PDDs, such as Asperger’s disorder and PDD NOS, has not been studied as thoroughly as that of autism.
Estimates, therefore, vary widely, though median figures from extant studies suggest a rate 1.5 to 2 times that of autism (2 ). Thus,
taken together, the PDDs may affect 15 to 25 per 10,000 school-aged children. Given that autism is a lifelong condition, the
prevalence in adults is likely to be similar to that found in children.

Family and Twin Studies

Family and twin studies help to determine the pattern and strength of the heritability of a disorder. The recurrence risk of autism
for siblings of autistic probands is approximately 4% to 5% (19 ), translating to a sibling relative risk (sibling recurrence
risk/population prevalence) of roughly 50 to 100. The risk to second- and third-degree relatives drops off dramatically to less than
1% (20 ). Twin studies, which compare concordance rates between monozygotic (MZ) and dizygotic (DZ) twins, estimate the
heritability for autism to be greater than 90% (21 ,22 ).

Gender Differences
All epidemiologic studies of autism demonstrate a male preponderance of the disorder. The overall ratio of males to females is
approximately 4:1, though this varies with IQ, approaching 6:1 in normal IQ groups and being less than 2:1 in moderate to severe MR
groups (2 ).

Associated Medical Conditions

A host of medical conditions have been reported to cause occasional cases of autism, including neurofibromatosis, tuberous sclerosis
(TS), phenylketonuria, rubella, cerebral palsy, trisomy 21, and epilepsy (23 ). For most of these disorders, however, whether they
occur in autism more frequently than expected by chance is unclear. TS has the strongest association; its population prevalence is
1/10,000, and up to 25% of individuals with TS meet diagnostic criteria for autism or PDD (24 ) (discussed in more detail below).
Overall, it has been estimated that approximately 5% of autistic individuals have an associated medical condition that may play an
etiologic role in the development of the disorder (2 ).
 P.551

Environmental Determinants
Investigators have repeatedly postulated that in utero events might predispose a fetus to the development of autism. Early twin studies, for example, suggested that obstetric
complications differentiated autistic twins from nonautistic co-twins (25 ). Subsequent examination of these and other data, however, has shown that the obstetric complications are
typically quite minor, the association between autism and complications is weak (26 ), and that the causality may be inverted—an impaired fetus may actually predispose to obstetric
complications instead of complications having affected the fetus (27 ). Similarly, some studies have reported associations between viral infections [i.e., rubella (28 ), cytomegalovirus]
during pregnancy or season of birth and the subsequent development of autism. The weight of evidence, however, either fails to support such associations or suggests that they
account for only a small minority of autism cases (29 ,30 ). Thus, although perinatal factors are reasonably inferred in rare instances (e.g., encephalitis), in most cases they appear to
have either a negligible effect or a minor effect of undetermined significance.

Chromosomal Abnormalities
Estimates of the frequency of chromosomal abnormalities in autism vary widely. Early studies reported rates as high as 20% (31 ), though recent surveys have reported lower
frequencies ranging from 3% to 8% (32–34; Wassink et al., submitted), with the fragile X mutation accounting for one-third to one-half of these. These rates may increase, however, as
more sophisticated molecular cytogenetic techniques are applied. Up to 10% of unexplained cases of MR, for example, have been found to be associated with cytogenetic abnormalities
detectable only by recently developed subtelomeric probes, and similar abnormalities may be found in autism as well. The most common chromosomal abnormalities currently
associated with autism include the fragile X mutation, other sex chromosome abnormalities, and abnormalities of 15q11-q13 (the Prader-Willi/Angelman syndrome (PW/AS) region).

Genetic Mechanisms
Thus, although a small proportion of cases of autism are due to chromosomal abnormalities or medical conditions, the vast majority are likely to be multifactorial, arising from an as
yet unknown environmental component superimposed on a strong genetic predisposition. The heritability for autism of 90% exceeds that of other common psychiatric disorders such as
schizophrenia, bipolar disorder, or alcoholism. The mode of heritability, like other psychiatric disorders, appears to be complex. Autism pedigrees have not been reported that
demonstrate mendelian segregation (unless the broader autism phenotype is included—see below), and the differential gender distribution across IQs suggests genetic heterogeneity.
The rapidly diminishing relative risk from first- to second- to third-degree relatives, combined with the >4:1 MZ:DZ concordance ratio, indicates that autism is likely to be due to
multiple genes interacting in variable combinations in additive, multiplicative, epistatic, or as yet unknown fashions (35 ). Estimates of the number of genes involved have ranged from
at least three (36 ) to more than 15 (37 ). Furthermore, other disorders composed of isolated components of the autism phenotype (e.g., specific language impairment) are themselves
considered to be due to multiple, interacting genes, making it likely that the genetics of autism will be complicated as well.

GENETIC INVESTIGATIONS OF AUTISM

Part of "41 - The Molecular and Cellular Genetics of Autism "

Early genetic investigations of autism were hampered by a number of constraints, including small sample sizes, inconsistent diagnostic criteria, and limited molecular tools. The
development of standardized diagnostic criteria and advanced molecular tools, such as high-quality, densely spaced genetic markers, FISH (fluorescent in situ hybridization)
chromosomal probes, and high-throughput sequencing, is beginning to overcome these constraints. Multicenter collaborations can now gather large, consistently characterized samples,
genome-wide screens are practical, sequence data are available for focused genetic investigations, and chromosomal studies are more exact and informative. These advances are
reflected in the recent surge of genetic investigations of autism, which are summarized below.

Genome-Wide and Focused Linkage and Association Studies

Four genome-wide linkage studies of autism have been published to date (37 ,38 ,39 and 40 ). All these studies have examined families containing at least two affected siblings
[affected sibling pair (ASP) families] and are summarized in Table 41.1 . The strongest single finding to emerge from these screens is a multipoint heterogeneity logarithm of odds (LOD)
score of 3.0 on chromosome 13q at 55.3 centimorgans (cM) reported by the Collaborative Linkage Study of Autism (CLSA) (39 ), whereas the most replicated finding consists of support
for linkage to chromosome 7q.

TABLE 41.1. GENOME-WIDE LINKAGE STUDIES OF AUTISM

Some caution must be taken when comparing these studies, however, because none of them report exactly the same statistic. The CLSA (39 ) reported a maximum multipoint LOD
score (MLS), calculated using the program GENEHUNTER (41 ) and based on a likelihood that allowed explicitly for heterogeneity (42 ). The Stanford group also reported an MLS (37 ),
but the underlying likelihood was parameterized in terms of a multiplicative model allowing for dominance variance, and calculated using ASPEX (43 ,44 ). The International Molecular
Genetic Study of Autism
 P.552

Consortium (IMGSAC) also used ASPEX to calculated the MLS, but under an additive model that assumed no dominance variance (38 ).
The Paris Autism Research International Sibpair Study (PARISS) used a related MLS, maximized over the “possible triangle” (45 ),
using MAPMAKER/SIBS (40 ). While all these statistical approaches are related to one another (Huang and Vieland, in press), they
may involve estimation of somewhat different numbers of parameters, or “degrees of freedom.” The most appropriate use of these
data in aggregate, therefore, is not to directly compare numerical results, but rather to look for regions that have either very strong
support for linkage within individual studies or that have recurrent support across studies.

Focused genetic studies have examined smaller chromosomal regions chosen for one of three reasons: (a) the region showed
evidence of linkage in a genome-wide screen; (b) the region contains a high rate of chromosomal abnormalities associated with
autism; or (c) a “candidate” gene of interest, chosen because of its potential biological or developmental relevance to autism, is
located in the region. The samples for these focused studies include both ASP families and trios (proband and both parents), and are
summarized in Table 41.2 .

TABLE 41.2. FOCUSED LINKAGE AND ASSOCIATION STUDIES OF AUTISM

Chromosome 7
The most replicated evidence for linkage is to chromosome 7q (Fig. 41.1 ). The IMGSAC, examining 87 ASPs, reported a LOD of 2.53
at D7S530 (134.6 cM), which increased to 3.55 in a subset of 66 United Kingdom ASPs (38 ). A recent second-stage analysis of 125
ASPs by this same group reported, in poster format, a multipoint MLS near D7S2533 (140.5 cM) of 3.63 (46 ). The CLSA, examining 75
ASPs, reported an MLS of 2.2 at D7S813 (104 cM) (39 ), whereas the PARISS (40 ) and Stanford (37 ) studies reported modestly
positive results (MLS = 0.83 and 0.93, respectively).

FIGURE 41.1. Chromosome 7 findings in autism. Rectangles represent chromosome abnormalities, black ovals indicate regions
with evidence for linkage or association in autism, and gray ovals indicate regions with evidence for linkage or association in
related disorders. MLS, maximum multipoint LOD score within the region indicated.

A subsequent focused examination of nine 7q markers was performed in 76 multiplex families and 32 trios by the Duke/University of
South Carolina (USC) group (47 ). They found a peak multipoint MLS of 1.77 at D7S2527 (129.0 cM) using an additive model
calculated with ASPEX. This group also found high rates of recombination throughout 7q and evidence of linkage disequilibrium at
D7S1824
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 P.554

(149.9 cM), reporting that the linkage, excess recombination, and linkage disequilibrium appeared to be due primarily to paternal
and not maternal effects.

Chromosome 15q11-Q13
Because of numerous reports of cytogenetic abnormalities (discussed below), chromosome 15q11-q13 has been intensively examined
in linkage and association studies. None of the currently published genome-wide screens, which all included tightly spaced 15q11-
q13 markers, identified linkage to the region. The Duke/USC group screened fourteen 15q11-q13 markers in their families and
reported a maximum MLS of 1.78 at D15S217 (48 ). Another group has reported highly significant linkage disequilibrium with the
marker GABRB3 155CA-2, a dinucleotide repeat polymorphism that was not typed in any of the genome-wide screens (Buxbaum et
al., submitted). This result was obtained when the data were pooled with a number of other focused studies that included this
marker.

Candidate Gene Studies

Genes tested as candidates for involvement in autism include genes involved in neurotransmission (i.e., HTT, TH, DBH), genes
contributing to related disorders (i.e., FMR-1, the fragile X syndrome (FXS) gene, and NF-1, a neurofibromatosis gene), and genes
thought to be involved in brain development (i.e., EN2, HRAS-1) (Table 41.2 ). Though positive results have occasionally been
reported, replication has been limited. Cook et al. (49 ), for example, reported preferential transmission of the small allele of the
HTT promoter polymorphism to autistic probands. Klauck et al. (50 ), attempting replication, reported preferential transmission of
the larger allele, and the IMGSAC reported no association with either allele (51 ). No other candidate genes tested thus far have
found consistent support.

CHROMOSOMAL ABNORMALITIES
Part of "41 - The Molecular and Cellular Genetics of Autism "

Studies of cytogenetic abnormalities can complement molecular approaches by identifying genes whose effects are either too small
to be detected by linkage or are obscured by epigenetic phenomena. The X chromosome and chromosome 15q11-q13, for example,
have both received scrutiny because they are frequent sites, relative to other regions, of cytogenetic abnormalities in individuals
with autism. Additionally, linked regions in complex disorders are typically broad, and chromosomal abnormalities occurring in
linked regions can help to pinpoint disease susceptibility genes. This can be done either by cloning the chromosomal break points
and identifying disrupted genes, or by overlaying deleted regions across individuals in order to delineate a minimal deleted region
that might harbor a disease susceptibility gene.

The prevalence of chromosomal abnormalities in autism has been discussed above (see Epidemiology and Genetic Mechanisms ). This
section, therefore, focuses on abnormalities in specific regions: 7q, 15q11-q13, the sex chromosomes, and the fragile X mutation.
 P.555

Chromosome 7 Cytogenetic Abnormalities

A number of chromosome 7 cytogenetic abnormalities in close proximity to the 7q linkage findings have been identified in
individuals with autism (Fig. 41.1 ). In one family, a paracentric inversion, inv(7 )(q22q31.2), is carried by two brothers, a sister, and
their mother (47 ). The brothers appear to have autistic disorder, the sister has expressive language disorder, and the mother has
neither of these abnormalities. Another autistic individual has been described with a translocation t(7;13)(q31.2;q21) (52 ). The
break points for these abnormalities have been cloned, and identification and screening of nearby candidate genes is in progress.
Two autistic twins have been found to have translocations t(7;q20)(q11.2;p11.2) (53 ). The chromosome 7 break point and the gene
it disrupts, named “autism-related gene 1” (ARG1), have been cloned; the gene is novel with an unknown function, spans an 800-
kilobase (kb) genomic region, and is highly expressed in fetal and adult brain (53 ). Lastly, one individual with autism and another
with a specific developmental disorder of speech and language (SDDSL) have been reported, both of whom have chromosomal
abnormalities involving 7q31 [autism, 46,XY, inv(7 )(p12.2q31.3); and SDDSL, 46,XY,t(2;7)(p23;q31.3)] (54 ).

Chromosome 15q11-Q13
Chromosome 15q11-13 is the most frequent site of autosomal abnormalities in autism. In a recent chromosomal survey, 6 (2.2%) of
278 autistic subjects referred for cytogenetic studies had a gross abnormality of chromosome 15 (Wassink et al., submitted). These
abnormalities most commonly involve duplication of maternal DNA, typically as either interstitial duplications (55 ,56 ,57 ,58 ,59 ,60
and 61 ) or inverted duplicated isodicentric marker chromosomes [inv dup(15 )] (62 ,63 ,64 and 65 ). The duplications that produce
illness typically extend into the Prader-Willi syndrome (PWS) and Angelman syndrome (AS) critical region, as smaller duplications are
generally asymptomatic (64 ,66 ). Complementing these data, individuals identified because of 15q11-q13 duplications frequently
have autistic features (67 ). Deletions of 15q11-q13, though less frequent, have also been reported in autistic individuals, with the
deleted material usually of paternal origin (68 ,69 ). Figure 41.2 summarizes these data, displaying the relevant genes and markers
as well as a putative autistic disorder region.

FIGURE 41.2. Schematic map of 15q11-q13 autism candidate region. The upper part of the figure is a low-resolution schematic
representation of the 15q11-q13 interval deleted in Prader-Willi syndrome (PWS) and Angelman syndrome (AS) and duplicated
in cases of autism. PWS and AS critical regions are indicated by arrows over the map, with relevant imprinted genes indicated
in their respective regions. A prioritized autism region excludes the imprinted PWS domain based on the apparent maternal
specificity of 15q11-q13 duplications [interstitial or inv dup(15) markers] in association with autism. The lower expanded
region reveals gene and marker order and transcriptional orientation within the candidate region. Large break-point regions
are depicted for the primary distal PWS/AS deletion break point, as well as a less common (˜10%) PWS/AS break point and a
break-point interval associated with inv dup(15) marker chromosomes.

In addition to these gross abnormalities, cytogenetic abnormalities at the molecular level are also being reported. Cook et al. (70 ),
while screening autism trios across 15q11-q13, identified a nonautistic mother in whom a duplication had arisen de novo on her
paternally derived homologue (70 ). The duplication was transmitted to one child who
 P.556

developed autism and a second child with atypical autism, whereas a third child who did not receive the duplication remained
unaffected. In the CLSA genomic screen, which examined 75 ASP families (152 affected individuals) and included two 15q11-q13
markers, six individuals (3.9% of probands) from four families (5.3% of families) were found to have either maternal duplications or
paternal deletions at one or both of these markers (Wassink et al., submitted). Forty-five unrelated autism trios were subsequently
screened using 12 polymorphic 15q markers, with three (6.1%) autistic probands found to have similar abnormalities (Wassink et al.,
submitted).

The apparent gender specificity of the 15q11-q13 abnormalities is presumably attributable to imprinting, an epigenetic mechanism
by which only one of a gene’s two inherited alleles is expressed, with expression determined by the allele’s gender of origin (71 ).
The two primary 15q11-q13 syndromes, PWS and AS, are oppositely imprinted mental retardation syndromes that bear some
phenotypic similarities to autism (72 ). In brain tissue (but not elsewhere), UBE3A, the AS gene, is expressed predominantly from the
maternally derived allele. Disrupted expression of the maternal UBE3A, therefore, produces AS, whereas disruption of the paternally
derived allele produces no discernible abnormal phenotype (73 ). PWS genes, conversely, are paternally expressed; the predominant
cause of PWS, therefore, is disrupted expression of the paternal copy of the small nuclear ribonucleoprotein polypeptide N (SNRPN)
gene and other contiguous genes (74 ). Another recently identified element of imprinting is the presence of antisense transcripts for
imprinted genes. These segments of RNA are oppositely imprinted complements to an imprinted gene’s coding sequence (75 ).
UBE3A, for example, has an antisense transcript that is expressed solely from the paternally derived allele (71 ). This antisense
transcript may play a role in the suppression of the nonexpressed allele, and mutations in this transcript, therefore, could
contribute to some cases of AS (76 ). Thus, just as imprinting plays a pivotal role in PWS and AS, it is likely to significantly influence
the effect of 15q11-q13 abnormalities in autism as well.

The 15q11-q13 abnormalities themselves may be due to the presence of repeated or duplicated homologous genomic segments that
exist in multiple copies throughout this region (77 ). Duplications of large genomic segments are associated with chromosomal
abnormalities in a number of specific syndromes (78 ,79 ). One such repeated segment (duplicon) appears near each of three most
common PWS/AS duplication breakpoints (80 ). Another, located centromeric to the PWS/AS critical region, is repeated an increased
and variable number of times in PWS/AS individuals (81 ). Though this does not imply a traditional repeat expansion mechanism, it
may be that these repeats predispose the region to recombination abnormalities or “mistakes” (77 ), a finding with support from
data showing increased rates of recombination across 15q11-q13 in subjects with either PWS/AS (82 ) or autism (48 ).

Interestingly, one other chromosomal region that shares many of these genomic features is 22q11 (77 ). Chromosome 22q11 contains
large repeated segments that contribute to a high rate of deletions and duplications (83 ). These chromosomal abnormalities are
associated with a constellation of syndromes grouped under the umbrella term CATCH-22 (84 ). One of these syndromes,
velocardiofacial syndrome (VCFS), is associated with a high rate of schizophrenia (85 ), a psychiatric disorder that, like autism, is
felt to arise from disturbed brain development. In addition, a small but significant percentage of individuals with schizophrenia have
now been shown to have microabnormalities of 22q11 (86 ). Thus, schizophrenia and autism may share a common genomic
mechanism for a subgroup of cases, and insights from one disorder may inform investigations into the other.

Fragile X
The association of autism with the fragile X syndrome (FXS) was first suggested nearly 20 years ago (87 ,88 ). The fragile X
phenotype is frequently characterized by behaviors that can resemble the core symptom domains of autism such as language
abnormalities, decreased nonverbal communication, social isolation, and repetitive motor behaviors such as rocking and hand biting
(89 ). In support of this association, early chromosomal investigations reported a rate of the fragile X mutation [fra(X)(q27.3)] in
autism that approached 20% (31 ,90 ).

Recent studies, however, have questioned the strength of the link between these two disorders. Current surveys estimate the
frequency of fragile X in autism to be 2% to 4% (91 ,92 and 93 ), similar to the rate of fragile X in the general MR population (94 ).
This is more common than other types of chromosomal abnormalities in autism, though not necessarily disproportionately so.
Likewise, there are subtle but significant differences between the behavioral phenotypes of the two disorders. Autism is
characterized by social indifference and deficits in the perception of emotion, whereas individuals with FXS experience social
anxiety and gaze avoidance with no attendant impairment of emotional perception (95 ).

Genetically, FXS is a disorder of unstable DNA caused by a trinucleotide repeat that expands as it is transmitted to successive
generations (96 ). The repeat is located at Xq27.3 in the 5′ UTR (untranslated region) of the FMR1 gene (89 ). Once this expanded
region crosses a threshold (approximately 200 repeats), it becomes susceptible to methylation, which inhibits transcription of FMR1.
FMRP, the FMR1 protein, is an RNA binding protein that appears to act as a chaperone for transport of RNA from the nucleus to the
cytoplasm (97 ). FMRP is expressed in numerous tissues including fetal brain. Intracellularly, it is found in the nucleus near the
nucleolus and in cytoplasm in association
 P.557

with ribosomes. It may function, therefore, as a chaperone molecule in the transportation of messenger RNA (mRNA) from the
nucleus to the cytoplasm (98 ). How dysfunction of this protein gives rise to FXS, however, remains unclear.

Other Sex Chromosome Abnormalities

The possibility of a sex chromosome–related genetic effect is suggested by the preponderance of males affected by autism (2 ). In
accord with this, a sizable number of sex chromosome abnormalities, in addition to the fragile X mutation, have been reported in
subjects with autism (99 ). In a recent survey of a clinical population, six out of 265 (2.3%) autistic individuals referred for
cytogenetic testing were found to have abnormalities of the sex chromosomes other than fragile X (Wassink et al., submitted). In
addition, two X-linked disorders, Turner syndrome and Rett syndrome, have phenotypic features that are similar to some of the core
features of autism. Skuse et al. (100 ) reported that Turner syndrome (45,X) females with maternally derived X chromosomes had
diminished verbal skills and social cognition compared to those with paternally derived Xs. Molecular studies implicated a paternally
imprinted disease locus that escapes X-inactivation in distal Xp22.3 (101 ). This paternal imprinting could explain why karyotypically
normal males (who have a maternally derived X) are more vulnerable to developmental disorders of language and social cognition,
such as autism, than females. Skuse et al. also reported three Turner syndrome females, all with maternally inherited X
chromosomes, who had been diagnosed with autism. Two more XO autistic individuals have recently been reported, one with a
maternally derived X (102 ) and the other with an X of unknown origin (Wassink et al., submitted).

The gene for Rett syndrome was recently identified on Xq28 (1 ). Rett syndrome, considered to be a subtype of PDD, is a disorder
occurring only in girls that is characterized by mental retardation, loss of speech, and stereotypic hand movements after 1 to 2
years of normal development. The gene for Rett syndrome, (MECP2), is widely expressed and codes for a DNA binding protein that
regulates gene expression (1 ).

Linkage screens of the X chromosome in autism have generally been negative, excluding genes of even small effect (37 ,103 ), and
have contributed to a reluctance to examine the sex chromosomes for autism disease genes. The evidence from sex chromosome
abnormalities and from X-linked disorders with phenotypic similarities, however, suggests that such pessimism is premature, and
that the X and Y chromosomes should continue to be a focus of attention in autism.

BROADER AUTISM PHENOTYPE

Part of "41 - The Molecular and Cellular Genetics of Autism "

In addition to describing the hereditary basis of autism, family and twin studies have demonstrated, in nonautistic relatives of
autistic probands, the presence of milder traits that are qualitatively similar to the defining features of autism. These collective
traits, referred to as the “broader autism phenotype” (BAP), were first observed by Kanner in parents of autistic children. Bailey et
al., replicating and extending findings from the original Folstein and Rutter (104 ) twin study, found a substantially higher
concordance rate for the presence of mild social and communication deficits in MZ versus DZ twin pairs (92% versus 10%) (21 ). These
results are supported by several family studies using the family history method of assessment (105 ,106 ). In the London Autism
Family Study, Bolton et al. (105 ) reported that familial aggregation of the BAP was associated with proband verbal IQ. In the Iowa
Autism Family Study (Piven and Palmer, submitted) familial aggregation of the BAP was higher in relatives from families with two
autistic siblings (multiple-incidence families) than in families ascertained through a single autistic child.

A more detailed examination of the BAP has been accomplished through direct assessment of relatives. Relatives from multiple-
incidence families, for example, were found to have (a) elevated rates of personality characteristics such as aloofness and rigidity,
(b) diminished pragmatic language and speech abilities, (c) fewer quality friendships, and (d) decreased scores on a number of
specific cognitive measures (107 ,108 and 109 ).

Investigation of the BAP may, by clarifying the range of phenotypic expression of the underlying genetic liability to autism, provide a
complementary approach to traditional linkage that increases power to detect genes by identifying more affected individuals,
thereby enabling extension of typically small autism pedigrees. Understanding the boundaries and nature of the BAP may also help
our efforts to detect genes in autism by enabling focused investigation of specific BAP components (e.g., language deficits,
ritualistic-repetitive behaviors, or cognitive deficits) that may map on to separate genes that together cause the full syndrome of
autism. This approach to disaggregating complex phenotypes has proved successful in dyslexia, where separate linkages were found
to single-word reading and phonemic awareness (110 ). Clearly, clarification of the genetically relevant aspects of both the autism
and the broader autism phenotype is an important strategy to pursue in our search for genes in this disorder.

RELATED DISORDERS
Part of "41 - The Molecular and Cellular Genetics of Autism "

Autism is characterized by dysfunction in three symptom domains: language; social interaction; and repetitive, stereotyped
movements and behaviors. As autism is a heterogeneous, genetically complex disorder, it may be that each of these domains has
unique, independent genetic determinants. Studying disorders that resemble these individual domains, therefore, may provide
insight into their etiology in
 P.558

autism. There are also related disorders, such as tuberous sclerosis, and domains of investigation, such as immunogenetics, that may
provide insight into autism.

Disorders of Language
Specific language impairment (SLI) is a disorder characterized by isolated impairment of language skills, and may be characterized
by grammatical impairment, word finding difficulties, or an underlying perceptual deficit (111 ). Though traditionally considered
distinct disorders, SLI has been found to be common in autistic individuals and to occur at higher rates than expected in their
nonautistic relatives (108 ). Conversely, an increased rate of autistic disorder has recently been found in siblings of children with SLI
(112 ). Tying this in to chromosome 7, an association study found significant associations between two 7q31 genetic markers and a
group of SLI trios (113 ). Also, a family has been identified with a severe speech and language disorder characterized by deficits in
grammar, expressive language, articulation, and coordination of orofacial musculature (114 ). A genome-wide screen of this three-
generation pedigree found a maximum LOD score of 6.62 at a marker in 7q31, with fine mapping narrowing the region to a 5.6-cM
interval (SPCH1 locus). These findings, therefore, may represent localizations of heritable components of the autism phenotype and
are of particular interest given the evidence for linkage of autism to this same region of chromosome 7.

Disorders of Repetitive and Stereotyped Behaviors

The phenomenologic overlap between autism, obsessive-compulsive disorder (OCD), and Tourette’s syndrome has led some to
wonder whether these disorders have etiologic mechanisms in common. For example, caudate volumes have been found to be
abnormal in both Tourette’s (115 ) and OCD (116 ). Therefore, we examined caudate volumes in an MRI study of autistic children and
found enlargement of the caudate that was correlated to ritualistic, stereotyped behaviors but not to social or communication
deficits (17 ). In an earlier family study, we reported higher familial aggregation of autism and the BAP in families ascertained
through a Kanner proband (more severe ritualistic behavior) versus more broadly defined (DSM, third edition, revised) probands
(117 ). Findings such as these suggest that traits such as stereotypies or ritualistic behavior may have unique genetic determinants
that, when combined with genes that give rise to other traits such as language or communication deficits, could give rise to the
syndrome of autism.

Disorders of Social Activity

Examples of disorders that involve significant social deficits include Turner syndrome and the fragile X syndrome, both of which have
been discussed above (see Other Sex Chromosome Abnormalities ). Additional research related to social deficits that may have
relevance to autism comes from studies of various neuropeptide systems. For example, nematode worms that lack receptors for
neuropeptide Y become strikingly isolated in situations where they would normally congregate with other worms (118 ). Genetic
variability in receptors for oxytocin/vasopressin in mice and other rodents is also associated with clear variability in social behavior
(119 ). Thus, though there is significant evolutionary distance between worms, rodents, and humans, these transmitter systems may
merit closer examination in individuals with autism.

Tuberous Sclerosis
Tuberous sclerosis complex (TSC) is a neurocutaneous disorder characterized by benign tumors affecting numerous organs, most
commonly the brain, eyes, skin, kidneys, and heart (120 ), with a population prevalence estimated at 1/10,000 (121 ). The
occurrence of autism and other behavioral and psychiatric disturbances in the context of TSC has long been recognized (122 ).
Clinic-based and epidemiologic studies of autism in TSC suggest that up to 25% of individuals with TSC meet diagnostic criteria for
autism and over 40% meet criteria for any PDD (24 ,123 ). Conversely, 1% to 3% of autistic individuals will have TSC (124 ), though
this rate approaches 10% for autistic individuals with seizure disorders (24 ,123 ).

TSC is an autosomal-dominant disorder caused by mutation in one of either two genes, TSC1 or TSC2 (125 ). TSC2 is located on
chromosome 16p13 and codes for the protein tuberin, whereas TSC1 is located on 9q34 and codes for the protein hamartin. Tuberin
has numerous functions, acting as a tumor suppressor or a chaperone, and having an influence on cell cycle passage. Dysfunction of
tuberin may result in constitutive activation of RAP1, a protein that regulates DNA synthesis and cellular transition, thereby
producing excessive proliferation and impaired differentiation of a variety of cell types. Hamartin is one of the proteins for which
tuberin acts as a cytosolic chaperone. Other than an ill-defined role in tumor suppression, the function of hamartin remains
unknown (126 ). Approximately two-thirds of TSC cases are sporadic and one-third familial. Half of the familial cases and 75% to 80%
of sporadic cases arise from mutations of TSC2, with the remainder attributed to TSC1. Two studies have shown that TSC due to
TSC2 mutations is more likely to be associated with either mental retardation or intellectual impairment than TSC due to TSC1
mutations (127 ,128 ). Despite the strong association between TSC and autism, however, the mechanistic link between the two
disorders remains unclear. Autism in the context of TSC may arise directly from the TSC mutations, from the tubers they produce, or
from some other as yet undiscovered mechanism. One group, for example, has reported an association between the presence of
temporal lobe tubers and autism (129 ), though this finding has not been replicated (24 ).
 P.559

Immunogenetics
A number of investigators have suggested that some cases of autism may be attributable to interactions between infections, the
immune system, and genetic factors (130 ). Subjects with autism have been shown to have deficits in the number and function of
various immune cell subtypes (131 ,132 ,133 ,134 ,135 and 136 ). A series of investigations, therefore, performed primarily by one
research group, has investigated specific components of the major histocompatibility complex (MHC) on chromosome 6p21 for
association with autism (130 ,137 ,138 ,139 and 140 ) (Table 41.2 ). The samples in these studies were generally small and
overlapping, and associations emerged primarily when probands were compared to a population control group as opposed to a
parent-based test. Nonetheless, the authors report consistently significant findings that await replication by others.

FUTURE DIRECTIONS
Part of "41 - The Molecular and Cellular Genetics of Autism "

Alternative Sampling Designs

As noted above, all of the genome-wide linkage screens performed to date have focused almost exclusively on affected sibling-pair
(ASP) pedigrees. This is in part due to the difficulty of gathering extended autism pedigrees. Also, however, because the mode of
inheritance for autism is unknown, many researchers appear to be more comfortable with the simple “model-free” linkage analysis
methods available for ASP data than with the more complicated methods required for the analysis of general pedigree structures.
Yet, limiting pedigrees solely to ASPs may be unnecessarily and detrimentally restrictive.

The optimal pedigree structure for the detection of linkage depends on the true, underlying genetic model for the trait, which in
this case is unknown. Large, multiplex pedigrees, for example, are optimal when a trait is transmitted as a rare dominant but tend
to be uninformative if the trait is a common recessive. Furthermore, an ASP data set contains only two primary pieces of
information—the number of sibling pairs sharing 1 allele identical by descent and the number of pairs sharing 2 alleles—thereby
limiting the complexity of any model that can be fit to ASP data. By limiting the families that are gathered to ASPs, the ability to
detect linkage under all possible modes of transmission may be similarly limited. It could be argued, therefore, that precisely
because the mode of inheritance for autism is unknown, the optimal strategy would be to ascertain all types of potentially
informative pedigree structures. This could include large multigenerational extended pedigrees and moderate-sized pedigrees with
more than just two affected individuals, in addition to ASPs.

The difficulty is that there are few larger pedigrees for autism, thus highlighting the potential benefit and utility of the broader
autism phenotype. As noted above, classifying individuals with familial autism–related traits as “affected” may increase the
prevalence of extended pedigrees and reveal patterns of segregation within those pedigrees beyond what is seen for autism itself.
Segregation of such traits (and their underlying genetic diatheses), however, will only be detected if extended pedigrees are sought
to begin with. A further benefit to using the BAP is that it enables us to diagnose parents, and possibly siblings, of ASPs as well. Thus
even within the nuclear families already collected through existing genomic screens, the amount of linkage information may be
greatly increased if indeed the BAP is genetically related to autism.

Another sampling design enabled by the BAP is discordant sibling pair (DSP) analysis. The BAP conceptualizes and measures the traits
related to autism along continua. Those subjects who exceed severity thresholds on these measures but do not meet criteria for
PDDs are considered to have some form of the BAP. These measures also indicate, however, family members who are very
unaffected, and therefore discordant, with their autistic siblings. If the traits measured by the BAP truly reflect underlying genetic
diatheses toward autism, one would expect reduced allele sharing between DSPs at susceptibility loci as opposed to the excess allele
sharing expected in ASPs (141 ).

The major advantage of the DSP method is that, theoretically, it may require far fewer sibling pairs than the ASP method under
some circumstances. Risch and Zhang (142 ) estimated that DSP analysis may provide the same power to detect disease genes as the
ASP method with a sample 10- to 40-fold smaller. The primary disadvantage of the DSP method is that DSPs are difficult to find, and
require an extensive screening effort to identify enough pairs.

Microarray Technology
DNA chip microarray technology, which enables the simultaneous analysis of tens of thousands of DNA or RNA sequences, may be of
significant benefit to autism research in two primary domains. First, an important focus of the current effort to sequence the human
genome is the identification of single nucleotide polymorphisms (SNPs) (143 ). SNPs are sites of single base pair substitutions, are
much more common than di-, tri-, or tetranucleotide polymorphisms, and, in contrast to current methodologies, can be easily and
rapidly genotyped using DNA chips (144 ). The hope, therefore, is that DNA SNP chips will be used to rapidly screen a dense marker
map, thereby enabling the performance of genome-wide association studies (145 ). Implementation of DNA chips for this purpose
awaits the development of such SNP maps as well as the statistical and computational tools with which to analyze and interpret the
resultant data.

The second potential use of microarray technology is to examine gene expression patterns in relevant tissues. DNA chips can be
created that recognize all possible mRNAs that a tissue is currently producing (146 ). Thus, the expression
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of thousands of genes from the brains of autistic individuals and controls can be compared in order to detect etiologically meaningful differences. The primary limitations of this
method are the collection of brain tissue, which must be done rapidly and according to exact protocols, and interpretation of the data generated from such experiments (147 ). Altered
gene expression, for example, may be due to medication effects as opposed to an etiologically relevant mechanism. The most meaningful studies of gene expression at this time,
therefore, may come from animals genetically designed to exhibit autistic behaviors, where the environment and timing of the tissue analysis can be controlled.

Refining the Phenotype Through Biological Correlates of Illness

The primary purpose for investigating the BAP is to enable identification of meaningful genetic subgroups based on the core symptom domains of autism. Similar strategies are being
pursued with various biological correlates of the disorder, including serotonin metabolism, minor physical anomalies, head circumference, brain morphology and function, and others.

CONCLUSION
Part of "41 - The Molecular and Cellular Genetics of Autism "

Numerous complementary strategies are currently being employed to attempt to locate autism disease genes. Linkage studies have identified a number of suggestive loci, most notably
distal 7q. Interest in this region is supported by findings from language-disorder families and from a small number of 7q chromosomal anomalies in individuals with autism.
Chromosomal anomalies also implicate 15q11-q13 as a region of interest, though linkage and association studies of the region have not been as impressive. These findings clearly
demonstrate progress in the effort to find regions harboring genes in autism. As sample sizes continue to grow through collaborative efforts and molecular and statistical methods
improve, and as complementary strategies such as the use of the BAP emerge, it seems plausible that in the near future more disease loci will be uncovered and specific autism disease
genes may be identified.

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42
Current and Emerging Therapeutics of Autistic Disorder and Related
Pervasive Developmental Disorders

Christopher J. McDougle

Christopher J. McDougle: Department of Psychiatry, Section of Child and Adolescent Psychiatry, Indiana University School of
Medicine; James Whitcomb Riley Hospital for Children, Indianapolis, Indiana.

 DIAGNOSIS OF PERVASIVE DEVELOPMENTAL DISORDERS

 PHARMACOTHERAPY OF PERVASIVE DEVELOPMENTAL DISORDERS
 CONCLUSION
 ACKNOWLEDGMENTS

DIAGNOSIS OF PERVASIVE DEVELOPMENTAL DISORDERS

Part of "42 - Current and Emerging Therapeutics of Autistic Disorder and Related Pervasive Developmental Disorders "

Pervasive developmental disorders (PDDs) are characterized by severe and pervasive impairment in several areas of development,
including reciprocal social interaction skills, communication skills, and the presence of stereotyped behavior, interests, and
activities (1 ). The qualitative impairments that define these disorders are abnormal relative to the individual’s developmental level
or mental age. These conditions are typically evident in the first 1 to 3 years of life and are usually associated with some degree of
mental retardation. The PDDs are sometimes observed among a diverse group of identifiable biological abnormalities (e.g.,
chromosomal abnormalities, congenital infections, structural abnormalities of the brain). In the majority of cases, however, a
specific etiologic factor is not found. Previously, terms like psychosis and childhood schizophrenia were used to refer to individuals
with these conditions. However, there is now considerable evidence to demonstrate that PDDs are distinct from schizophrenia.
There are five subtypes of PDD in the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) (1 ). They
include autistic disorder, Rett syndrome, childhood disintegrative disorder, Asperger’s syndrome, and PDD not otherwise specified
(NOS) (Table 42.1 ).

TABLE 42.1. DSM-IV PERVASIVE DEVELOPMENTAL DISORDERS (1)

Autistic Disorder
The essential features of autistic disorder are the presence of markedly abnormal or impaired development in social interaction and
communication and a markedly restricted repertoire of activity and interests. The clinical presentation of the disorder varies
significantly depending on the developmental level and chronologic age of the individual. In the past, autistic disorder was referred
to as early infantile autism, childhood autism, or Kanner’s syndrome.

The clinical features must include delays or abnormal functioning in social interaction, in language as used in social communication,
or in symbolic or imaginative play prior to the age of 3 years. There is usually no period of clearly normal development, although 1
or 2 years of relatively normal progression can occur. In some instances, regression in language development, typically manifest as a
complete loss of speech after a child has acquired from five to ten words, has been reported. If there is a period of normal
development, it cannot extend past the age of 3 years.

In approximately 75% of cases, there is an associated diagnosis of mental retardation, commonly in the moderate range (IQ 35 to 50).
A number of behavioral symptoms, including hyperactivity; inattention; impulsivity; aggression toward self, others, or property; and
interfering repetitive thoughts and behavior are often present. The disorder is sometimes observed in association with an
identifiable medical condition (e.g., herpes encephalitis, phenylketonuria, tuberous sclerosis, fragile X syndrome, anoxia during
birth, maternal rubella). Seizures may develop, particularly in adolescence, in up to 25% to 33% of cases. The disorder is four to five
times more common in males than in females, although females often have a more severe cognitive disability. Epidemiologic studies
have identified rates of autistic disorder of two to five cases per 10,000. Language skills and IQ are the strongest predictors of
eventual outcome.

Rett Syndrome
Rett syndrome differs from autistic disorder in its characteristic sex ratio and distinctive pattern of abnormal developmental
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progression. The syndrome is much less common than autistic disorder and has been diagnosed almost exclusively in females.
Following apparently normal prenatal and perinatal development, and a period of normal psychomotor development through the
first 5 months of life, there is a characteristic pattern of head growth deceleration, loss of previously acquired purposeful hand skills,
intermittent hyperventilation, and the appearance of ataxic gait or trunk movements. Individuals with Rett syndrome may exhibit
difficulties in social interaction, particularly during the preschool years, but these may improve somewhat over time. Severe or
profound mental retardation, seizures, and significant expressive and receptive language impairment are typical. Recently, a
mutation in the gene (MECP2) encoding X-linked methyl-CpG-binding protein 2 (MeCP2) has been identified as the cause of some
cases of Rett syndrome (2 ).

Childhood Disintegrative Disorder

Childhood disintegrative disorder contrasts with autistic disorder in that there is a distinctive pattern of developmental regression
following at least 2 years of normal development. In autistic disorder, some developmental abnormalities are usually noted within
the first year of life. After the first 2 years of life (but before the age of 10 years), the child with childhood disintegrative disorder
has a clinically significant loss of previously acquired skills in at least two of the following areas: expressive or receptive language,
social skills or adaptive behavior, bowel or bladder control, play, or motor skills. The onset, in most cases, is between the ages of 3
and 4 years and may be insidious or abrupt. To date, an underlying pathologic mechanism has not been identified. The disorder has
been reported in association with metachromatic leukodystrophy and Schilder’s disease, in some cases. Childhood disintegrative
disorder is usually associated with severe mental retardation. It appears to be very rare, much less frequent than autistic disorder,
and more common among males. The disorder has also been termed Heller’s syndrome, dementia infantilis, or disintegrative
psychosis.

Asperger’s Syndrome
Asperger’s syndrome can be distinguished from autistic disorder by the lack of delay in language and cognitive development, in
addition to no significant abnormality in the development of age-appropriate self-help skills, adaptive behavior (other than in social
interaction), and curiosity about the environment in childhood. Motor milestones may be delayed, and motor clumsiness is often
observed. The syndrome appears to be more common in males. Asperger’s syndrome is usually recognized somewhat later than
autistic disorder, frequently in the context of school. All-encompassing preoccupations or circumscribed interests are typically
present and can contribute to significant functional impairment.

Pervasive Developmental Disorder Not Otherwise Specified

PDD NOS is diagnosed when there is a severe and pervasive impairment in the development of reciprocal social interaction or verbal
and nonverbal communication skills, but the criteria are not met for a specific PDD, schizophrenia, schizotypal personality disorder,
or avoidant personality disorder. Stereotyped behavior, interests, and activities are often present. This category includes
presentations associated with late age at onset, atypical or subthreshold symptoms, or both.

PHARMACOTHERAPY OF PERVASIVE DEVELOPMENTAL DISORDERS

Part of "42 - Current and Emerging Therapeutics of Autistic Disorder and Related Pervasive Developmental Disorders "

The treatment of autistic disorder and related PDDs is multimodal and largely based on educational interventions and behavior
management principles. Speech therapy is usually indicated, and physical and occupational therapy are often needed as well.
Despite educational and behavioral strategies, many children, adolescents, and adults with PDDs remain significantly impaired.
Under these circumstances, pharmacologic treatment is often appropriate and warranted.

Adequate drug treatment studies that have been focused on subjects with particular subtypes of PDD, other than autistic disorder,
have not been conducted. Many investigations have included mixed samples of subjects with autistic disorder, Asperger’s syndrome,
and PDD NOS. Because of the extreme rarity of Rett syndrome and childhood disintegrative disorder, essentially no systematic
psychopharmacologic treatment research has occurred in subjects with these subtypes of PDD. More recently, researchers have been
conducting drug studies in adults with PDDs, in addition to those in children and adolescents with these disorders. The results from
these investigations have allowed for some assessment of the impact of developmental factors on drug efficacy and tolerability.

Drugs that have consistent, primary effects on the core social disability of autistic disorder have not yet been developed. Studies in
laboratory animals have identified particular
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neurochemical systems that mediate some elements of affiliative behavior (3 ,4 ). The translation of these findings into
investigational applications in humans, however, has not yet occurred. The pharmacotherapy of autistic disorder currently involves
the identification and treatment of symptoms including motor hyperactivity (primarily in prepubertal autistic individuals);
inattention; aggression directed toward self, others, or the environment; and interfering repetitive thoughts and behavior. With
reduction in these associated target symptoms, improvement in some aspects of social behavior can result secondarily.

Following a brief review of earlier drug studies, results from more current investigations, including those of atypical antipsychotics
and serotonin reuptake inhibitors (SRIs), will be presented in some detail. For a more comprehensive review of drug treatment of
PDDs, the reader is referred to other sources (5 ,6 ).

Early Drug Treatment Studies

Beginning in the 1960s, numerous agents, including lysergic acid diethylamide, methysergide, levodopa, triiodothyronine,
imipramine, and 5-hydroxytryptophan were studied in autistic disorder. Many of these investigations were limited by a lack of
diagnostic precision and inadequate methodologic design. In general, these initial studies identified no drug that resulted in
consistent target symptom reduction.

Elevated levels of whole blood serotonin (5-hydroxytryptamine, 5-HT) have long been associated with autistic disorder in a large
minority of subjects (7 ). Following reports that fenfluramine, an indirect 5-HT agonist, decreased blood and brain 5-HT in animals,
this drug received extensive investigation. Despite early enthusiasm generated by small open-label reports, most controlled studies
found no consistent efficacy for fenfluramine as a treatment for autistic disorder (8 ). Furthermore, increasing evidence of possible
neurotoxic effects of the drug on 5-HT neurons in animals and the association of fenfluramine with primary pulmonary hypertension
and (in combination with phentermine) valvular heart disease have eliminated its use as a safe agent.

Most of the available typical antipsychotic drugs were studied in heterogeneous groups of children that included autistic subjects.
Many of these early investigations suffered from the lack of adequate diagnostic methods and nonstandardized outcome measures.
Most of these trials were direct comparisons of two drugs, usually low-potency antipsychotics, and did not include a placebo control.
A number of these agents were found to be effective for behavioral symptoms including motor hyperactivity, agitation, and
stereotypies. Due to significant sedation and adverse cognitive effects secondary to the low-potency drugs, however, studies of
higher potency conventional antipsychotics were next pursued.

Campbell and co-workers (9 ,10 ,11 and 12 ) conducted several well-designed controlled studies of haloperidol in autistic children.
In doses of 1.0 to 2.0 mg per day, haloperidol was found to be more efficacious than placebo for withdrawal, stereotypy,
hyperactivity, affective lability, anger, and temper outbursts. However, acute dystonic reactions along with withdrawal and tardive
dyskinesias were not infrequent.

Beginning in the late 1980s, the opioid antagonist naltrexone was investigated as a potential treatment for the associated behavioral
symptoms of autistic disorder, as well as the core social deficits. Again, results from initial open-label reports and small controlled
studies suggested possible effectiveness for naltrexone. More recent large well-designed controlled investigations involving children,
adolescents, and adults with autistic disorder, however, have failed to demonstrate improvement in the majority of target
symptoms or social behavior (13 ,14 ). The most consistent findings from these controlled studies were that naltrexone is well
tolerated and may be effective for reducing motor hyperactivity.

A number of other drugs have been studied in autistic disorder, although most of the trials were either uncontrolled or contained a
small number of subjects (5 ,6 ). For example, β-adrenergic antagonists have been reported to reduce aggression and self-injury in
some small open-label pilot trials in adults with autistic disorder. Hypotension and bradycardia were common dose-related adverse
effects. Case reports and small open-label studies have described mixed results with the 5-HT1A partial agonist buspirone. Controlled
investigations of mood stabilizers, including lithium, valproic acid, carbamazepine, and gabapentin, have not been reported in well-
defined groups of autistic subjects.

The pharmacologic management of motor hyperactivity and impaired attentional mechanisms in individuals with PDDs has proven
particularly challenging to clinicians and researchers. These symptoms are most prominent in younger-aged autistic children. Thus,
these symptoms are largely present during a time when educational programming and interventions are most critical. The
psychostimulants, such as methylphenidate and dextroamphetamine, are effective treatments for these symptoms in individuals
with attention-deficit/hyperactivity disorder (ADHD). Early controlled studies of these agents in autistic children, however,
produced mixed results at best (15 ,16 ). In a more recent double-blind crossover study of methylphenidate and placebo, ten autistic
children, ages 7 to 11 years, received doses of 10 or 20 mg twice daily for 2 weeks (17 ). Statistically significant improvement was
seen on the Conners Teacher Questionnaire (18 ) and on the hyperactivity factor, irritability factor, and total score of the Aberrant
Behavior Checklist (19 ). Adverse effects were minimal. The authors’ impression was that the effects of methylphenidate were
modest. Following completion of the study, it was necessary to add haloperidol to the treatment regimen of two of the ten children
due to continued symptoms of aggression. Anecdotal reports from physicians in clinical practice and in
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academic centers commonly describe the onset or exacerbation of irritability, insomnia, and aggression in individuals with PDDs with
psychostimulant treatment.
The α2-adrenergic agonist clonidine has been shown to be an effective treatment for some individuals with ADHD. In a small double-
blind, placebo-controlled study of clonidine (4 to 10 μg per kg daily) in eight children with autistic disorder, statistically significant
improvement was recorded in hyperactivity and irritability on some teacher and parent ratings (20 ). No significant drug–placebo
differences were identified on clinician ratings of videotaped observations, however. Adverse effects included hypotension, sedation,
and irritability. In contrast, transdermal clonidine (5 μg per kg daily) was reported to be effective in a double-blind, placebo-
controlled crossover study (4 weeks in each treatment phase) involving nine males (ages 5 to 33 years) with autistic disorder (21 ).
Significant improvement was seen on the Clinical Global Impression Scale (CGI) (22 ), and hyperactivity and anxiety were also
reduced. The most common adverse effects were sedation and fatigue.
Guanfacine is an α2-adrenergic agonist with a longer half-life than clonidine that may be less sedating and cause less pronounced
hypotension (23 ). No open-label or controlled studies have been published on the use of guanfacine in PDDs.
To more rigorously address the pharmacotherapy of symptoms of hyperactivity and inattention in PDD, the National Institute of
Mental Health (NIMH)-sponsored Research Units on Pediatric Psychopharmacology (RUPP) Autism Network is planning a controlled
investigation of a methylphenidate vs. placebo in children with PDDs.

Current Drug Treatment Studies

Atypical Antipsychotics
Over the past 5 to 10 years, considerable interest has been generated by the introduction of the atypical antipsychotics (24 ). These
drugs appeared to have potential as a treatment for autistic disorder for a number of reasons. Initial studies in schizophrenia
indicated that these agents were better tolerated and had a lower risk of acute and tardive dyskinesias compared with conventional
antipsychotics. In addition, these drugs were shown to improve both the “positive” (hallucinations and delusions) and “negative”
symptoms of schizophrenia. The negative symptoms include blunted affect, emotional and social withdrawal, lack of interest in
interpersonal relationships, difficulty in abstract thinking, lack of spontaneity and flow of conversation, and stereotyped thinking
(25 ). A number of investigators suggested that the negative symptoms of schizophrenia were comparable to those that characterize
the social impairment of autistic disorder. To date, reports have appeared in which clozapine, risperidone, olanzapine, or
quetiapine was used in the treatment of autistic disorder and other PDDs. The reader is referred to a recent publication that
provides a comprehensive review of atypical antipsychotics in autistic disorder (26 ).

Clozapine
Clozapine has been shown to be effective for treatment-refractory schizophrenia (27 ). The drug’s ability to block 5-HT2A, 5-HT2C, 5-
HT3, and dopamine D1-D4 receptors has been proposed as its mechanism of action. There has been only one report to date describing
the use of clozapine in autistic disorder (28 ). Three children with significant hyperactivity or aggression were given clozapine after
they had not responded to typical antipsychotics. Improvement was observed in the three subjects after 3 months’ treatment at
dosages up to 200 mg per day. The scarcity of reports describing the use of clozapine in autistic disorder might reflect concern
regarding the risk of agranulocytosis or seizures in children or adolescents that are associated with the drug. Because autistic
individuals typically have an impaired ability to communicate effectively and often a high pain threshold, infections secondary to a
decreased white blood cell count may not be identified in a timely manner. Additionally, as mentioned above, many individuals with
autistic disorder have comorbid seizure disorders. Furthermore, the necessary frequent blood draws are not ideal for children,
particularly those with autistic disorder.

Risperidone
Risperidone is a highly potent 5-HT2A/D2 antagonist that has been shown in controlled trials to improve both the positive and negative
symptoms of schizophrenia (29 ). A number of open-label reports describing improvement in aggression, self-injury, ritualistic
behavior, irritability, impulsivity, hyperactivity, and social relatedness in children, adolescents, and adults with autistic disorder
have appeared (26 ). Only one controlled study of risperidone, or any atypical antipsychotic for that matter, has been published in
individuals with autistic disorder and related PDDs (30 ) (Table 42.2 ). In that study, 31 adults (mean age, 28.1 years) with autistic
disorder (n = 17) or PDD NOS (n = 14) entered the 12-week trial. For subjects completing the study, eight (57%) of 14 treated with
risperidone [mean ± standard deviation (SD) dose, 2.9 ± 1.4 mg daily; range 1 to 6 mg per day] were categorized as responders
compared with none of 16 in the placebo group based on the CGI. Nine (60%) of 15 subjects who received open-label risperidone
following the double-blind placebo phase responded. Specifically, risperidone was effective for reducing interfering repetitive
behavior, as well as aggression toward self, others, and property.

TABLE 42.2. CONTROLLED TRIALS OF

CURRENT DRUG TREATMENTS FOR
AUTISTIC DISORDER

Although risperidone was better than placebo for decreasing the overall maladaptive behaviors of autistic disorder, as measured by
the Ritvo-Freeman Real Life Rating Scale overall score (31 ), this finding was largely accounted
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for by significant changes in sensory motor behaviors (e.g., rocking, flapping), affectual reactions (e.g., temper outbursts), and to
some extent sensory responses (e.g., spinning objects, sniffing self or objects). Significant differences between risperidone and
placebo were not captured on subscales of the Ritvo-Freeman Scale that measure social relationships to people and language. For
many subjects, however, clinicians, parents, and other members of the treatment teams had the impression that anxiety associated
with social interactions was reduced, allowing for enhanced social function. It may be that the rating scales used to assess social
relatedness in this study were not sensitive enough to detect changes in this complex aspect of behavior.

In general, risperidone was well tolerated. Thirteen (87%) of 15 subjects randomized to risperidone had at least one adverse effect,
although this included only mild, transient sedation in five subjects, compared with five (31%) of 16 subjects given placebo
(agitation in all five cases). Interestingly, the weight gain that has been observed with risperidone in the treatment of some children
and adolescents with PDDs did not occur to the same degree in this study of adults.

Based on these results and other clinical, preclinical, and safety data, the RUPP Autism Network chose risperidone as the first drug
to study in children and adolescents with autistic disorder (26 ). When completed, this investigation will be the largest controlled
drug trial conducted to date in autistic disorder.

Olanzapine
Three case reports and an open-label case series have described positive responses to the atypical antipsychotic olanzapine in
subjects with PDDs. In the case series, six of seven children, adolescents, and adults with autistic disorder and other PDDs (mean ±
SD age, 20.9 ± 11.7 years; range 5 to 42 years) who completed the 12-week open-label trial were responders based on the CGI (32 ).
Significant improvement in overall symptoms of autistic disorder, motor restlessness or hyperactivity, social relatedness, affectual
reactions, sensory responses, language usage, self-injurious behavior, aggression, irritability or anger, anxiety, and depression was
observed. Significant changes in repetitive behavior did not occur for the group. The mean ± SD dose of olanzapine was 7.8 ± 4.7 mg
daily (range 5 to 20 mg per day). The drug was well tolerated, with the most significant adverse effects being increased appetite
and weight gain in six subjects and sedation in three.

Quetiapine
Only one report of quetiapine in the treatment of autistic disorder has been published (33 ). Six males with autistic disorder, 6.2 to
15.3 years of age (mean ± SD age, 10.9
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± 3.3 years), entered a 16-week open-label trial of quetiapine. The mean ± SD daily dose of quetiapine was 225 ± 108 mg (range 100
to 350 mg per day). Overall, there was no statistically significant improvement for the group as a whole on various behavioral rating
scales. Two subjects completed the entire 16-week trial; both were considered responders based on CGI scores. However, only one
of these two subjects continued to benefit from longer term treatment with the drug. The other four subjects dropped out due to
lack of response and sedation (three subjects), and a possible seizure during the fourth week of treatment (one subject). Other
significant side effects included behavioral activation, increased appetite, and weight gain (range 0.9 to 8.2 kg). The authors
concluded that quetiapine was poorly tolerated and ineffective in most subjects, although the sample size was small. More studies
of quetiapine in autistic disorder and related PDDs are needed before definitive conclusions about its effectiveness and safety can
be made.

Summary
The atypical antipsychotics have potent effects on 5-HT and dopamine neuronal systems, both of which have been implicated in the
pathophysiology of PDDs. Unlike the typical antipsychotic, haloperidol, which has been shown to be effective for reducing many of
the maladaptive behaviors associated with PDDs, the atypical agents’ 5-HT2A/D2 ratio of receptor blockade appears to produce a
lower risk of acute and chronic extrapyramidal side effects, as well as enhanced efficacy for the “negative” symptoms of PDD.
Because most of the studies of this class of drugs have been short-term open-label trials in small samples, larger scale controlled
investigations are needed to confirm these preliminary results. Due to the possibility that many children and adolescents who
demonstrate short-term benefit from these drugs will remain on them indefinitely, the longer term safety and efficacy of atypical
antipsychotics in this population also needs to be determined.

Serotonin Reuptake Inhibitors

In Kanner’s 1943 landmark description of 11 autistic children, the repetitive natures of behavior, speech, and modes of social
interaction were designated as core clinical elements of the syndrome (34 ). Verbal and motor rituals, obsessive questioning, a rigid
adherence to routine, a preoccupation with details, and an anxiously obsessive desire for the maintenance of sameness and
completeness were all noted. Religious and somatic obsessions, repetitive hand washing, and tics were identified in family members.

Results from a more recent study indicated that adults with autistic disorder and obsessive-compulsive disorder (OCD) can be
distinguished on the basis of their current types of repetitive thoughts and behavior (35 ). Compared with the OCD group, the
autistic subjects were significantly less likely to have aggressive, contamination, sexual, religious, symmetry, and somatic thoughts.
Repetitive ordering, hoarding, telling or asking (trend), touching, tapping, or rubbing, and self-damaging or self-mutilating behaviors
were reported significantly more frequently in the autistic subjects, whereas cleaning, checking, and counting behaviors were less
common in the autistic group compared with the OCD subjects.

The clear efficacy of potent SRIs in OCD and their differential effects when directly compared to drugs that potently inhibit
norepinephrine (NE) uptake support the hypothesized importance of 5-HT in the treatment of obsessive-compulsive symptoms (36 ).
Consistent with these drug response data is the hypothesis that a dysregulation of 5-HT function might contribute to the
pathophysiology of at least some individuals with OCD (37 ). Abnormalities in 5-HT function have also been identified in subjects
with autistic disorder and other PDDs (38 ). Based on the efficacy of SRIs in the treatment of OCD, the high prevalence of interfering
repetitive thoughts and behavior in subjects with PDD, and evidence indicating that a dysregulation in 5-HT neurotransmission may
contribute to the pathophysiology of some individuals with autistic disorder, researchers have been studying the clinical response
and side effect profile of SRIs in children, adolescents, and adults with PDDs.

Clomipramine
Clomipramine, a tricyclic antidepressant (TCA) and potent, but nonselective, inhibitor of 5-HT uptake, has been shown to be more
efficacious than the relatively selective NE uptake inhibiting TCA desipramine in the treatment of children and adolescents with OCD
(39 ). In the first controlled investigation of clomipramine in autistic disorder, the drug was found to be more efficacious than
desipramine and placebo on standardized ratings of autistic disorder and anger, as well as ratings of repetitive and compulsive
behaviors (40 ). Seven subjects with autistic disorder, ages 6 to 18 years (means age, 9.6 years), completed the 10-week double-
blind crossover trial of clomipramine (mean dose, 129 daily) and desipramine (mean dose, 111 mg daily) following a 2-week single-
blind placebo phase. In general, the side effects were relatively minor and did not differ between the two drugs. Mild sleep
disturbance, dry mouth, and constipation were observed, and one patient developed a minor tremor on clomipramine. Two subjects
taking desipramine developed uncharacteristic and severe irritability and temper outbursts. The parents of all seven subjects chose
to have their children continue on clomipramine after completion of the study.

As a follow-up to this pilot study, a larger double-blind comparison of clomipramine, desipramine and placebo was conducted in
children and adolescents with autistic disorder (41 ). Following a 2-week single-blind placebo phase, 12 subjects completed a 10-
week double-blind crossover comparison of clomipramine and placebo, and 12 different subjects completed a similar comparison of
clomipramine and
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desipramine. The latter study included data from the seven subjects who participated in the original pilot study described above.
Clomipramine (mean dose, 152 mg daily) was superior to both placebo and desipramine (mean dose, 127 mg daily) on ratings of
autistic symptoms, including stereotypies, anger, and ritualized behaviors, with no difference between desipramine and placebo.
Clomipramine was equal to desipramine and both drugs were superior to placebo for reducing motor hyperactivity. One child
developed prolongation of the corrected QT interval (0.45 seconds) and another developed severe tachycardia (resting heart rate,
160 to 170 beats per minute) during clomipramine treatment. A third child experienced a grand mal seizure.

Subsequent open-label studies of clomipramine have been published with mixed results and increased recognition of adverse effects.
Clomipramine treatment of five young adults (ages 13 to 33 years) with autistic disorder led to ratings of “much improved” on the
CGI in four patients, with improvement seen in social relatedness, obsessive-compulsive symptoms, aggression, and self-injurious
behavior (42 ). In another study, 11 consecutively referred children and adolescents with developmental disabilities and chronic
stereotypies or self-injurious behavior were treated with clomipramine (43 ). Four of the subjects (ages 13 to 20 years) had been
diagnosed with autistic disorder and of them, three had a significant reduction in stereotypic, self-injurious behavior with
clomipramine at doses of 50 to 125 mg daily. Adverse effects included constipation, aggression, rash, and enuresis. In another open-
label study, clomipramine 200 mg daily, was associated with decreased abnormal motor movements and compulsions in five autistic
boys ages 6 to 12 years (44 ).

A large prospective open-label study of clomipramine (mean dose, 139 mg daily) treatment of 35 adults diagnosed with different
subtypes of PDD was described (45 ). Of the 33 subjects who completed the 12-week study, 18 (55%) were judged responders on the
CGI with improvement seen in aggression, self-injury, interfering repetitive thoughts and behavior, and social relatedness. Thirteen
of the 33 subjects had significant adverse effects including seizures (in three patients, including two who had preexisting seizure
disorders stabilized on anticonvulsants), weight gain, constipation, sedation, agitation, and anorgasmia.

A number of studies have suggested that younger children may tolerate clomipramine less well and show a decreased response
compared to adolescents and adults with PDDs. In one report, eight children (ages 3.5 to 8.7 years) were given clomipramine (mean
dose, 103 mg daily) for 5 weeks in a prospective open-label manner (46 ). Among the seven children who completed the trial, only
one child was rated as moderately improved on a clinical global consensus measure. Adverse effects were frequent and included
urinary retention requiring catheterization, constipation, drowsiness, and increased aggression and irritability. In a follow-up report
to the study described above, in which five autistic children had an initial positive response to clomipramine (44 ), it was noted that
the drug was eventually discontinued in all cases due to adverse effects or continued maladaptive behavior (47 ). Adverse effects
included the serotonin syndrome, increased seizure frequency, and exacerbation of agitation and aggressiveness that required
hospitalization.

Because of their better side effect profile compared with clomipramine, including their lower propensity to decrease the seizure
threshold, selective SRIs (SSRIs) have been receiving increasing attention as a potential treatment for the interfering symptoms
associated with autistic disorder and other PDDs.

Fluvoxamine
To date, only one double-blind, placebo-controlled study of an SSRI in subjects with autistic disorder has been published (48 ).
Fluvoxamine (mean dose, 276.7 mg daily) or placebo was given to 30 autistic adults for 12 weeks. Eight of 15 subjects who received
fluvoxamine vs. none who received placebo were categorized as “much improved” or “very much improved” on the CGI.
Fluvoxamine was significantly more effective than placebo for reducing repetitive thoughts and behavior, maladaptive behavior, and
aggression. In addition, fluvoxamine reduced inappropriate repetitive language usage. Adverse effects included nausea and sedation,
which were transient and of minor severity.

In contrast to the encouraging results from this study of fluvoxamine in autistic adults, a 12-week double-blind, placebo-controlled
study in children and adolescents with autistic disorder and other PDDs found the drug to be poorly tolerated with limited efficacy
at best (McDougle and co-workers, unpublished data). Thirty-four patients (five female, 29 male; age range 5 to 18 years, mean age,
9.5 years), 12 of whom met criteria for autistic disorder, eight for Asperger’s syndrome, and 14 for PDD NOS, participated. Of the 16
subjects randomized to placebo, none demonstrated any significant change in target symptoms. Adverse events that occurred in the
placebo-treated subjects included increased motor hyperactivity (n = 2), insomnia (n = 2), dizziness and/or vertigo (n = 1), agitation
(n = 1), diarrhea (n = 1), decreased concentration (n = 1), and increased self-stimulation (n = 1). Eighteen of the subjects were
randomized to fluvoxamine (range 25 to 250 mg daily; mean dose, 106.9 mg per day). The drug was begun at 25 mg every other day
and increased by 25 mg every 3 to 7 days as tolerated. Only one of the fluvoxamine-treated children demonstrated a significant
improvement with the drug. Fourteen of the children randomized to fluvoxamine demonstrated adverse effects [insomnia (n = 9),
motor hyperactivity (n = 5), agitation (n = 5), aggression (n = 5), increased rituals (n = 2), anxiety (n = 3), anorexia (n = 3), increased
appetite (n = 1), irritability (n = 1), decreased concentration (n = 1), and increased impulsivity (n = 1)].

The marked difference in efficacy and tolerability of fluvoxamine in children and adolescents with autistic disorder
 P.572

and other PDDs in this study, compared with that of autistic adults, underscores the importance of developmental factors in the
pharmacotherapy of these subjects. This differential drug response is consistent with the hypothesis that ongoing brain development
has a significant impact on the subjects’ ability to tolerate and respond to a drug, at least with respect to fluvoxamine and possibly
other SSRIs. Developmental changes in brain 5-HT function may contribute to these widely varying clinical responses between
subjects with autistic disorder and other PDDs of different age groups.

Fluoxetine
Several case reports have described fluoxetine treatment of autistic subjects although, to date, no controlled studies have appeared.

In a large case series, Cook and associates (49 ) found fluoxetine (10 to 80 mg daily), given in an open-label manner, effective in 15
of 23 subjects (ages 7 to 28 years) with autistic disorder as determined by the CGI (49 ). Intolerable side effects, including
restlessness, hyperactivity, agitation, elated affect, decreased appetite, and insomnia, occurred in six of 23 subjects.

In a retrospective investigation, fluoxetine (20 to 80 mg daily) and paroxetine (20 to 40 mg daily) were found to be effective in
approximately one-fourth of adults (mean age, 39 years) with “intellectual disability” and autistic traits (50 ). The sample included
all intellectually disabled subjects who had been treated with an SSRI over a 5-year period within a health care service in Great
Britain. The mean duration of treatment was 13 months. Target symptoms were perseverative behaviors, aggression, and self-injury.
Six of 25 subjects treated with fluoxetine and three of 12 subjects given paroxetine were rated as “much improved” or “very much
improved” on the CGI.

In another study, 37 children (ages 2.25 to 7.75 years) with autistic disorder were treated with fluoxetine in an open-label fashion at
doses ranging from 0.2 to 1.4 mg per kg daily (51 ). Eleven of the children had an “excellent” clinical response and 11 others had a
“good” response. Improvement was seen in behavioral, cognitive, affective, and social areas. Interestingly, language acquistion
seemed to improve with fluoxetine treatment. Drug-induced hyperactivity, agitation, and aggression were frequent causes of
discontinuation of fluoxetine.

Sertraline
To our knowledge, no controlled studies of sertraline in subjects with autistic disorder or other PDDs have been published, although
a number of open-label reports have appeared. In a 28-day trial of sertraline (at doses of 25 to 150 mg daily) in nine adults with
mental retardation (five of whom had autistic disorder), significant decreases in aggression and self-injurious behavior occurred in
eight as rated on the CGI severity rating (52 ). In a case series of nine autistic children (ages 6 to 12 years) treated with sertraline
(25 to 50 mg daily), eight showed significant improvement in anxiety, irritability, and “transition-induced behavioral deterioration”
or “need for sameness” (53 ). In three of the responders, a return of symptoms occurred after 3 to 7 months. Two children
demonstrated agitation when the dose was raised to 75 mg daily.

A large prospective open-label study of 42 adults with PDDs (including subjects with autistic disorder, Asperger’s syndrome, and PDD
NOS) found sertraline (mean dose, 122 mg per day) effective for improving aggression and interfering repetitive behavior, but not
impaired social relatedness as assessed by various rating scales over the course of the 12-week study (54 ). As determined by a CGI
global improvement item score of “much improved” or “very much improved,” 15 of 22 subjects with autistic disorder, none of six
with Asperger’s syndrome, and nine of 14 with PDD NOS were judged responders. Those subjects with autistic disorder and PDD NOS
showed significantly more benefit from sertraline than those with Asperger’s syndrome; the authors hypothesized that this might
have been because those diagnosed with Asperger’s syndrome were less symptomatic at baseline. Three of the 42 subjects dropped
out of the study due to intolerable agitation and anxiety.

Paroxetine
Only a few reports, none of them controlled, have appeared on the use of paroxetine in autistic disorder. Paroxetine 20 mg per day
decreased self-injury in a 15-year-old boy with “high-functioning” autistic disorder (55 ). In another report, paroxetine resulted in a
reduction of irritability, temper tantrums, and interfering preoccupations in a 7-year-old boy with autistic disorder (56 ). The
optimal dose of paroxetine was 10 mg daily; an increase of paroxetine to 15 mg per day led to agitation and insomnia. As described
earlier, a retrospective case analysis found paroxetine to be effective in approximately 25% of adults with PDD NOS (50 ).

In a 4-month open-label study of 15 adults with severe and profound mental retardation (seven with PDD), paroxetine at doses of 20
to 50 mg daily was effective for symptoms of aggression at 1 month, but not at 4-month follow-up (57 ). The investigators
hypothesized that adaptive changes may have occurred in 5-HT receptor density, availability of 5-HT, or in 5-HT transporter
sensitivity.

Citalopram
To date, there have been no published reports on the effects of citalopram, an SSRI that has been recently introduced in the United
States, in patients with autistic disorder or other PDDs.

Summary
Recent work has determined that the types of repetitive phenomena associated with autistic disorder are different from those that
characterize OCD. Nevertheless, these signs
 P.573

and symptoms can interfere with the autistic individual’s quality of life. Studies of SRIs, the mainstay of treatment for the
obsessions and compulsions of OCD, have yielded mixed results in autistic disorder. To date, only three controlled studies of SRIs in
autistic disorder have been published, as reviewed above (Table 42.2 ). All three of these studies found the SRI to be helpful for the
interfering repetitive phenomena associated with autistic disorder, as well as for aspects of aggression, self-injury, and impaired
social relatedness. On the other hand, results from an unpublished controlled study of fluvoxamine in children and adolescents with
autistic disorder and other PDDs indicated that the drug was poorly tolerated and of limited efficacy. The results from that study are
consistent with those from a number of open-label reports suggesting that SRIs may be less well tolerated and less effective in
younger (prepubertal) autistic subjects compared with autistic adolescents and adults (postpubertal). Although this developmental
difference in tolerability and response to SRIs may be a dose-related phenomena, other factors need to be considered. Recent data
indicate that significant changes in measures of 5-HT function occur during puberty in autistic individuals. For example, McBride and
co-workers (58 ) found that mean platelet 5-HT levels were significantly higher in prepubertal autistic children than prepubertal
normal controls, but no significant difference was found between postpubertal male autistic subjects and postpubertal normal
controls (58 ). Furthermore, Chugani and associates (59 ) reported results from a positron emission tomography brain imaging study
showing that changes in brain 5-HT synthesis capacity that normally occur in developing humans are disrupted in autistic children
(59 ). Thus, pre- and postpubertal autistic subjects may have significant differences in brain 5-HT function that influence their
ability to tolerate and respond to SRIs. Pharmacogenetic differences among autistic individuals, which may affect SRI tolerability
and responsivity, will also require more investigation (60 ).

Novel Therapeutic Strategies

Secretin
Secretin is a polypeptide hormone secreted primarily by the endocrine cells in the upper gastrointestinal (GI) tract that is involved
in regulating pancreatic exocrine secretion. A synthetic form of secretin is Food and Drug Administration (FDA) approved for use in
the diagnosis of particular GI diseases. In 1998, Horvath and co-workers (61 ) published a report that described marked improvement
in language and social behavior in three children with autistic disorder who received secretin as part of a routine diagnostic workup
for GI problems.

These encouraging yet preliminary results, coupled with enthusiastic media reports, led to widespread excitement and optimism
among many family members of autistic individuals about a potential “cure” for the disorder. In response, researchers began
conducting controlled studies of secretin in autistic children. A double-blind, controlled study of single-dose intravenous secretin
(0.4 μg per kg) or placebo administration was conducted in 60 children, ages 3 to 14 years, of whom 40 had autistic disorder and 20
had PDD NOS (62 ). No significant differences were found between secretin and placebo on primary outcome measures that assessed
changes in autistic behaviors and adaptive functioning at days 1 and 2 and weeks 1, 2, and 4 following the infusion. No significant
difference was found in adverse effects between secretin and placebo. Two additional controlled studies have reported similar
findings (63 ,64 ). Based on the results of these systematic investigations, secretin cannot be recommended as a treatment for the
target symptoms associated with autistic disorder.

Glutamatergic Agents
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is central to developmental processes including neuronal migration,
differentiation, and plasticity (65 ). Disturbances in glutamatergic function, via reduced neurotropic actions of glutamate or
excessive neurotoxic effects, could alter neurodevelopment substantially (66 ). During the past 5 to 10 years, significant advances
have been made in the identification of potential pharmacotherapies affecting glutamatergic function for a number of
neuropsychiatric disorders (67 ).

Hypotheses regarding glutamatergic dysfunction in autistic disorder have been proposed (68 ). In addition, preliminary results from
studies of drugs that modulate glutamate neurotransmission in autistic disorder have been published. Lamotrigine is a drug that
attenuates some forms of cortical glutamate release via inhibition of sodium channels, P- and N-type calcium channels, and
potassium channels. In one study, eight of 13 children and adolescents with autistic disorder given lamotrigine for intractable
epilepsy showed a decrease in “autistic symptoms” (69 ). Another report described improvement in self-injurious behavior,
irritability, and disturbed sleep in an 18-year-old woman with profound mental retardation and a generalized seizure disorder who
was given open-label lamotrigine (70 ). Interestingly, the subject showed improvement in measures of “fixed facial expression, lacks
emotional responsivity,” “resists any form of physical contact,” and “inactive, never moves spontaneously.” The authors suggested
that these changes might represent a “prosocial” effect of the drug. In a double-blind, placebo-controlled study, 39 subjects with
autistic disorder, ages 5 to 19 years old, were given placebo or the NMDA receptor antagonist amantadine (71 ). The design included
a single-blind placebo lead-in, followed by a single daily dose of amantadine (2.5 mg per kg) or placebo for the next week, and then
twice daily dosing for the subsequent 3 weeks. No significant difference was found between drug
 P.574

and placebo on parent ratings, although clinician-rated measures of hyperactivity and inappropriate speech showed statistically
significant improvement. A trend toward greater response in the amantadine group, based on CGI ratings, occurred. Amantadine was
well tolerated.

Based on these preliminary results, and reports that the “negative” symptoms of schizophrenia can be improved with drugs active at
the NMDA receptor (72 ), additional research with these and other agents affecting the glutamatergic systems appears warranted.
The group II/III metabotropic-glutamate receptor (mGluR II/III) agonists (73 ) and the positive allosteric modulator of α-amino-3-
hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, CX516 (Ampakine) (74 ) may hold promise in this regard. Interestingly,
one mechanism of action underlying the relative efficacy of atypical antipsychotics, such as risperidone, for autistic disorder (30 )
may be the suppression of glutamate release via 5-HT2A antagonism (75 ).

Neuroimmune Modulation
Neuroimmunologic dysfunction has been implicated in the pathophysiology of autistic disorder (76 ) and other neuropsychiatric
conditions (77 ). To date, no consistent immunologic abnormalities have been found in autistic disorder, although viral and
autoimmune hypotheses, among others, have been posited (76 ). Neurovirologic disease and other insults to the immune system can
lead to increased production of catabolites of tryptophan, including quinolinate and kynurenate, which can cause significant
neurotoxicity via activity at the NMDA receptor complex (78 ). Thus, neuroimmune dysregulation in autistic disorder would not be
inconsistent with altered glutamatergic function, as described above. Results from small open-label studies of intravenous
immunoglobulin have suggested that this intervention may be helpful in only a limited minority of subjects, if at all (79 ). Controlled
studies of agents that have direct effects on immune function, however, have not been conducted in autistic disorder. Such research
on neuroimmune interactions may yield important data on pathophysiology, if not etiology, in a subset of autistic subjects.

CONCLUSION
Part of "42 - Current and Emerging Therapeutics of Autistic Disorder and Related Pervasive Developmental Disorders "

Significant progress in the neuropsychopharmacology of autistic disorder and related PDDs has been made since the fourth edition of
this text (80 ). Future research in this area should include controlled studies of atypical antipsychotics in children and adolescents
with autistic disorder and other PDDs, such as that being conducted by the NIMH-sponsored RUPP Autism Network (26 ). Longitudinal
efficacy and safety data will need to be gathered on atypical antipsychotics in this population, as well. Larger double-blind,
placebo-controlled trials of SRIs in pre- vs. postpubertal individuals with autistic disorder, as well as studies designed to determine
the effects of these drugs on the target symptoms of subjects with different subtypes of PDD, including Asperger’s syndrome, are
also needed. In these studies, the optimal dosage for age and developmental level and the duration of adequate treatment should
be determined. In addition, genetic predictors of treatment response, such as 5-HT transporter protein genotype, should be sought
(60 ). The scientific community needs to continue to respond to reports of putative “cures” for autistic disorder, such as those that
surrounded secretin, by conducting controlled studies of such agents. Accepting this responsibility will contribute to ensuring the
continued safety of autistic subjects and provide family members with data on which to base informed decisions regarding their
child’s medical care. Finally, exploration of promising novel therapeutic strategies, such as those affecting glutamatergic and
neuroimmune function, may provide new insights into the neurobiology and treatment of this devastating group of disorders.

ACKNOWLEDGMENTS
Part of "42 - Current and Emerging Therapeutics of Autistic Disorder and Related Pervasive Developmental Disorders "

This work was supported by an independent investigator award (Seaver Foundation Investigator) from the National Alliance for
Research in Schizophrenia and Depression, the Theodore and Vada Stanley Research Foundation, a research unit on Pediatric
Psychopharmacology Contract, no. N01MH70001 from the National Institute of Mental Health, and the State of Indiana Division of
Mental Health.

Dr. McDougle has received research support from Pfizer, Eli Lilly, and Janssen Pharmaceutica, and has served on speakers’ bureaus
and/or as a consultant for Pfizer, Eli Lilly, Janssen, and Solvay Pharmaceuticals.

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43
Pathophysiology of Attention-Deficit/Hyperactivity Disorder

Stephen V. Farone

Joseph Biederman

Stephen V. Farone: Pediatric Psychopharmacology Unit, Child Psychiatry Service, Massachusetts General Hospital; Harvard Medical
School; Massachusetts Mental Health Center; Harvard Institute of Psychiatric Epidemiology and Genetics, Boston, Massachusetts.

Joseph Biederman: Pediatric Psychopharmacology Unit, Child Psychiatry Service, Massachusetts General Hospital; Harvard Medical
School, Boston, Massachusetts.

Attention-deficit/hyperactivity disorder (ADHD) is a childhood-onset, clinically heterogeneous disorder of inattention, hyperactivity,

and impulsivity. Its impact on society is enormous in terms of its financial cost, stress to families, adverse academic and vocational
outcomes, and negative effects on self-esteem (1 ). Children with ADHD are easily recognized in clinics, in schools, and in the home.
Their inattention leads to daydreaming, distractibility, and difficulties in sustaining effort on a single task for a prolonged period.
Their impulsivity makes them accident prone, creates problems with peers, and disrupts classrooms. Their hyperactivity, often
manifest as fidgeting and excessive talking, is poorly tolerated in schools and is frustrating to parents, who can easily lose them in
crowds and cannot get them to sleep at a reasonable hour. In their teenage years, symptoms of hyperactivity and impulsivity
diminish, but in most cases the symptoms and impairments of ADHD persist. The teen with ADHD is at high risk of low self-esteem,
poor peer relationships, conflict with parents, delinquency, smoking, and substance abuse (1 ).

The validity of diagnosing ADHD in adults has been a source of much controversy (2 ). Some investigators argue that most cases of
ADHD remit by adulthood (3 ), a view that questions the validity of the diagnosis in adulthood. Others argue that the diagnosis of
ADHD in adults is both reliable and valid (2 ). These investigators point to longitudinal studies of children with ADHD, studies of
clinically referred adults, family-genetic studies, and psychopharmacologic studies. Longitudinal studies have found that as many as
two thirds of children with ADHD have impairing ADHD symptoms as adults. Studies of clinically referred adults with retrospectively
defined childhood-onset ADHD show them to have a pattern of psychosocial disability, psychiatric comorbidity, neuropsychological
dysfunction, familial illness, and school failure that resemble the well known features of children with ADHD.

Throughout the life cycle, a key clinical feature observed in patients with ADHD is comorbidity with conduct, depressive, bipolar,
and anxiety disorders (4 ,5 ). Although spurious comorbidity can result from referral and screening artifacts (5 ), these artifacts
cannot explain the high levels of psychiatric comorbidity observed for ADHD (4 ). Notably, epidemiologic investigators find
comorbidity in unselected general population samples (6 ,7 ), a finding that cannot be caused by the biases that inhere in clinical
samples. Moreover, as we discuss later, family studies of comorbidity dispute the notion that artifacts cause comorbidity; instead,
they assign a causal role to etiologic relationships among disorders.

 NEUROPSYCHOPHARMACOLOGY
 BRAIN ABNORMALITIES
 GENETICS
 ENVIRONMENTAL RISK FACTORS
 SUMMARY AND CONCLUSIONS
 DISCLAIMERS

NEUROPSYCHOPHARMACOLOGY
Part of "43 - Pathophysiology of Attention-Deficit/Hyperactivity Disorder "

Pharmacotherapy
Any pathophysiologic theory about ADHD must address the large pharmacotherapy literature about the disorder. The mainline
treatments for ADHD are the stimulant medications methylphenidate, pemoline, and dextroamphetamine. These compounds are
safe and effective for treating ADHD in children, adolescents, and adults (8 ,9 ). In addition, to improving ADHD’s core symptoms of
inattentiveness, hyperactivity, and impulsivity, stimulants also improve associated behaviors, including on-task behavior, academic
performance, and social functioning in the home and at school. In adults, occupational and marital dysfunction tend to improve with
stimulant treatment. There is little evidence of a differential response to methylphenidate, pemoline, and dextroamphetamine. The
average response rate for each is 70%.

Stimulants enhance social skills at home and in school.

 P.578

They also improve maternal-child and sibling interactions. Children with ADHD who are treated with stimulants have increased
abilities to perceive peer communications and situational cues and to modulate the intensity of their behavior. They also show
improved communication, greater responsiveness, and fewer negative interactions. Neuropsychological studies show that stimulants
improve vigilance, cognitive impulsivity, reaction time, short-term memory, and learning of verbal and nonverbal material in
children with ADHD.

Although stimulants are the mainstay of anti-ADHD pharmacotherapy, tricyclic antidepressants (TCAs) also are effective anti-ADHD
agents. TCAs include secondary and tertiary amines with a wide range of receptor actions, efficacy, and side effects. Secondary
amines are more selective (noradrenergic) with fewer side effects. Most studies of TCAs have found either a moderate or robust
response rate of ADHD symptoms (8 ,9 and 10 ). These studies show anti-ADHD efficacy for imipramine, desipramine, amitriptyline,
nortriptyline, and clomipramine. Both short- and long-term studies show that TCAs produce moderate to strong effects on ADHD
symptoms. In contrast, neurocognitive symptoms are do not respond well to TCA treatment. Because of rare reports of sudden death
among TCA-treated children, these drugs are not a first-line treatment for ADHD and are only used after carefully weighing the risks
and benefits of treating or not treating a child who does not respond to other agents.

Other noradrenergic agents help to control ADHD symptoms. Bupropion hydrochloride, which has both dopaminergic and
noradrenergic effects, is effective for ADHD in children (11 ,12 )as well as in adults (13 ). Although they are rarely used because of
their potential for hypertensive crisis, several studies suggested that monoamine oxidase inhibitors may be effective in juvenile and
adult ADHD (14 ). The experimental noradrenergic compound tomoxetine showed efficacy in a controlled study of adults with ADHD
(15 ) and in an open study of children with ADHD (16 ).

In contrast to the beneficial effects of stimulants and TCAs, there is only weak evidence that either α2-noradrenergic agonists or
serotonin reuptake inhibitors effectively combat ADHD (17 ). A controlled clinical trial showed that transdermal nicotine improved
ADHD symptoms and neuropsychological functioning in adults with ADHD (18 ). Consistent with this finding, a controlled study found
the experimental compound ABT-418 to treat adult ADHD effectively (19 ). ABT-418 is a potent and selective agonist for α4β2-subtype
central nervous system neuronal nicotinic receptors.

Catecholamine Hypothesis
As the foregoing review shows, effective medications for ADHD act in noradrenergic and dopaminergic systems. Stimulants block the
reuptake of dopamine and norepinephrine into the presynaptic neuron and increase the release of these monoamines into the
extraneuronal space (20 ). Solanto suggested that stimulants may also activate presynaptic inhibitory autoreceptors and may lead to
reduced dopaminergic and noradrenergic activity (21 ). The maximal therapeutic effects of stimulants occur during the absorption
phase of the kinetic curve, within 2 hours after ingestion. The absorption phase parallels the acute release of neurotransmitters into
synaptic clefts, a finding providing support for the hypothesis that alteration of monoaminergic transmission in critical brain regions
may be the basis for stimulant action in ADHD (22 ). A plausible model for the effects of stimulants in ADHD is that, through
dopaminergic or noradrenergic pathways, these drugs increase the inhibitory influences of frontal cortical activity on subcortical
structures (22 ).

Human studies of the catecholamine hypothesis of ADHD that focused on catecholamine metabolites and enzymes in serum and
cerebrospinal fluid produced conflicting results (23 ,24 ). Perhaps the best summary of this literature is that aberrations in no single
neurotransmitter system can account for the available data. Of course, because studies of neurotransmitter systems rely on
peripheral measures, which may not reflect brain concentrations, we cannot expect such studies to be completely informative.
Nevertheless, although such studies do not provide a clear profile of neurotransmitter dysfunction in ADHD, on balance, they are
consistent with the idea that catecholaminergic dysregulation plays a role in the origin of at least some cases of ADHD.

The catecholamine hypothesis of ADHD finds further support from animal studies. One approach has been the use of 6-
hydroxydopamine to create lesions in dopamine pathways in developing rats. Because these lesions created hyperactivity, they were
thought to provide an animal model of ADHD (25 ). Disruption of catecholaminergic transmission with chronic low-dose N-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, creates an animal model of ADHD in monkeys. In this latter work, MPTP
administration to monkeys caused cognitive impairments on tasks thought to require efficient frontal-striatal neural networks. These
cognitive impairments mirrored those seen in monkeys with frontal lesions (26 ,27 ). Like children with ADHD, MPTP-treated
monkeys show attentional deficits and task impersistence. Methylphenidate and the dopamine D2 receptor agonist LY-171555
reversed the behavioral deficits but not the cognitive dysfunction (28 ,29 ).

Several investigators used the spontaneously hypertensive rat (SHR) as an animal model of ADHD because of the animal’s locomotor
hyperactivity and impaired discriminative performance. Studies using the SHR have implicated dopaminergic and noradrenergic
systems. For example, the dopamine D2 receptor agonist, quinpirole, caused significantly greater inhibition of dopamine release
from caudate-putamen
 P.579

but not from nucleus accumbens or prefrontal cortex slices in SHR compared with control mice (30 ). In another study, dopamine
release secondary to electrical stimulation was significantly lower in caudate-putamen and prefrontal cortex slices of SHR compared
with control mice. These findings were attributed to increased autoreceptor-mediated inhibition of dopamine release in caudate-
putamen slices but not in the prefrontal cortex. Another study showed that the altered presynaptic regulation of dopamine in SHR
led to the down-regulation of the dopamine system (31 ). The authors hypothesized that this may have occurred early in
development as a compensatory response to abnormally high dopamine concentrations.

Other SHR studies implicated an interaction between the noradrenergic and dopaminergic system in the nucleus accumbens, but
they ruled out the idea that a dysfunctional locus ceruleus and A2 nucleus impairs dopaminergic transmission in the nucleus
accumbens through α2-adrenoceptor–mediated inhibition of dopamine release (32 ). Papa et al. used molecular imaging techniques
to assess the neural substrates of ADHD-like behaviors in the SHR rat (33 ). Their data showed the corticostriatopallidal system to
mediate these behaviors. King et al. showed that exposure to excess androgen levels early in development led to decreased
catecholamine innervation in frontal cortex and enhanced expression of ADHD-like behaviors (34 ). Carey et al. used quantitative
receptor autoradiography and computer-assisted image analysis to show a higher density of low-affinity D1 and D5 dopamine
receptors in the caudate-putamen, the nucleus accumbens, and the olfactory tubercle of SHR (35 ). Stimulant treatment normalized
these receptors by decreasing the number of binding sites and increasing affinity to the control level.

In contrast to the large body of evidence implicating dopaminergic and noradrenergic systems in ADHD, evidence implicating
serotonergic systems is mixed. Although the tertiary amines (imipramine and amitriptyline) are more selective for the serotonin
transporter than the norepinephrine transporter (36 ), the secondary amines (desipramine, nortriptyline, and protriptyline) are more
selective for the norepinephrine transporter (36 ). Moreover, measures of serotonin metabolism appear minimally related to the
clinical efficacy of the stimulants (22 ), a finding consistent with the lack of efficacy of serotonergic drugs for treating ADHD. This
suggests that the anti-ADHD efficacy of the TCAs stems from their actions on catecholamine reuptake, particularly that of
norepinephrine.

Despite these equivocal findings, work by Gainetdinov et al. suggests that we cannot rule out a role for serotonergic systems in the
pathophysiology of ADHD (37 ). These authors studied knockout mice lacking the gene encoding the dopamine transporter (DAT).
These mice have elevated dopaminergic tone, are hyperactive, and show decreased locomotion in response to stimulants.
Gainetdinov et al. showed that the effects of stimulants were mediated by serotonergic neurotransmission (37 ).

The anti-ADHD efficacy of nicotine and ABT-418 suggests that nicotinic dysregulation may also play a role in the pathophysiology of
ADHD. Patients with ADHD are more likely to smoke and have an earlier age of onset of smoking than persons who do not have ADHD
(38 ,39 and 40 ). In addition, maternal smoking during pregnancy appears to increase the risk of ADHD in the children (41 ), and in
utero exposure to nicotine in animals confers a heightened risk of an ADHD-like syndrome in the newborn (42 ,43 ). That nicotine
dysregulation could play an important role in the pathophysiology of ADHD is not surprising considering that nicotinic activation
enhances dopaminergic neurotransmission (44 ,45 ).

BRAIN ABNORMALITIES
Part of "43 - Pathophysiology of Attention-Deficit/Hyperactivity Disorder "

Satterfield and Dawson were among the first to propose that ADHD symptoms were caused by frontolimbic dysfunction (46 ). These
investigators suggested that weak frontal cortical inhibitory control over limbic functions could lead to ADHD. A review of the
neurologic literature showing similarities in disinhibited behavior between adult patients with frontal lobe damage and children with
ADHD provided further evidence that the frontal lobes could be involved in the pathophysiology of the disorder (47 ). Two sources of
data have tested the frontolimbic hypothesis of ADHD: neuropsychological studies and neuroimaging studies.

Neuropsychological Studies
Neuropsychological tests indirectly assess brain functioning by assessing features of human perception, cognition, or behavior that
have been clinically or experimentally linked to specific brain functions (48 ). Although limited in their ability to localize brain
dysfunction, these tests have several advantages. Many of these tests have been standardized on large populations, thus making it
straightforward to define deviant performance. Because of the extensive use of these tests in brain-damaged populations,
performance on many of these tests can lead to hypotheses, albeit weak, about the locus of brain dysfunction. Being noninvasive
and inexpensive, neuropsychological tests are frequently used to generate hypotheses about brain dysfunction.

Given that inattention is a one of the defining clinical features of ADHD, many neuropsychological studies of the disorder have
assessed the attention of children with ADHD. The most commonly used measure of attention is the continuous performance test,
which requires subjects to sustain their attention to subtle sensory signals, to avoid being distracted by irrelevant stimuli, and to
maintain alertness for the duration of the session. Most of these studies
 P.580

find children with ADHD to be impaired on this measure (1 ).

Children with ADHD also perform poorly on tasks requiring inhibition of motor responses, organization of cognitive information,
planning, complex problem solving, and the learning and recall of verbal material (49 ). Examples of tests that measure these
functions are the Stroop Test, the Wisconsin Card Sorting Test, the Rey-Osterrieth Test, the Freedom from Distractibility factor from
Wechsler’s Tests of Intelligence, and the California Verbal Learning Test.

Some studies suggest that the impairments found in children with ADHD cannot be accounted for by psychiatric comorbidity (50 ).
Moreover, having a family history of ADHD may predict a greater degree of neuropsychological impairment. This latter finding
suggests that familial ADHD and neuropsychological impairment identify a more biologically based type of ADHD. In contrast,
nonfamilial cases of ADHD with lesser neuropsychological impairments may have other etiologic factors. Children with ADHD do not
appear to be impaired on simple motor speed, verbal fluency, or visual spatial accuracy, findings that suggest that observed
neuropsychological impairments are caused by specific, not generalized, deficits (51 ).

Notably, neuropsychological studies have consistently found adults with ADHD to be impaired on measures of vigilance using the
continuous performance test (52 ,53 ). These studies have also shown adults with ADHD to be impaired in other functions known to
affect children with ADHD. These include the following: perceptual-motor speed as assessed by the digit symbol/coding tests
(54 ,55 ); working memory as assessed by digit span tests (53 ,56 ); verbal learning, especially semantic clustering (52 ,56 ); and
response inhibition as assessed by the Stroop Color-Word Test (57 ,58 ). Because neuropsychological tests are free of the potential
biases of self-reported symptoms, the finding that the neurocognitive profiles of adults with ADHD are similar to those of children
with ADHD suggests that the diagnosis of ADHD is valid as applied in adulthood.

Our description of neuropsychological dysfunction in ADHD describes trends that have emerged in the research literature, not
findings that have been consistently replicated. Although there are inconsistencies among studies, it is notable that the pattern of
deficits that has emerged is similar to what has been found among adults with frontal lobe damage. Thus, the neuropsychological
data tend to support the hypothesis that the frontal cortex or regions projecting to the frontal cortex are dysfunctional in at least
some children with ADHD.

Because neuropsychological tests provide indirect measures of brain function, we must be cautious in using them to make inferences
about the locus of brain impairment in ADHD. Yet because many of these tests have been standardized on normative populations and
administered extensively to brain-damaged populations, observed deficits tests can stimulate hypotheses about the role of specific
brain regions in the pathophysiology of ADHD.

With this considerations in mind, we view the pattern of neuropsychological impairment in children with ADHD as consistent with
Satterfield and Dawson’s (46 ) idea that symptoms of ADHD derive from abnormalities of prefrontal cortex or its neural connections
to subcortical structures. This inference derives from the clinical and behavioral features that have been linked to regions of the
prefrontal cortex (59 ). Notably, orbital frontal lesions predict social disinhibition and impulsivity, and dorsolateral lesions affect
organizational abilities, planning, working memory, and attention. Studies of children with ADHD find impairment in all these
neuropsychological domains. Thus, the neuropsychological test data—along with the clinical features of the disorder—implicate both
orbitofrontal and dorsolateral prefrontal dysfunction in ADHD. In contrast, the mesial prefrontal region, where lesions predict
dysfluency and the slowing of spontaneous behavior, is not implicated in ADHD.

Given the complexity of prefrontal circuitry (60 ), along with the limitations of neuropsychological inference, we cannot endorse a
simple lesion model of ADHD. The “prefrontal” abnormalities in ADHD may result from abnormalities of prefrontal cortex, but they
may also reflect the dysfunction of brain areas with projections to prefrontal cortex. Given the known role of subcortical networks
as modulators of prefrontal functioning, the term frontosubcortical seems appropriate for ADHD. This term denotes a behavioral or
cognitive dysfunction that looks “frontal” but may be influenced by subcortical projections.

The neuropsychological findings in ADHD provide a fertile resource for speculations about the role of subcortical structures. For
example, the cingulate cortex influences motivational aspects of attention and in response selection and inhibition. The brainstem
reticular activating system regulates attentional tone and reticular thalamic nuclei filter interference. Working memory deficits
implicate a distributed network including anterior hippocampus, ventral anterior and dorsolateral thalamus, anterior cingulate,
parietal cortex, and dorsolateral prefrontal cortex. Moreover, the attentional problems of children with ADHD may implicate a wider
distribution of neural networks. A system mainly involving right prefrontal and parietal cortex is activated during sustained and
directed attention across sensory modalities. The inferior parietal lobule and superior temporal sulcus are polymodal sensory
convergence areas that provide a representation of extrapersonal space and play an important role in focusing on and selecting a
target stimulus.

Neuroimaging Studies
Fortunately, hypotheses based on neuropsychological inference can be tested with neuroimaging paradigms. Because neuroimaging
studies provide direct assessments of brain
 P.581

structure and function, they are ideal for testing hypotheses about the locus of brain dysfunction. Table 43.1 reviews 18 structural
neuroimaging studies of children, adolescents, and adults with ADHD that used computed tomography or magnetic resonance
imaging. Among these studies, the most consistent findings implicated frontal cortex, usually limited to the right side, cerebellum,
globus pallidus, caudate, and corpus callosum. Several other regions were less consistently implicated. Consistent with these
findings, the I/LnJ mouse strain shows total callosal agenesis along with behavioral features that resemble ADHD (61 ). These mice
show learning impairments, impulsiveness, and hyperactivity. Metabolic mapping studies suggest that their behavioral deficits are
associated with lower 2-deoxyglucose uptake in the left striatum and the frontal and parietal cortex (61 ).

TABLE 43.1. STRUCTURAL NEUROIMAGING STUDIES OF ADHD

Table 43.2 reviews 14 functional neuroimaging studies of ADHD using regional cerebral blood flow, positron emission tomography,
single photon emission tomography, functional magnetic resonance imaging, or electroencephalography. The most consistent
findings were hypoactivity of frontal cortex and subcortical structures, usually on the right side. Because Ernst et al. found
significant brain dysfunction for girls, but not boys, with ADHD (62 ), and Baving et al. found gender differences in lateralization
(63 ), future studies will need to assess gender differences and to determine how they may be related to the male predominance of
the disorder.

TABLE 43.2. FUNCTIONAL NEUROIMAGING STUDIES OF ADHD

Ernst et al. noted that findings of frontal hypoactivity are stronger in adult ADHD compared with adolescent ADHD (64 ). They
offered two explanations for this finding. First, the adolescent samples studied may have been more heterogeneous than the adult
samples. Although all the adults had persistent ADHD, some of the adolescent cases may have remitted by adulthood. Thus, frontal
dopaminergic hypoactivity may be associated with persistent ADHD only. Alternatively, Ernst et al. speculated that, because of brain
maturation, the locus of ADHD’s dopamine abnormality may shift from the midbrain in childhood to the prefrontal cortex in adults.

Anterior cingulate cortex, lying on the medial surface of the frontal lobe, has strong connections to dorsolateral prefrontal cortex.
Bush et al. used a Stroop task to compare anterior cingulate cortex activation in adults with ADHD and those who did not have ADHD
(65 ). In contrast to controls, the adults with ADHD failed to activate the anterior cingulate cortex. Notably, in the prior study by
Zametkin et al. (66 ), cingulate cortex was one of only four (of 60) regions evaluated that still showed regional hypoactivity after
global normalization.

The neurochemical basis of brain dysfunction in ADHD was studied by Dougherty et al. (67 ). They measured DAT density by single
photon emission computed tomography with the radiopharmaceutical iodine 123–labeled altropane. Their findings were consistent
with the catecholamine hypothesis
 P.582

of ADHD in showing the DAT to be elevated by about 70% in adults with ADHD.

The functional studies are consistent with the structural studies in implicating frontosubcortical system in the pathophysiology of
ADHD. Taken together, the brain imaging studies fit well with the idea that dysfunction in frontosubcortical pathways occurs in
ADHD. They are also consistent with the report of a father and son, both having methylphenidate-responsive ADHD secondary to
frontal lobe epilepsy (68 ). Notably, the frontosubcortical systems that control attention and motor behavior are rich in
catecholamines, which have been implicated in ADHD by the mechanism of action of stimulants.

In a novel approach to assessing brain regions implicated in ADHD, Herskovits et al. used magnetic resonance imaging to assess the
spatial distribution of lesions in children who developed ADHD after closed-head injuries (69 ). Compared with head-injured children
who did not develop ADHD, the children with ADHD had more lesions in the right putamen and a trend for more lesions in the right
caudate nucleus and right globus pallidus.

Very little is known about when ADHD-related brain abnormalities emerge. To address this issue, Nopoulos et al. assayed four brain
abnormalities believed to occur before birth: neural migration anomalies, corpus callosum agenesis or partial agenesis, enlarged
cavum septi pellucidi, and malformations of the posterior fossa (70 ). Neural migration anomalies and malformations of the posterior
fossa were more common among patients with ADHD compared with control subjects. Both these abnormalities were rare. However,
given that several other studies showed partial agenesis of the corpus callosum or anomalies of the cerebellar vermis (also formed
before birth), it seems reasonable to conclude that at least some children with ADHD have a very early onset of brain abnormalities.

GENETICS
Part of "43 - Pathophysiology of Attention-Deficit/Hyperactivity Disorder "

Family Studies
Figure 43.1A shows rates of hyperactivity among the siblings of hyperactive probands (71 ,72 ,73 ,74 and 75 ). Figure 43.1B shows an
elevated prevalence of ADHD among mothers and fathers of children with ADHD that provides further support for the familiality of
the disorder and evidence that the adult diagnosis is valid. These studies leave no doubt that ADHD
 P.583

is familial. Moreover, studies of more distant relatives are consistent with this idea as well (76 ).

FIGURE 43.1. ADHD in relatives of ADHD and controls children. A: ADHD in siblings. B: ADHD in fathers. C: ADHD in mothers.

Family studies of ADHD suggest that its psychiatric comorbidities may help to clarify its genetic heterogeneity. The
Harvard/Massachusetts General Hospital (Boston) ADHD family project studied two independent samples of children with attention-
deficit disorder (ADD) as defined by the DSM-III (74 ) and ADHD as defined by the DSM-III-R (77 ). These data show that (a) ADHD and
major depression share common familial vulnerabilities (78 ,79 ), (b) children with ADHD who have conduct (80 ,81 ) and bipolar
(82 ,83 ) disorders may comprise a distinct familial subtype of ADHD, and (c) ADHD is familially independent of anxiety disorders (84 )
and learning disabilities (85 ). Thus, stratification by conduct and bipolar disorders may cleave the universe of children with ADHD
into more familially homogeneous subgroups. In contrast, major depression may be a nonspecific manifestation of different ADHD
subforms. In a sample of 132 ADHD sib-pair families, Smalley et al. reported further evidence that ADHD with conduct disorder is a
distinct subtype (86 ). These investigators also examined comorbidity with learning disability, but these data produced equivocal
results.

Faraone et al. proposed that stable or persistent ADHD may be a useful subtype of ADHD for genetic studies (87 ). These
investigators reasoned that cases that remit before adolescence could have a smaller genetic component to their disorder than
persistent cases. Evidence supporting this hypothesis derives from several studies. In a prospective follow-up study, Biederman et al.
showed that by midadolescence, 85% of boys with ADHD continued to have ADHD; 15% remitted (88 ). The prevalence of ADHD
among parents was 16.3% for the persistent ADHD probands and 10.8% for the remitted ADHD probands. For sibs, the respective
prevalences were 24.4% and 4.6%. Thus, these data suggest that children with persistent ADHD have a more familial form of ADHD
than those whose ADHD remits by adolescence.

Consistent with this finding, Biederman et al.(188 ) showed that children of parents with clinically referred, childhood-onset, ADHD
were at high risk of meeting diagnostic criteria for ADHD: 84% of the adults with ADHD who had children had at least one child with
ADHD, and 52% had two or more children with ADHD (89 ). The 57% rate of ADHD among children of adults with ADHD was much
higher than the more modest 15% risk for ADHD in siblings of referred children with this disorder. These findings were consistent
with a prior study by Manshadi et al. (72 ). They studied the siblings of 22 alcoholic adult psychiatric patients who met DSM-III
criteria for ADD, residual type. The authors compared these patients with 20 patients matched for age and comorbid psychiatric
diagnoses. Forty-one percent of the siblings of the adult ADD probands were diagnosed with ADHD compared with 0% of the non-
ADHD comparison siblings.

In another retrospective study, Biederman et al.(188 ) compared adolescents with ADHD having retrospectively reported childhood-
onset ADHD with children with ADHD (90 ). These investigators found that the relatives of adolescent probands had higher rates of
ADHD compared with the relatives of child probands. Thus, a prospective study of children and retrospective studies of adolescents
and adults suggested that, when ADHD persists into adolescence and adulthood, it is highly familial. This idea is consistent with one
of Ernst’s explanations for the finding that frontal dopaminergic hypoactivity is stronger in adult ADHD compared with adolescent
ADHD; that is, frontal dopaminergic hypoactivity may be associated with persistent ADHD.

Twin and Adoption Studies

Although family studies provide much useful information, they cannot disentangle genetic from environmental sources of
transmission. To do so, we must turn to twin and adoption studies. There are two types of twins: identical or monozygotic twins
share 100% of their genes in common. In contrast, fraternal or dizygotic twins are no more genetically alike than siblings and
therefore share only 50% of
 P.584

their genes. Thus, the occurrence of twinning creates a natural experiment in psychiatric genetics (91 ). If a disorder is strongly
influenced by genetic factors, then the risk to co-twins of ill probands should be greatest when the twins are monozygotic. The risk
to dizygotic twins should exceed the risk to controls but should not be greater than the risk to siblings.

Twin data are used to estimate heritability, which measures the degree to which a disorder is influenced by genetic factors.
Heritability ranges from zero to one, with higher levels indicating a greater degree of genetic determination. Figure 43.2 presents
heritability data from 11 twin studies of ADHD. These data attribute about 80% of the origin of ADHD to genetic factors.

FIGURE 43.2. Heritability of ADHD.

Goodman and Stevenson found the heritability of hyperactivity to be 64% (92 ,93 ). In a repeat analysis of these data, Stevenson
reported that the heritability of mother-reported activity levels was 75%, and the heritability of a psychometric measure of
attention was 76% (94 ). In a study of ADHD in twins who also had reading disability, Gilger et al. estimated the heritability of
attention-related behaviors as 98% (95 ). In a study of 288 male twin pairs, Sherman et al. examined inattentive and impulsive-
hyperactive symptoms using both mother and teacher reports (96 ). Within both raters, the heritability of the impulsivity-
hyperactivity dimension exceeded that of the inattention dimension; however, mothers’ ratings showed higher heritability than did
teachers’ ratings. Specifically, mothers’ ratings produced a heritability of 91% for impulsivity and hyperactivity and 69% for
inattention. Teachers’ ratings yielded a heritability of 69% for impulsivity and hyperactivity and 39% for inattention. Using the Child
Behavioral Checklist as a dimensional measure, Hudziak and colleagues found a similar heritability (60% to 68%) for mother-reported
attention problems (97 ).

Other studies of inattentive and hyperactive symptoms found a high heritability and minimal impact of the shared environment
(98 ,99 ). Rhee et al. examined gender differences in heritability using twin and sibling pairs from Australia (99 ). Specific genetic
and environmental influences were highly similar for boys and girls. Slight differences that emerged were related to more influence
of the shared environment in girls and some evidence genetic dominance in boys.

Several twin studies examined the genetic contribution to the comorbidity of ADHD and other disorders. Data from Gilger et al. (95 )
were consistent with a prior family study (85 ) in suggesting that ADHD and reading disability were genetically independent; however,
the existence of a genetically mediated subtype of both disorders could not be excluded. In contrast, two twin studies suggested
that ADHD and reading disability share some genes in common (100 ,101 ). That this relationship may be complex is suggested by the
report by Willicutt et al. of genetic overlap between reading disability and inattention but not between reading disability and
hyperactive impulsive symptoms (102 ).

Nadder et al. examined whether ADHD and comorbid conduct and oppositional defiant disorder symptoms shared genetic risk factors
(98 ). These investigators found that 50% of the correlation between the ADHD and comorbid conduct was the result of shared genes.
Similarly, the twin study of Silberg et al. found that genes influencing variation in hyperactivity scores were also responsible for
variation in conduct problems (103 ). Between 76% and 88% of the correlation between hyperactivity and conduct scores were
attributed to genes. These investigators concluded that the results were consistent with the existence of a biologically based group
of children who manifest both hyperactivity and conduct disturbances. Further evidence that the ADHD plus comorbid conduct
subgroup may be etiologically meaningful comes from a study showing differences in serotonergic functioning between aggressive
and nonaggressive children with ADHD (104 ).

Like twinning, adoption provides another useful experiment for psychiatric genetics (91 ). Whereas parents can confer a disease risk
to their biological children by both biological and environmental pathways, to adoptive children they can confer risk only by an
environmental pathway. Thus, by examining both the adoptive and the biological relatives of ill probands, we can disentangle
genetic and environmental sources of familial transmission.

Adoption studies of ADHD also implicate genes in its origin. The adoptive relatives of children with ADHD are less likely to have ADHD
or associated disorders than are the biological relatives of children with ADHD (105 ,106 ). Biological relatives of children with ADHD
also do more
 P.585

poorly on standardized measures of attention than do adoptive relatives of children with ADHD (107 ).

Segregation Analysis Studies

Segregation analysis provides evidence of genetic transmission by demonstrating that the pattern of illness in families is consistent
with known genetic mechanisms. An early approach to this was reported by Morrison and Stewart, who concluded that polygenic
inheritance was a likely mode of transmission for ADHD (108 ). Contrasting data were presented by Deutsch et al. (109 ). They found
preliminary evidence for a single dominant gene regulating the transmission of ADHD and minor physical anomalies in 48 families.
Similarly, Faraone et al. reported that the familial distribution of ADHD was consistent with the effects of a single major gene (75 ).
Similar results were since reported in a twin study by Eaves et al. (110 ) and in a pedigree study by Hess et al. (111 ). Consistent
findings also emerged from South America (112 ). Based on a sample of families from Colombia, the only models of inheritance that
could not be rejected were those of dominant and codominant major gene effects. Finally, when families of ADHD probands were
ascertained by the father’s diagnosis of substance abuse, Maher et al. found that a sex-dependent mendelian codominant model was
the best explanation for the pattern of transmission of ADHD (113 ).

Although the segregation analyses of ADHD suggest that a single gene of major effect is involved in the origin of ADHD, the
differences in fit among genetic models was modest. This was especially true for the comparison of multifactorial and single gene
inheritance. Several interpretations of these results are possible. If ADHD had more than one genetic cause, then the evidence of
any single mode of transmission could be relatively weak. Alternatively, ADHD may be caused by several interacting genes of modest
effect. This latter idea is consistent with ADHD’s high population prevalence (2% to 7% for ADHD) and high concordance in
monozygotic twins but modest recurrence risks in first-degree relatives.

The studies by Deutsch et al. and Faraone et al. predicted that only about 40% of children carrying the putative ADHD gene would
develop ADHD. This finding and other features of the genetic epidemiology of ADHD suggest that such a gene likely interacts with
other genes and environmental factors to produce ADHD. Moreover, the segregation studies indicated that about 2% of people
without the ADHD gene would develop ADHD, a finding suggesting that nongenetic forms of ADHD may exist.

Chromosomal Anomalies and Molecular Genetic Studies

Anomalies in the number or gross structure of chromosomes usually lead to very early-onset disorders having severe clinical
manifestations (e.g., mental retardation, gross physical anomalies). No systematic studies of gross chromosomal anomalies in ADHD
have been conducted, but there are several reports that such anomalies cause hyperactivity and inattention. Examples include the
fragile X syndrome, duplication of the Y chromosome in boys, and loss of an X chromosome in girls. These associations are intriguing
but rare. Thus, they can account for only a very small proportion of cases of ADHD.

Molecular genetic studies use the methods of linkage and association to search for aberrant genes that cause disease. Such studies of
ADHD are relatively new and far from definitive. Hauser et al. demonstrated that a rare familial form of ADHD is associated with
generalized resistance to thyroid hormone, a disease caused by mutations in the thyroid receptor-β gene (114 ). The thyroid
receptor-β gene cannot, however, account for many cases of ADHD because the prevalence of generalized resistance to thyroid
hormone is very low among patients with ADHD (1 in 2,500) (115 ), and, among pedigrees with generalized resistance to thyroid
hormone, the association between ADHD and the thyroid receptor-β gene has not been consistently found (116 ).

Several research teams have examined candidate genes in dopamine pathways because, as discussed earlier, animal models,
theoretic considerations, and the effectiveness of stimulant treatment implicate dopaminergic dysfunction in the pathophysiology of
this disorder. Several groups have reported an association between ADHD and dopamine D4 receptor gene (DRD4) gene
(117 ,118 ,119 ,120 ,121 ,122 and 123 ). Notably, each study showed the 7-repeat allele of DRD4 to be associated with ADHD despite
the use of different diagnostic systems (DSM-IIIR and DSM-IV) and measures of ADHD (rating scales and structured interviews).
However, like many findings in psychiatric genetics (91 ), these positive findings are offset by some negative studies
(124 ,125 ,126 ,127 and 128 ).

The positive DRD4 findings could be caused by another gene in linkage disequilibrium with DRD4 or another variant within DRD4.
However, because the DRD4 7-repeat allele mediates a blunted response to dopamine, it is a biologically reasonable risk factor for
ADHD (129 ). The 7-repeat allele has also been implicated in novelty seeking, a personality trait related to ADHD (130 ,131 ).
Moreover, both norepinephrine and dopamine are potent agonists of DRD4 (132 ).

When the D4 gene is disabled in a knockout mouse model, dopamine synthesis increases in the dorsal striatum, and the mice show
locomotor supersensitivity to ethanol, cocaine, and methamphetamine. (133 ). D4 knockout mice also show reduced novelty-related
exploration (134 ), a finding consistent with human data suggesting a role for D4 in novelty-seeking behaviors.

Cook et al. reported an association between ADHD and the 480-bp allele of the DAT gene using a family-based association study
(135 ). This finding was replicated by Gill et al. (136 ), Daly et al. (126 ), and Waldman et al. (137 ), but
 P.586

not in other studies (124 ,138 ). In the study by Waldman et al. (137 ), hyperactive-impulsive symptoms but not inattentive
symptoms were related to the number of DAT high-risk alleles. Further support for a link between the DAT gene and ADHD comes
from a study that relates this gene to poor methylphenidate response in children with ADHD (139 ) and from the neuroimaging study
(Table 43.2 ) showing that DAT activity in the striatum is elevated by 70% in adults with ADHD (67 ).

In mice, eliminating DAT gene function leads to several features suggestive of ADHD: hyperactivity, deficits in inhibitory behavior,
and a paradoxical response to stimulants (i.e., stimulants reduce hyperactivity) (37 ,140 ). Studies of this knockout mouse model
show the potential complexities of gene–disease associations. The loss of the DAT gene has many biological effects: increased
extracellular dopamine, a doubling of the rate of dopamine synthesis (141 ), decreased dopamine and tyrosine hydoxylase in
striatum (142 ), and a nearly complete loss of functioning of dopamine autoreceptors (143 ). Because ADHD is believed to be a
hypodopaminergic disorder, the decreased striatal dopamine may be most relevant to the disorder.

Gainetdinov et al. showed that enhancement of serotonergic transmission mediates the mouse’s paradoxical response to stimulants
(37 ). These researchers attributed this to the effects of stimulants on the serotonin transporter. To complicate matters further,
Bezard et al. showed that DAT knockout mice did not experience MPTP-induced dopaminergic cell death (144 ), and another study
found a gradient effect such that mice with zero, one, and two functional DAT genes showed increasing susceptibility to MPTP (145 ).
These latter findings suggest that individual differences in the DAT gene may mediate susceptibility to neurotoxins having an affinity
for the DAT.

A population-based association study has also implicated the A1 allele of the dopamine D2 receptor gene in ADHD (146 ). Absence of
the D2 gene in mice leads to significantly reduced spontaneous movements, a finding suggesting that D2 plays a role in the
regulation of activity levels (147 ,148 ). Mice without D2 genes also show decreased striatal DAT functioning (149 ), a finding that
illustrates the potential effects of gene–gene interaction on simple phenotypes such as locomotion in mice. In addition, Calabresi et
al. used the D2 knockout mouse to study the role of the D2 receptor in striatal synaptic plasticity (150 ). In these mice, these
researchers found abnormal synaptic plasticity at corticostriatal synapses and long-term changes in synaptic efficacy in the striatum.

The only human study of the D3 receptor gene found no evidence of an association with ADHD (151 ). However, homozygous mice
lacking D3 receptors displayed increased locomotor activity, and heterozygous mice showed less pronounced hyperactivity. These
results led Accili et al. to conclude that D3 receptors play an inhibitory role in the control of certain behaviors (152 ).

Four human studies of ADHD have examined the catechol-O-methyltransferase (COMT) gene, the product of which is involved in the
breakdown of dopamine and norepinephrine. Although one study found that ADHD was associated with the Val allele (153 ), others
have found no association between the COMT polymorphism and ADHD in Irish (154 ), Turkish (155 ), and Canadian (156 ) samples.
Despite the negative finding, the positive finding is intriguing because the Val allele leads to high COMT activity and an increased
breakdown of catecholamines.

Another study found an association with the DXS7 locus of the X chromosome, a marker for monoamine oxidase that encode enzymes
that metabolize dopamine and other neurotransmitters (157 ). Finally, Comings and colleagues found associations and additive
effects of polymorphisms at three noradrenergic genes (the adrenergic α2A, adrenergic α2C, and dopamine-β-hydroxylas) on ADHD
symptoms in a sample of patients with Tourette syndrome (158 ), but they found no association between the tyrosine hydroxylase
gene and ADHD in this sample (159 ).

Some investigators have used the coloboma mouse model to investigate the genetics of ADHD. These mice have the coloboma
mutation, a hemizygous, 2-centimorgan deletion of a segment on chromosome 2q. The mutation leads to spontaneous hyperactivity
(which is reversed by stimulants), delays in achieving complex neonatal motor abilities, deficits in hippocampal physiology that may
contribute to learning deficiencies, and deficits in Ca2+-dependent dopamine release in dorsal striatum (160 ).

The coloboma deletion region includes the gene encoding SNAP-25, a neuron-specific protein implicated in exocytotic
neurotransmitter release. Hess et al. suggested that interference with SNAP-25 may mediate the mouse’s hyperactivity (161 ). As
predicted by this hypothesis, when these investigators bred a SNAP-25 transgene into coloboma mice, the animals’ hyperactivity was
reduced. Moreover, other work suggested that reduced SNAP-25 expression leads to striatal dopamine and serotonin deficiencies,
which may be involved in hyperactivity (162 ).

Hess et al. tested the idea that the human homologue of the mouse coloboma gene could be responsible for ADHD by completing
linkage studies of families with ADHD by using markers on human chromosome 20p11-p12, which is syntenic to the coloboma
deletion region (111 ). These investigators used five families for which segregation analysis suggested that ADHD was the result of a
sex-influenced, single gene. However, no significant linkage was detected between ADHD and markers on chromosome 20p11-p12.

ENVIRONMENTAL RISK FACTORS

Part of "43 - Pathophysiology of Attention-Deficit/Hyperactivity Disorder "

Although genetic studies of ADHD unequivocally show that genes are risk factors for the disorder, they also show
 P.587

that the environment has a strong influence on the emergence of the disorder. This conclusion follows from studies of identical
twins, which show that when one twin has ADHD, the probability of the other, genetically identical, twin’s having ADHD is only
about 60%. This less than perfect identical twin concordance implicates environmental risk factors. The nature of these risk factors
has emerged from studies assessing features of the biological and psychosocial environment that may increase the risk of ADHD.

Biological Adversity
The idea that certain foods could cause ADHD received much attention in the popular press after claims were made that ADHD could
be cured by eliminating food additives from the diet. The Feingold diet for ADHD was popularized by the media and was accepted by
many parents of ill children. Systematic studies, however, showed the diet was not effective and concluded that food additives do
not cause ADHD (163 ). Another popular theory posited that excessive sugar intake would lead to ADHD symptoms. Although some
positive studies supported this idea, the bulk of systematic, controlled research did not (164 ).

In contrast to the mostly negative studies of dietary factors, some toxins have been implicated in the origin of at least some cases of
ADHD. Several groups have shown that lead contamination leads to distractibility, hyperactivity, restlessness, and lower intellectual
functioning (165 ). However, many children with ADHD do not show lead contamination, and many children with high lead exposure
do not develop ADHD. Thus, lead exposure cannot account for the bulk of cases of ADHD.

The literature examining the association of ADHD with pregnancy and delivery complications (PDCs) presents conflicting results; it
tends to support the idea that PDCs can predispose children to ADHD (166 ,167 and 168 ), although some investigators do not (169 ).
The PDCs implicated in ADHD frequently lead to hypoxia and tend to involve chronic exposures to the fetus, such as toxemia, rather
than acute, traumatic events, such as delivery complications.

For example, Conners reported that mothers of children with ADHD had high rates of toxemia during pregnancy (166 ). Hartsough
and Lambert described eight PDCs associated with ADHD: maternal illness, toxemia, eclampsia, older maternal age, parity of child,
fetal postmaturity, duration of labor, and fetal distress during labor or birth (170 ). Nichols and Chen found that hyperactivity was
significantly associated with low birth weight (171 ), and Chandola et al. reported antepartum hemorrhage, maternal age, length of
labor, sex, and 1-minute Apgar scores to be significant prenatal and perinatal risk factors for subsequent referral for hyperactivity
(172 ).

Sprich-Buckminster et al. showed that the association between ADHD and PDCs was strongest for children with ADHD who had
psychiatric comorbidity (168 ). PDCs were also elevated among children with ADHD who had no family history of ADHD. These
investigators concluded that PDCs may be more common among those children with ADHD having a weaker genetic predisposition,
but this hypothesis was not confirmed in another study by the same group (167 ). The latter study found that children with ADHD
and a history of PDCs showed more school failure and psychometric evidence of cognitive impairment than other children with ADHD.
In addition to confirming the etiologic role of medical complications, this study showed that psychosocial stress during pregnancy
predicted subsequent ADHD and poor cognitive performance in children. Notably, catecholamines are secreted in response to stress,
and mouse studies showed that catecholamine administration produces uterine vasoconstriction and fetal hypoxia (173 ).

One extensively studied risk factor has been maternal smoking during pregnancy. By exposing the fetus to nicotine, maternal
smoking can damage the brain at critical times in the developmental process. The smoking mother is at increased risk of antepartum
hemorrhage, low maternal weight, and abruptio placentae (173 ). Her fetus is at risk of low birth weight (173 ,174 ), and because
smoking increases carboxyhemoglobin levels in both maternal and fetal blood, it places the fetus at risk of hypoxia (175 ).
Consistent with these effects, maternal smoking during pregnancy predicts behavioral and cognitive impairment in children and
ADHD (41 ,176 ).

Animal studies in pregnant mice and rats have shown a positive association between chronic exposure to nicotine and hyperactivity
in offspring (42 ). Neonatal nicotine exposure prevents the development of low-affinity nicotine receptors (177 ), and chronic
exposure results in tolerance to the drug and an increase in brain nicotinic receptors (178 ,179 ,180 and 181 ). Because nicotinic
receptors modulate dopaminergic activity and dopaminergic dysregulation may be involved in the pathophysiology of ADHD, it is
theoretically compelling to consider maternal smoking as a risk factor for ADHD.

Little is known about the potential role of in utero exposure to viral infections. Because maternal viral infections can affect the
fetus and can have an adverse impact on the developing brain, viral infections could be associated with later psychopathology.
Because viral infections occur more commonly in winter than in other seasons, season-of-birth data have been used to implicate in
utero viral infection for several disorders including schizophrenia (182 ), autism (183 ), and dyslexia (184 )

Although Mick et al. found no evidence of a strong seasonal pattern of birth in children with ADHD (185 ), they did find statistically
significant peaks for September births in children with ADHD who had comorbid learning disabilities and in children with ADHD who
had no additional psychiatric comorbidity. Thus, it is possible that winter infections during the first trimester of pregnancy may
account
 P.588

for some subtypes of ADHD. Mick et al. found no evidence favoring the idea that putative viral exposure led to a nonfamilial form of
ADHD. In contrast, they found a weak trend toward an increase in winter births for children with ADHD who have a positive family
history of ADHD. If replicated, this finding suggests that a seasonally mediated infection at birth may be an environmental “trigger”
for the genetic predisposition to the disorder.

Psychosocial Adversity
The delineation of psychosocial features in the child’s environment associated with more impaired outcome in children with ADHD
has potentially important clinical, scientific, and public health implications. Such efforts can help to identify etiologic risk factors
associated with more impaired outcome in ADHD and can characterize early predictors of persistence and morbidity of this disorder.
Moreover, finding environmental risk factors for ADHD could help to design improved preventive and therapeutic intervention
programs.

The classic studies by Rutter et al. of the Isle of Wight and the inner borough of London provide a compelling example of how
psychosocial risk factors influence child psychopathology (186 ). Compelling examples of how psychosocial risk factors affect child
psychopathology, these studies examined the prevalence of mental disorders in children living in two very different geographic areas.
This research revealed six risk factors within the family environment that correlated significantly with childhood mental
disturbances: (a) severe marital discord, (b) low social class, (c) large family size, (d) paternal criminality, (e) maternal mental
disorder, and (f) foster placement. This work found that it was the aggregate of adversity factors, rather than the presence of any
single one, that impaired development. Other studies also found that as the number of adverse conditions accumulated, the risk of
impaired outcome in the child increased proportionally (187 ). Biederman et al. found a positive association between Rutter’s index
of adversity and ADHD, measures of ADHD-associated psychopathology, impaired cognition, and psychosocial dysfunction (188 ).

Other cross-sectional and longitudinal studies have identified variables such as marital distress, family dysfunction, and low social
class as risk factors for psychopathology and dysfunction in children. For example, the Ontario Child Health Study in Canada showed
that family dysfunction and low income predicted persistence and onset of one or more psychiatric disorders over a 4-year follow-up
period (189 ). Other work implicated low maternal education, low social class, and single parenthood as important adversity factors
for ADHD (171 ,190 ). These studies suggested that the mothers of children with ADHD had more negative communication patterns,
more conflict with their children, and a greater intensity of anger than did control mothers.

Biederman et al. showed that long-term conflict, decreased family cohesion, and exposure to parental psychopathology, particularly
maternal psychopathology, were more common in ADHD-affected families compared with control families (191 ). The differences
between children with ADHD and control children could not be accounted for by either socioeconomic status or parental history of
major psychopathology. Moreover, increased levels of family-environment adversity predicted impaired psychosocial functioning.
Measures indexing long-term family conflict showed a more pernicious impact on the exposed child than those indexing exposure to
parental psychopathology. Indeed, marital discord in families has consistently predicted disruptive behaviors in boys (192 ). This
research shows that the extent of discord and overt conflict, regardless of whether the parents are separated, predicts the child’s
risks of psychopathology and dysfunction (193 ).

Thus, dysfunctional family environments appear to be a nonspecific risk factor for psychiatric disorders and psychological distress.
Reid and Crisafulli reported a metaanalysis of the impact of marital discord on the psychological adjustment of children and found
that parental conflict significantly predicted a variety of child behavior problems (194 ). The Ontario Child Health Study provided a
prospective example of the impact of parental conflict on children’s mental health: family dysfunction (and low income) predicted
persistence and onset of one or more psychiatric disorders over a 4-year period (189 ).

Low maternal warmth and high maternal malaise and criticism were previously associated with ADHD in children (195 ), and an
epidemiologic study examining family attributes in children who had undergone stressful experiences found that children’s
perceptions of mothers, but not fathers, differentiated stress-resilient and stress-affected children (196 ).

An extensive literature documents maternal depression as a risk factor for psychological maladjustment and psychiatric disorder in
children (197 ). This is consistent with the known familial link between ADHD and depression (79 ). Some investigators have
suggested that depressed mood may lead mothers to perceive their children as more deviant than warranted by the child’s behavior.
Richters, however, reviewed 22 studies of this issue and concluded that, owing to methodologic problems with research in the area,
there was no empiric foundation for this claim (198 ).

Other data revealed a link between maternal depression and child functioning that was independent of the mother’s perceptions.
These data suggested that depressed mothers accurately perceive symptomatic behavior but react to it in a negative manner that
worsens the condition of the child. This conclusion was echoed by Gelfand and Teti (197 ). Their comprehensive review of relevant
literature found many studies to document the assertion that depressed mothers have attitudes of insensitivity, disengagement,
disapproval, and hostility toward their children. They also
 P.589

found maternal depression to be associated with undesirable parenting practices such as intrusiveness, unresponsiveness, and inept
discipline. In addition, their review supported the idea that depressed mothers had negative perceptions of their children.

Other work shows that ADHD in children predicts depression in mothers, but maternal depression provides no additional information
for predicting ADHD in siblings of ADHD probands. This finding suggests that maternal depression is a heterogeneous disorder. It may
be that some mothers have a disorder that is genetically linked to ADHD, whereas others may experience depression resulting from
the stress of raising a child with ADHD (and perhaps living with an ADHD-affected or antisocial husband). Furthermore, it is possible
that maternal depression exacerbates family conflict and poor parenting, both of which could exacerbate ADHD symptoms.

Notably, although many studies provide strong evidence of the importance of psychosocial adversity for ADHD, these factors tend to
emerge as universal predictors of children’s adaptive functioning and emotional health, not predictors that are specific to ADHD.
Thus, they can be conceptualized as nonspecific triggers of an underlying predisposition or as modifiers of the course of illness.

SUMMARY AND CONCLUSIONS

Part of "43 - Pathophysiology of Attention-Deficit/Hyperactivity Disorder "

It is not yet possible to describe the origin and pathophysiology of ADHD completely. Nevertheless, converging evidence from the
studies reviewed in this chapter supports several empiric generalizations, which should be useful in guiding future research and
theory.

Catecholamine Hypothesis
Much research supports the idea that catecholaminergic systems mediate the onset and expression of ADHD symptoms. The key data
supporting this idea are as follows: (a) anti-ADHD medications have noradrenergic and dopaminergic effects; (b) lesion studies in
mouse and monkey models implicate dopaminergic pathways; (c) the SHR rat shows deficits in catecholaminergic systems; (d) D2, D3,
and D4 knockout mice studies show that these genes regulate locomotor activity; and (e) human studies implicate the DRD4 and DAT
genes in the origin of ADHD.

Although the role of catecholamine systems cannot be disputed, future work must also consider other neurotransmitter systems that
exert upstream effects on catecholamines. Two prime candidates are nicotinic and serotonergic systems. Nicotinic agonists help to
control the symptoms of ADHD, and nicotinic activation enhances dopaminergic neurotransmission. Serotonergic drugs have not been
shown to be effective anti-ADHD agents, but knockout mice studies suggest that the paradoxical effects of stimulants on
hyperactivity are mediated by serotonergic neurotransmission. Moreover, SNAP-25, which has been implicated in studies of the
coloboma mouse, leads to striatal dopamine and serotonin deficiencies. These data call for further studies of serotonergic and
nicotinic systems.

Brain Systems
Several types of study provide information about the locus of ADHD’s pathophysiology in the brain: neuropsychological studies,
neuroimaging studies, and animal models. Taken together, these studies support the idea that ADHD arises from the dysregulation of
frontal cortex, subcortical structures, and networks connecting them. This idea fits with the pharmacotherapy of ADHD because a
plausible model for the effects of stimulants is that, through dopaminergic or noradrenergic pathways, these drugs increase the
inhibitory influences of frontal cortical activity on subcortical structures.

Additional data supporting frontal-subcortical involvement in ADHD are as follows: (a) neuropsychological studies implicate
orbitofrontal and dorsolateral prefrontal cortex or regions projecting to these regions; (b) the monkey model of ADHD implicates
frontal-striatal neural networks; (c) studies of the SHR rat implicate caudate, putamen, nucleus accumbens, and frontal cortex;
patients with frontal lobe damage show ADHD-like behaviors; (d) structural neuroimaging implicates frontal cortex, usually limited
to the right side, cerebellum, globus pallidus, caudate, and corpus callosum; (e) the I/LnJ mouse strain shows total callosal agenesis
along with behavioral features that resemble ADHD; (f) functional neuroimaging finds hypoactivity of frontal cortex, anterior
cingulate cortex, and subcortical structures, usually on the right side; (g) ADHD secondary to brain injury shows lesions in right
putamen, right caudate nucleus, and right globus pallidus; (h) disabling the D4 gene in mice leads to increased dopamine synthesis
in dorsal striatum; (i) mice without D2 genes also show decreased striatal DAT functioning, abnormal synaptic plasticity at
corticostriatal synapses, and long-term changes in synaptic efficacy in the striatum; and (j) the coloboma mouse shows deficient
dopamine release in dorsal striatum.

Etiologic Factors
In a word, the origin of ADHD is complex. Although rare cases may have a single cause such as lead exposure, generalized resistance
to thyroid hormone, head injury, and frontal lobe epilepsy, most cases of ADHD are probably caused by a complex combination of
risk factors.

From the many twin studies of ADHD, we know for certain that genes mediate susceptibility to ADHD. Molecular genetic studies
suggest that two of these genes may be the DRD4 gene and the DAT gene. To confirm these findings, we need much more work
because, even if the positive
 P.590

studies are correct, they may implicate neighboring genes instead of those targeted by the studies. It seems unlikely that a single
“ADHD gene” causes ADHD with certainty. Instead, it seems likely that several genes act together to form the genetic substrate of
the disorder.
When the ADHD-related variants of these genes are discovered, they will probably be “normal” variants and will most certainly not
have the devastating effects seen in knockout mouse models. For example, suppose future work confirms that the 7-repeat allele is
a risk factor for ADHD. We would consider this a normal variant because about 20% of people who do not have ADHD carry this
version of the DRD4 gene. Most of these people do not develop ADHD despite the blunted dopaminergic transmission associated with
that allele, and many patients with ADHD do not carry the allele. Thus, the 7-repeat allele cannot be a necessary or sufficient cause
of the disorder. Instead, it acts in concert with other genes and environmental risk factors to bring forth ADHD.
Like genetic studies, studies of environmental risk factors suggest that most of these risks exert small but significant influences on
the origin of ADHD. For example, most children with a history of PDCs do not develop ADHD, and most children with ADHD do not
have a history of ADHD. Nevertheless, research suggests that such complications are more common among children with ADHD.
These considerations lead us to conclude that the origin of ADHD is multifactorial. A simple multifactorial model posits ADHD to
arise a pool of genetic and environmental variables—each of small effect—that act in concert to produce vulnerability to ADHD. If a
person’s cumulative vulnerability exceeds a certain threshold, he or she will manifest the signs and symptoms of ADHD. According to
the multifactorial model, no single factor is a necessary or sufficient cause for ADHD, and each of the etiologic factors is
interchangeable (i.e., it does not matter which factors one has; only the total number is important). Whether risk factors combine
in an additive or interactive manner is unknown.
The mouse models of ADHD we described provide examples of multifactorial causation in a simple system. One model showed that
individual differences in the DAT gene could directly produce a hypodopaminergic state; these studies showed that dopamine
transporter variants differ in their affinity for neurotoxins. Thus, dopamine transporter abnormalities could interact with
environmental toxins to produce hyperactivity. Another line of work shows that catecholamines are secreted in response to stress,
and catecholamine administration produces fetal hypoxia. Human studies implicate both stress during pregnancy and fetal hypoxia
as risk factors for ADHD.
These simple examples suggest that unraveling the complexities of multifactorial causation will be a difficult task for ADHD
researchers. However, because technological developments in neuroscience and molecular genetics are moving at a rapid pace, the
next decade of work should provide us with more accurate assessments of the brain along with a complete sequence of the human
genome. These advances should set the stage for breakthroughs in our understanding of the neurobiology of ADHD and in our ability
to treat affected persons.

DISCLAIMERS
Part of "43 - Pathophysiology of Attention-Deficit/Hyperactivity Disorder "
Dr. Biederman receives research support from Shire Laboratories, Gliatec, Cephalon, Novartis Pharmaceuticals, and Eli Lilly &
Company. In addition, he serves on speaking bureaus for SmithKline Beecham, Eli Lilly & Company, and Pfizer Pharmaceuticals.

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44
Learning Disorders

C. Keith Conners

Ann C. Schulte

C. Keith Conners: Behavioral Neurology Department, Durham, North Carolina.

Ann C. Schulte: Department of Psychology, North Carolina State University, Raleigh, North Carolina.

 NOSOLOGY AND CLASSIFICATION

 PREVALENCE
 COMORBIDITY
 PHONOLOGIC PROCESSING
 GENETICS
 NEUROIMAGING
 EDUCATIONAL MANAGEMENT
 PSYCHOPHARMACOLOGY
 SUMMARY AND CONCLUSIONS

NOSOLOGY AND CLASSIFICATION

Part of "44 - Learning Disorders "

Current conceptualizations of learning disorders (LDs), formerly referred to as “academic skills disorders” (1 ), follow the traditional
approach of classifying learning by specific academic skills. These skills include reading, mathematics, and written expression. In
each case, the skills are measured by standardized tests whose scores must fall substantially below the level expected from
chronologic age, intelligence, and age-appropriate education. The deficits must significantly interfere with academic or daily living
activities requiring the skills. When LDs result from sensory, medical, or neurologic conditions, they are coded on Axis III (medical
conditions) within the DSM-IV nomenclature.

Commonly associated features of LDs include low selfesteem and demoralization, social skills deficits, school dropout, and
difficulties in employment or social adjustment. Patients with conduct disorder, oppositional disorder, attention-
deficit/hyperactivity disorder (ADHD), major depression, dysthymic disorder, and Tourette syndrome all have substantially elevated
rates of LD. Academic skills in pervasive developmental disorders are often not discrepant from the measured intelligence and
language abilities associated with the pervasive development disorder. Communication disorders and motor skills disorders are also
common in LDs, including expressive language disorders, phonologic disorder, and stuttering. Spelling disorders are usually not
considered separate from other reading- and writing-related deficits.

Although this approach to classification of LDs is useful in a practical context and allows for an operational definition for detection
and remediation, it has several drawbacks from a theoretic and scientific point of view. Reading, mathematics, and writing comprise
many processing skills, giving rise to subtypes with different underlying mechanisms. Thus, reading at the word level may involve
visual, lexical, or semantic processes (2 ,3 ), with correspondingly different neuroanatomic circuitry and computational mechanisms
within the brain. There are subtypes characterized both by the pattern of skills deficits (e.g., reading and spelling, but no
mathematics disorder) and by different patterns of neuropsychological function, such as the relative strength of verbal and
nonverbal factors on intelligence tests (4 ). There are also important developmental changes in LD, such that variables
characterizing the disorder at earlier ages may be different from those seen in older patients (5 ). Advances in the genetics and
neuroimaging of LDs will depend on more homogeneous clinical definitions at the symptomatic level (6 ).

PREVALENCE
Part of "44 - Learning Disorders "

The DSM-IV reports prevalence estimates of 2% to 10% for LDs, depending on the nature of ascertainment and the definitions applied
(1 ). In most prevalence studies, a diagnosis of LD has been made on the basis of a significant discrepancy between IQ and
achievement in one or more areas (7 ), with studies varying in terms of the manner in which a discrepancy has been determined and
the cutoff score for considering a discrepancy “severe.” One study of the prevalence of regression-based ability/achievement
discrepancies using the co-norming sample from the Wechsler Intelligence Scale for Children III and the Wechsler Individual
Achievement Scales found that 17% of the norming group had ability/achievement discrepancies at the .05 significance level in one
or more areas of achievement (8 ). This figure can probably be considered the upper limit for LD prevalence estimates based on
ability/achievement discrepancies, given that a diagnosis of LD would also require determining both that the discrepancy was not
the result of poor instruction and that it interfered with daily functioning.

Several researchers have questioned the conceptual and empiric basis for the use of ability/achievement discrepancies in the
diagnosis of LDs, as well as current operationalizations of the exclusionary criteria. Reasons for concern on
 P.598

the use of ability/achievement discrepancies are (a) findings that the cognitive profiles of children with low achievement are similar
regardless of whether they evidence an ability/achievement discrepancy (9 ), (b) findings that the same deficits that lead to poor
achievement may also lower IQ (10 ), and (c) the finding that the use of such definitions prevents early identification and treatment
because the underlying cognitive deficits that cause the disability must retard growth in academic skills before intervention can
begin (11 ). Alternate proposals for identification include simply using a low achievement criterion (e.g., academic functioning 1 to
2 standard deviations [SD] below the mean), using a definition that combines the ability/achievement discrepancy and low
achievement approaches, and use of domain-specific rather than general cognitive ability tests as predictors of academic
achievement. In general, these alternate procedures are likely to raise prevalence rates.

There is some indication that more rigorous operationalization of the exclusionary criteria in the LD definition could substantially
reduce LD prevalence rates. For example, when Vellutino and his colleagues used daily tutoring as a “first cut” diagnostic criterion
to distinguish between children who had reading difficulties caused by cognitive deficits and those whose deficits were the result of
poor instruction, they found that two thirds of their sample scored within the average range in reading (thirtieth percentile and
higher) after one semester of one-to-one tutoring (12 ). This relatively stringent criterion for establishing an “adequate educational
environment” resulted in a drop in the prevalence rate of reading disorders (RDs) from 9% to 3%. Geary used failure to respond to
short-term intensive remedial instruction as a diagnostic criterion for mathematics disability (MD) and noted a marked drop in
prevalence (13 ). Clearly, the definition of caseness in these studies has implications for how phenotypes are characterized in
genetic and neurobiological investigations. The use of the more conservative methods of case definition are clearly more costly for
selecting subjects, but they may prove more valid and useful in finding biological markers of LD.

COMORBIDITY
Part of "44 - Learning Disorders "

Many psychiatric and medical conditions include LD as an associated deficit. The most common childhood condition comorbid with
LD is ADHD. Estimates of comorbidity range from 20% to 90%, with the lower figures appearing in epidemiologic samples and the
higher figures appearing in clinically referred samples. The high degree of overlap in clinical samples suggests that common
mechanisms may be at work in the neurologic basis for both disorders. LDs were once considered a necessary criterion for minimal
brain dysfunction. Although some studies suggest that ADHD may simply be the result of an LD, most studies indicate that when both
conditions are present, characteristics of each are found, whereas in LDs alone, only symptoms of LD, not those of ADHD, are
present, and vice versa (14 ). The high degree of overlap has the practical implication that when one disorder is identified, it is
always prudent to expect the presence of the other and to make appropriate diagnostic probes.

PHONOLOGIC PROCESSING
Part of "44 - Learning Disorders "

As noted earlier, the present classification approach to LD subdivides the disorder on the basis of impairment in specific academic
areas (reading, math, written expression). However, given that performance in each of these area draws on numerous cognitive
processes, it is likely that the present classification system will eventually be replaced by one that focuses on the specific cognitive
deficits that underlie poor academic performance and their impact on the development and execution of specific subskills within
and across academic areas.

The greatest progress in specifying the cognitive and neuropsychological dysfunctions underlying LDs has occurred in reading.
Numerous investigations using longitudinal, intervention, genetic, and neuroimaging methods have produced strong and converging
evidence that deficits in phonologic processing are the proximal cause of reading difficulties in a large proportion of children with
RDs (see ref. 15 and ref. 16 for reviews). Deficits in phonologic processing also appear to affect spelling, written expression, and
mathematics.

Phonologic processing refers to the ability to use and manipulate the sound structure of one’s oral language (17 ). Although
conceptualizations of phonologic processing and its components vary, within the Wagner and Torgesen model of phonologic
processing, it consists of three related abilities: phonologic awareness, phonologic memory, and rapid naming (18 ,19 ). Phonologic
awareness refers to the understanding that words can be broken down into phonemes and the ability to identify phonemes and
manipulate them in words (16 ). Phonemes are the smallest sound unit that changes the meaning of a word (e.g., tap and lap differ
by one phoneme). Phonologic awareness is a critical ability in learning to read because it allows beginning readers to link letters and
letter combinations in text to sound strings in oral language (20 ). Knowledge of these links allows readers to discover the
regularities in written text so written words can be rapidly translated into their spoken equivalents. Such recoding allows the reader
to access the semantic code (or meaning) for the letter string. The repeated pairing of the visual letter string and its spoken
equivalent is thought eventually to allow the reader to develop direct visual word recognition strategies that bypass the phonologic
code (10 ,21 ).

Phonologic memory is an individual’s ability to represent verbal information in working memory in terms of a sequence
 P.599

of sounds or a phonetic code. When children have difficulty with phonologic coding, reading acquisition is impaired because of
difficulty in performing the rapid comparison and blending needed to identify unfamiliar written words. Difficulties in verbal short-
term memory are also hypothesized to be a major factor underlying MDs (22 ), and they may affect the acquisition of foreign
languages (20 ).

Rapid naming is the ability to access phonologic information that is stored in long-term memory rapidly. It is typically assessed by
asking children to name well-known items as rapidly as possible (e.g., presentation of a series of colored squares with the child
naming the color of each square as fast as possible). Such tasks are thought to tap many of the same cognitive processes required in
skilled reading, such as rapid scanning, sequencing and processing of serially presented visual stimuli, and rapid access to strings of
phonemes (e.g., color names) (16 ). There is debate about whether the difficulty with rapid naming tasks observed in many children
with RDs is a reflection of a core deficit in phonologic processing or whether it represents a deficit in a second set of processes that
impairs reading. If this is the case, there may be “double-deficit” readers who are impaired in both phonologic and rapid naming
processes (23 ). Such disabled readers would be less responsive to interventions that address phonologic processing and would
require additional interventions targeted toward increasing language and reading fluency.

Efforts are also under way to understand more fully the core cognitive deficits underlying other types of LD. For example, Berninger
et al. proposed a model of the cognitive processes underlying written language and writing disabilities (24 ). Geary proposed that
there are three subtypes of MDs, with corresponding deficits in semantic memory, procedural knowledge of mathematics, and
visuospatial processes (22 ).

GENETICS
Part of "44 - Learning Disorders "

It has been known for decades that LDs run in families. In the 1990s, family aggregation studies, twin studies, and genetic linkage
analyses confirmed the strong hereditary influences on RD and MD (Table 44.1 ). The genetic studies also confirm the heterogeneity
of the phenotype, with both orthographic and phonologic traits implicated but not having identical sources of genetic influence. A
genetic link between RD and MD was confirmed in several studies. A strong link of Tourette syndrome, ADHD, and LD has been
suggested by studies of patients who have Tourette syndrome with and without ADHD. Evidence has accumulated that locations on
the short arm of chromosome 6
P.600
 P.601

(6p21.3) and the short arm of chromosome 15 are involved. Odds for linkage to chromosome 15 are reported as being 1,000 to 1,
with evidence that 30% of an extended series of families showed linkage to chromosome 15 polymorphisms. Some variations in
results may reflect sampling methods or trait markers. The excess of affected males with LDs identified in clinic and referred
samples disappears in research-based samples (25 ).

TABLE 44.1. GENETICS OF LEARNING DISORDERS

NEUROIMAGING
Part of "44 - Learning Disorders "

The neuroanatomic and functional pathways in the brain involved in LDs were greatly clarified in the 1990s by a variety of
neuroimaging techniques. Reviews of neuroimaging of LDs describe rapid progress in identifying the brain regions and functional
pathways involved (20 ,26 ,27 ,28 and 29 ). However, these reviews also call attention to discrepancies in findings, possibly the
result of small cohorts, variations in sampling, and heterogeneity of the LDs. Table 44.2 provides selected studies from several
hundred investigations, mainly of RDs. Many studies confirm earlier findings of abnormalities of the microstructure of the planum
temporale from autopsy studies, but conflicting data emerge, possibly related to the method employed or the sampling techniques
and definition of the RD (30 ). Although most studies implicate abnormalities in left temporal-parietal anatomic areas, additional
findings have identified white matter, right hemisphere anomalies, motor cortex, cingulate gyrus, and the splenium of the corpus
callosum.
 P.602

TABLE 44.2. NEUROIMAGING IN LEARNING DISORDERS

P.603
 P.604

One of the older controversies regarding the functional brain basis of dyslexia is whether dyslexia represents a visual (orthographic)
disorder or a language-based (phonologic processing) disorder. Neuroimaging studies now appear to provide evidence that brain
structures involving both the
 P.605

striate visual magnocellular pathways and specific phonologic processing pathways in the left hemisphere are involved in dyslexia, a
finding possibly reflecting different subtypes at the behavioral level. As noted earlier, cognitive behavioral analysis suggests that
distinctive mechanisms for visual, lexical, and semantic processing are required to explain normal human reading (2 ,3 ). Pathologic
studies indicate that each of these mechanisms can be affected separately in acquired dyslexias. For example, in deep dyslexia, it is
primarily the semantic aspects of reading that are disturbed, whereas orthography and lexicality are preserved. Thus, a patient may
read “spirit” as “whiskey,” or “church” as “priest.” Evidence suggests that, unlike the more typical left-hemisphere–based
phonologic and visual deficits in dyslexia, deep dyslexia may reflect a right-hemisphere—based processing mechanism (31 ).

Whereas some investigators interpret functional magnetic resonance imaging studies as giving strong support to the hypothesis that
dyslexia represents a disorder of the language system, involving the segmentation and synthesis of phonemes (20 ), others find
evidence that magnocellular pathways without involvement of phonologic regions occur in dyslexia (32 ,33 ). As noted by Filipek,
cognitive neuroscience identifies specific computational tasks that should be used to provide more homogeneous samples at the
behavioral level for further advances in the neurobiology of developmental disorders (28 ). For example, rather than using classic
clinical criteria for dyslexia, which leads to samples with diverse subtypes, neuroimaging studies may do better to select samples by
visual, lexical, and semantic criteria first.

EDUCATIONAL MANAGEMENT
Part of "44 - Learning Disorders "

Various educational treatments have been developed for LD. In general, the most effective treatment approach is one that involves
careful delineation of the specific academic deficits evidenced by the child and intensive instruction in the skill areas in which
deficiencies are documented (34 ). Response to treatment varies by individuals, so it is important that careful monitoring take place
throughout treatment to ensure that an intervention is effective for a particular child (35 ). In this section, we briefly summarize
the educational treatment literature by academic area and then summarize research related to treatment monitoring or formative
evaluation of interventions.

Reading
Considerable progress has been made in the development of preventive and early intervention approaches for beginning readers.
Several studies have demonstrated that explicit instruction in phonologic awareness (generally combined with letter identification
and reading instruction) in preschool and early elementary years can reduce the overall rate of RDs (36 ,37 ) and can improve
outcomes for children who are at high risk of RD (38 ,39 ). One metaanalysis reported a combined effect size for phonologic
awareness training of 1.16 for phonologic awareness skills and .40 for reading skills across studies that used samples of normal
readers and .54 and .60 for studies that used samples of students who were either at risk of, or had shown evidence of, reading
difficulty (40 ).

The difference in training effect on phonologic awareness between normal and impaired readers appears to reflect the difficulty
many poor readers have in mastering phonologic processing, even when they are provided with intensive instruction to address these
difficulties (40 ). Torgesen examined results from five large-scale early reading intervention studies and concluded that even with
use of the best current methods of early reading remediation, 2% to 6% of children would still evidence inadequate reading skills in
the early elementary grades (41 ). Such findings point to the need for the development of even more powerful intervention
techniques to facilitate the acquisition of early reading skills.

Current models of reading skill acquisition characterize phonologic awareness as a necessary, but not sufficient condition for the
development of skilled reading (15 ). Fluent reading requires the development of orthographic reading skills or the ability to
recognize words by sight (41 ). Impaired readers generally show deficits in this area that persist into adulthood (41 ,42 ).
Interventions to improve fluency are less well developed than interventions for the development of decoding skills (i.e., phonologic
awareness interventions). The repeated readings technique, which involves multiple readings of the same passages, is the most
researched approach to improving fluency (43 ), and it has shown limited but positive effects on fluency (44 ). The increased
attention to issues of fluency in reading research has resulted in the development of new, comprehensive intervention approaches
that ultimately may be more effective than existing techniques in addressing fluency deficits (23 ). At present, however, fluency
deficits remain one of the most persistent and intransigent symptoms of RD (20 ).

Although most children with RDs show deficits in word recognition skills, comprehension deficits are also common. These may occur
alone or in the presence of impaired word recognition skills (45 ). When impaired word recognition is the primary source of the
comprehension deficit, decoding and fluency interventions such as those discussed earlier can improve reading comprehension (46 ).
However, interventions have also been developed to address comprehension deficits directly. Two metaanalyses found substantial
improvements for disabled readers who receive intensive instruction in reading comprehension (47 ,48 ). In both studies,
metacognitive approaches (e.g., self-questioning, comprehension monitoring) produced the largest effect sizes.
 P.606

Math
Geary characterized research in the area of MDs as “primitive” in comparison with studies of RDs (22 ). Nevertheless, effective
remediation techniques for MDs have been developed. Mastropieri et al. presented a comprehensive review of mathematics
instructional techniques that have been effective for students with LDs (49 ). However, Cawley et al. questioned the efficacy of
available math computation instructional techniques (50 ). In a metaanalysis of math word problem interventions, Xin and Jitendra
found that instruction in problem representation was an effective remedial strategy for addressing this type of difficulty in children
with a range of mild disabilities (51 ). These investigators also found that problem representation instruction was most effective
when it was presented in a computer-assisted format. Long-term interventions (i.e., more than 1 month) resulted in better
maintenance and generalization of training.

Written Expression
Difficulties with composition and writing fluency are common in children with LDs. Several researchers have shown that cognitive
strategy instruction is effective in improving the composition skills of children with written language deficits (52 ,53 and 54 ).
Generally, such interventions provide students with explicit instruction in thinking and problem-solving strategies that allow them to
break down the complex task of composing written text into manageable substeps.

Difficulties with handwriting fluency appear not only to impair the speed with which children can take notes or copy but also to
affect compositional fluency and quality (55 ). For example, Berninger et al. found that instruction in handwriting increased
students’ scores on a writing composition test (56 ).

With more widespread use of computers in classrooms, word processing tools are increasingly being used to address the writing
problems of children with LDs (57 ). When writing fluency is a problem, word processing may be used as a text entry strategy on its
own, or it can be combined with word prediction programs (58 ). Voice recognition software has improved to the point that it may
be a practical text entry strategy for many students with writing disabilities (59 ). However, research on the efficacy of these tools
remains sparse. In one of the few studies to compare the efficacy of different word processing strategies for improving writing
fluency, accuracy, and composition in students with LDs, Lewis et al. compared groups of students after a year of writing instruction
using either keyboarding, keyboarding with word prediction software, or keyboarding with word prediction and synthesized speech
software (60 ). All groups using word processing tools showed decreases in speed of text entry over handwriting, although the
keyboarding with text prediction group showed the smallest decrease. There were no improvements in composition skills in any of
the treatment groups.

Treatment Monitoring
The unexpected results of the foregoing study by Lewis et al. reinforce the need to monitor response to treatment and to verify that
interventions for children with LDs achieve their intended results. Curriculum-based measurement (CBM) is a relatively new
development in special education and provides a useful tool for continuous monitoring of children’s response to treatment in a
number of academic areas (61 ,62 ,63 and 64 ). CBM involves the collection of brief samples of students’ performance on basic skills
on a weekly or monthly basis. For example, CBM procedures in reading involve the administration of short reading probes (e.g.,
passages of 200 words) to children once or twice per week. The number of correct responses per passage is charted, and slope is
then used as an indicator of a child’s response to treatment. Slopes that do not differ from zero are an obvious indicator of the need
for a new treatment approach. However, estimates of typical response to treatment for students with LDs are also available and can
be used as a basis for deciding whether a given treatment is producing sufficient progress (65 ). When formative evaluation
strategies such as CBM are incorporated into treatment strategies, outcomes for students with disabilities improve markedly (66 ).

PSYCHOPHARMACOLOGY
Part of "44 - Learning Disorders "

Psychostimulants
Early studies of psychostimulants in children with LDs suggested strong immediate effects in enhancing reading, spelling, and
arithmetic as well as in laboratory measures of learning (67 ,68 ,69 ,70 and 71 ). However, reviews concluded that lasting
educational gains resulting from psychotropic drugs have not been demonstrated (72 ,73 ). Stimulant drug effects have generally
been dose related, with linear increases in performance with higher doses (74 ,75 ,76 and 77 ). Drug-induced changes reflect
increased output, accuracy, efficiency, and improved learning acquisition. There is also evidence of increased effort and self-
correcting behaviors (78 ). Some studies suggest a positive effect of stimulants on memory consolidation that is not accounted for by
concomitant effects on acquisition (79 ). Because most studies involve students with comorbid ADHD, measures of specific effects of
stimulants on LDs are rare. However, because improvement in learning acquisition occurs in both clinical cases and neurologically
normal persons treated with amphetamine (80 ), it seems likely that stimulant effects on learning are nonspecific with respect to
diagnosis.

Stimulants have been widely used in rehabilitation of memory and LDs in brain injuries and encephalopathies
 P.607

secondary to medical X-irradiation of the brain. Animal models of selective exposure to X-irradiation during infancy show enhanced
learning from amphetamine treatment (81 ).

Nootropics
Piracetam (Nootropil, Nootropyl, 2-oxo-l-pyrrolidone acetamide) was originally developed as a molecular analogue of γ-aminobutyric
acid (GABA) for the purpose of altering vestibular function in motion sickness, but it is probably neither a GABA receptor agonist nor
antagonist. Numerous analogues of the piracetam molecule are currently under study, including oxiracetam (Neuromet),
pramiracetam, etiracetam, nefiracetam, aniracetam, and rolziracetam. This group of nootropics is commonly referred to as the
“racetams.” Piracetam has virtually no detectable peripheral effects at any dose in animals or humans and does not affect cerebral
blood flow, unlike other putative cerebral enhancers. It appears to alter cellular brain metabolism, however, because it increases
the concentration ratio of brain adenosine triphosphate. In neurologically normal volunteers, a single dose of piracetam was found
to change brain global functional state as measured by multichannel electroencephalographic recordings (82 ). Investigators have
suggested that the defining characteristics of nootropics should include lack of peripheral effect, absence of action on blood flow,
and an increase in brain metabolism (83 ).

Animal research indicates that memory deficits induced by epileptogenic kindling procedures are prevented by pretreatment with
piracetam (84 ). Piracetam (100 mg/kg, IP) and oxiracetam (10 mg/kg, IP) prevented the negative effects of microwaves on memory
processes in exposed rats (85 ). Hypobaric hypoxia of pregnant rats is followed by the reduction of weight gain of the newborn pups,
delayed impairment of memory (passive and active tasks), and changes of extrapolative water escape. Piracetam (200 mg/kg/d)
administered at early postnatal period (from the eighth to the twentieth day of life) corrected behavioral disturbances and physical
development in rats (86 ). Piracetam (800 mg/kg) administered orally once daily for 5 days before training completely antagonized
the scopolamine-provoked amnesia in step-through–trained mice, and piracetam (600 mg/kg) administered orally once daily for 5
days before training abolished the memory-impairing effect of clonidine in shuttle-box–trained rats and the amnestic effect of
methergoline in step-down–trained rats.

Early studies by Dimond and Brouwers suggested that piracetam could facilitate transfer of information across the callosal pathways
and hence is a “superconnector” drug (87 ). Numerous studies with neurologically normal and dyslexic adults indicated that the drug
could enhance verbal learning. These studies were reviewed by Wilshire (88 ). An early report on reading involved 16 dyslexic men
matched with 14 student volunteers for a 21-day trial of piracetam. It was found, using a double-blind crossover technique, that the
dyslexic men significantly increased their verbal learning by approximately double that of control students (89 ). Early uncontrolled
trials with a broader group of LDs were followed by a series of systematic studies of learning, memory, and reading (90 ).

Studies of 60 dyslexic boys 8 to 14 years old, who were carefully selected for exclusion of intellectual, sensory, psychiatric, and
neurologic impairment and educational deprivation, were conducted to determine the efficacy of piracetam, over a 12-week period,
in improving reading and other related skills (91 ). There were no changes at the end of 12 weeks to distinguish the groups in
accuracy or comprehension of prose reading. Short-term memory gains, however, were recorded for the treated group on two
different tests, digit span, and a test (Neimark) of immediate and delayed recall. The mean digit span scaled score for the entire
group was 1 SD below their mean IQ. Considering only the performance of children whose digit span scaled scores were 1 SD or
below the mean (7 or less), the treated group made a significant gain at the end of 12 weeks. On the Neimark test, the treated
group was significantly superior to the untreated group on first trial learning, and they also lost significantly fewer object names
after a delay. Improved retrieval from long-term storage could be demonstrated for the treated group on the rapid automatized
naming test. Although there was no significant difference between the groups at screening, the treated group was significantly
faster on letter naming at the end of the drug trial. The treated group also improved their single word reading on the Wide Range
Achievement Test (WRAT).

After previous research suggested that piracetam improves performance on tasks associated with the left hemisphere, a 12-week,
double-blind, placebo-controlled study of developmental dyslexics was conducted. Six study sites treated 257 dyslexic boys between
the ages of 8 and 13 years who were significantly below their potential in reading performance. The children were of at least normal
intelligence, had normal findings on audiologic, ophthalmologic, neurologic, and physical examination, and were neither
educationally deprived nor emotionally disturbed. Piracetam was found to be well tolerated in this study population. Children
treated with piracetam showed improvements in reading speed. No other effects on reading were observed. In addition,
improvement in auditory sequential short-term memory was observed in those piracetam-treated patients who showed relatively
poor memory at baseline (92 ).

Piracetam was given in a 3,300-mg daily dose to half of a group of 55 dyslexic boys aged 8 to 13 years, in a 12-week, double-blind,
placebo-controlled study. The other half of the subjects received placebo. Compared with the placebo control group, the boys
treated with piracetam did not show statistically significant improvements above their baseline scores on measures of perception,
memory, language, reading accuracy or comprehension, or writing accuracy. However, reading speed and numbers of words written
in a timed
 P.608

period were significantly enhanced in subjects treated with piracetam as compared with placebo. Effective reading and writing
ability, taking both rate and accuracy into consideration, were also significantly improved in the piracetam group as compared with
the placebo treatment group (93 ).

Two hundred twenty-five dyslexic children between the ages of 7 years 6 months and 12 years 11 months whose reading skills were
significantly below their intellectual capacity were enrolled in a multicenter, 36-week, double-blind, placebo-controlled study.
Piracetam-treated children showed significant improvements in reading ability (Gray Oral Reading Test) and reading comprehension
(Gilmore Oral Reading Test). Treatment effects were evident after 12 weeks and were sustained for the total period (36 weeks)
(94 ).

The neurophysiologic mechanisms involved in the effects of piracetam were examined in studies using event-related potentials.
Eight- to 12-year-old dyslexic boys were randomly assigned to 3.3 g of piracetam or matching placebo per day in two divided doses
over a 12-week period. Children performed a vigilance task in which they pressed a key when two alphabetic letters or shapes
occurred in sequence. Event-related potentials to letters and shapes, for active and passive responses, were recorded at the vertex
and left and right parietal areas of the scalp. Performance measures included letter and form hits, misses, commission errors, and
reaction times. Piracetam increased the amplitude of a late positive component (believed to correspond to P300) at the vertex for
letter hits. Piracetam also increased the latency of this component in both hemispheres, but only for active responses (letter hits) in
the left hemisphere and passive responses (correct rejections and misses) in the right hemisphere. Reaction time to letter hits was
significantly correlated with the latency of the P300 component, a finding suggesting that letters created increased effort or
attentional demand on the subjects compared with forms. An early event-related potential component (P225) also showed increased
amplitude to piracetam in both hemispheres, and effects were limited to form hits. These effects were thought possibly to reflect
slow negative potentials arising from stimulus anticipation in the CNV-like paradigm. The results were cautiously interpreted as
indicating a facilitation of verbal processing mechanisms responsible for analyzing the verbal significance of visual stimuli (95 ).

In a subsequent study, 29 dyslexic children (aged 7 to 12 years) were assigned to piracetam or matching placebo for 36 weeks.
Event-related potentials were obtained at the end of treatment from a vigilance paradigm that required a response to letter or form
matches. The drug group showed a significant advantage in letter hits compared with placebo and a reduced variance in reaction
time. The drug increased the amplitude of three factors from a principal components analysis of event-related potentials and was
interpreted as increasing a processing negativity when stimuli were letters. Piracetam was interpreted as enhancing feature analysis
and increasing attentional resources among dyslexic children when the stimuli are recognized as having linguistic significance (96 ).
These effects were shown to be dose related in a subsequent study (97 ).

One negative study examined the interaction of piracetam and tutoring (98 ). Sixty children with dyslexia (41 boys, 19 girls; ages 9
to 13 years) were enrolled in a 10-week summer tutoring program that emphasized word-building skills. They were randomly and
blindly assigned to receive either placebo or piracetam. The children were subtyped as “dysphonetic” or “phonetic” on the basis of
scores from tests of phonologic sensitivity and phoneme-grapheme correspondence skills. Of the 53 children who completed the
program, 37 were classified as dysphonetic and 16 as phonetic. The phonetic group improved significantly more in word-recognition
ability than the dysphonetic group. Overall, the children taking medication did not improve more than the nonmedicated children in
any aspect of reading. However, within the medication-treated group, the phonetic subgroup gained most in word recognition.

SUMMARY AND CONCLUSIONS

Part of "44 - Learning Disorders "

Significant difficulties continue to bedevil the definition of LD, including problems surrounding various criteria such as an
IQ/learning discrepancy or low absolute achievement level. Approaches that define the disorder by resistance to high-quality
instruction may be the most valid for purposes of identifying persons with LDs in genetic, neuroimaging, and pharmacologic studies.
The high degree of comorbidity with many psychiatric disorders raises further issues for studies requiring a homogeneous symptom
pattern, and it seems likely that further advances will require replacing broad clinical patterns with more specific processing
deficits based on cognitive neuroscience. Despite these limitations, existing research is encouraging regarding the possibility of
precise genetic and neuroanatomic localization of LDs, particularly for RDs. Again, however, subtyping issues at the phenotypic level
require elucidation before further progress is likely.

Much of the pharmacologic work has been confounded by the comorbidity of LD with ADHD and other childhood disorders. Evidence
generally supports the finding that psychostimulants (e.g., dextroamphetamine and methylphenidate) have positive effects on
immediate learning performance but less impact on long-term academic gains. Work with nootropic drugs shows intriguing effects
on verbal learning, single-word reading, and left-hemisphere processing of alphabetic stimuli. Good controlled trials indicate that
piracetam may be a safe and effective enhancer of reading in school-aged children, with gains double the rate expected in seriously
impaired readers. LD remains a large public health problem, is significantly undertreated, has devastating lifetime outcomes, and
therefore merits greater
 P.609

research efforts to understand its neurobiology and treatment needs.

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functional and clinical significance. Brain Res Brain Res Rev 1999;29:26–49.
 P.613

45
Psychosis in Childhood and Its Management

Paramjit T. Joshi

Kenneth E. Towbin

Paramjit T. Joshi: Division of Behavioral Medicine, Department of Psychiatry and Behavioral Sciences, George Washington
University School of Medicine Children’s National Medical Center, Washington, DC.

Kenneth E. Towbin: Complex Developmental Disorders Clinic, Department of Psychiatry and Behavioral Sciences, George
Washington University School of Medicine Children’s National Medical Center, Washington, DC.

The appearance of psychotic symptoms in childhood, albeit rare, is an important clinical entity. This importance extends beyond
their clinical prevalence and has begun to influence our understanding of the principal psychotic conditions. The term psychosis is
generally categoric and includes subgroups within it. It is clear that the peak onset of the most common psychotic disorders,
schizophrenia and bipolar disorder, is in adolescence (1 ,2 ). This points directly toward developmental events in biological, social,
and psychological domains of late childhood and adolescence that set the stage for activating psychotic disorders. However, in
addition, it appears increasingly likely that certain early childhood characteristics and developmental deficits may presage psychosis
and are related to the outcome of psychotic disorders.

For the purposes of this chapter, psychosis is defined as the presence of disruptions in thinking, accompanied by delusions or
hallucinations, along with an alteration in the thought processes, termed a thought disorder. Delusions and hallucinations are
considered to be positive psychotic symptoms. Delusions are fixed, false, idiosyncratic beliefs that the child cannot be deterred
from, with logical reasoning, whereas hallucinations are percepts that arise in the absence of external sensory stimulus. Psychotic
symptoms always encompass a broad range of conditions, but it is particularly so when they appear in children and adolescents.
Psychotic symptoms in children present distinctive diagnostic and clinical challenges because of the powerful influences of
immaturity and the moving target produced by development.

Although there may at one time have been confusion about whether children are capable of having psychotic symptoms, it is now
certain that children, like adults, can and do experience psychoses (i.e., disruptions in the form of mental life). Children and
adolescents experience the same range and types of psychotic symptoms as do adults. They can lose the connections between their
thoughts (formal thought disorder) and have perceptions without external stimuli (hallucinations). The term psychosis as described
by McHugh and Slavney (3 ) is intended simply to indicate that mental life has been disrupted in its capacities or forms, as a result
of a process that generates new forms of psychological experience.

Psychotic symptoms can be considered as general or nonspecific phenomena emerging with different disorders and etiologic
possibilities. Modern psychiatry eschews the misleading dichotomy of functional versus organic causes and recognizes that some of
these disorders stem from known brain or metabolic disorders, whereas for other conditions the pathophysiologic sources have yet
to be discovered. The interplay between environmental and biological forces is at work across the spectrum of these conditions. The
psychiatrist who must determine whether a young patient suffers from a psychotic disorder faces a challenging array of possibilities,
more extensive than when the patient is an adult. The influences of development, environment, and cognition are greater for young
or developmentally immature patients than for adults. Nonbiological events are clearly more influential because, in most respects,
children are more vulnerable to their surroundings. Immaturity makes children more susceptible to environmental stressors and
cognitive distortions. Children routinely have intrusions of fantasy into ordinary mental life; determining when this becomes
pathologic can be a matter of degree. Children learn and experiment with imitation, and they can acquire habits and strategies used
by those around them. They have not developed the cognitive abilities that permit them to observe and compare their experiences
in an objective manner. The range of normal functioning is greater in childhood, so the child’s behavior may simply be a result of
immaturity, rather than a deviation from a normal pathway.
 P.614

With the advent of categoric classification of psychiatric disorders, the criteria for psychotic disorders have become more stringent,
and the concepts have been defined more narrowly. When one examines a 5-year old child who claims that he is “superman and can
fly,” the challenge is to determine whether the child has a delusion. Similarly, in a child who complains about hearing a voice telling
her to “do bad things,” one must determine whether she is talking about her conscience or is experiencing auditory hallucinations.
This must be distinguished from make-believe (e.g., having an imaginary friend). Children can describe this makebelieve
phenomenon, and clinicians need to discern the differences as they work with children with symptoms of psychosis. Such
characteristics are sought by the clinician in the child’s answers to particular questions. The task and challenge as child and
adolescent psychiatrists are to ask the right questions, to differentiate delusions and hallucinations from other forms of thought,
such as a vivid imagination in a young child.

 HISTORY
 COGNITIVE ASPECTS
 CLINICAL AND DEVELOPMENTAL CONSIDERATIONS
 MANAGEMENT AND TREATMENT
 CONCLUSIONS

HISTORY
Part of "45 - Psychosis in Childhood and Its Management "

Interest in childhood psychosis can be traced to the nineteenth century, when Maudsley first wrote a description of the “insanity of
early life” in 1874 in his textbook, Physiology and Pathology of Mind (4 ). He took a developmental approach by noting that the
mental faculty of children was not organized, and hence the insanity in children must be of the simplest kind, influenced more by
“reason of bad descent or of baneful influences during uterine life.” However, De Sanctis may be credited first with setting out
childhood schizophrenia as different from mental deficiency and from certain neurologic disorders, such as epilepsy or
postinfectious encephalopathy (5 ). It was not until 1919, that Kraeplin introduced the concept of dementia praecox and noted its
onset in late childhood and adolescence (6 ). Given the insidious onset of the disorder, Kraeplin cautiously suggested that 3.5% of
patients with schizophrenia had the onset of their illness before the age of 10 years. This led to an increased interest in
understanding the developmental aspects of psychosis. Historically, despite this early description of the syndrome by Kraeplin that is
now recognized as schizophrenia, other diagnostic terms were put forward as well. These included dementia praecossima (5 ) and
dementia infantilis (7 ). Potter offered clearer descriptions of schizophrenia, with consideration of the child’s developmental age,
and offered specific diagnostic criteria for children (8 ).

Despite efforts to recognize childhood schizophrenia as a distinct clinical entity, during the decades between 1920 and 1970, the
term childhood psychosis comprised all forms of severe mental disorders in children, including schizophrenia and autism. Kanner’s
description of early infantile autism catalyzed an alternative view of the conceptualization of these disorders.

Beginning with the works of Loretta Bender (9 ), Leo Kanner (10 ), and others (11 ), all considered childhood schizophrenia to fall
under the broader category of childhood psychoses. Nevertheless, there came an acknowledgment and new awareness of major
developmental differences in the perception of reality (12 ) and that developmentally or culturally appropriate beliefs (e.g.,
imaginary playmates and fantasy figures) did not, of themselves, suggest psychosis. This cluster of syndromes, including infantile
autism, was defined by developmental lags in the maturation of language, perception, and motility (11 ). Although psychotic speech
and thoughts were initially considered inherent components of childhood schizophrenia, hallucinations and delusions were not
required criteria (6 ,13 ,14 and 15 ). DSM-II adopted this nosology and grouped all childhood psychoses under childhood
schizophrenia. As a result of this broad grouping, the literature regarding childhood schizophrenia from this period overlaps with
that of autism and does not differentiate autism from other psychotic disorders. With further development of psychiatric taxonomy
and elucidation of the phenomenology (course, onset, family history, and associated features), the distinctiveness of the various
childhood psychoses and the similarity between child and adult schizophrenia were demonstrated (16 ,17 ). This change had a
pronounced influence on the nosology of these disorders and led eventually to changes with the DSM-III (18 ). Schizophrenia arising
in childhood and infantile autism came to be recognized as distinct clinical syndromes, each with its unique and distinct
psychopathologic phenomenology, theories about causes, and longitudinal course. Research since the advent of DSM-III generally
validated this decision (19 ,20 ). This distinction has had an impact on how children with these disorders are currently evaluated,
managed, and treated.

COGNITIVE ASPECTS
Part of "45 - Psychosis in Childhood and Its Management "

Although children do not describe disorders, they nevertheless may complain of changes in their mental and cognitive states. To
these changes, clinicians add signs, based on observations derived from the mental state examination of the children and data
obtained from laboratory or cognitive tests. Subsequently, a distinctive pattern may emerge over the course of the child’s illness. A
collection of symptoms and signs occurring in a certain temporal pattern is then used to categorize the child’s problem. Psychotic
symptoms can be attributed to distinct mental illnesses (functional psychoses), which are contrasted with the psychotic symptoms
that usually result from a demonstrable underlying pathologic mechanism and organic origin (organic psychoses), such as delirium.
Cognitive impairments, particularly impaired concentration and ability to focus, usually accompany psychosis in children. However,
when the psychosis is secondary to an organic origin, there is often accompanying
 P.615

impairment in the sensorium presenting as confusion and disorientation, as is typical of delirium.

From a cognitive and developmental standpoint, certain clinical features in children create diagnostic challenges. One problem is
distinguishing true psychotic phenomena in children from nonpsychotic idiosyncratic thinking, perceptions caused by developmental
delays, exposure to disturbing and traumatic events, and overactive and vivid imaginations. Furthermore, because the onset of
childhood schizophrenia is insidious, with a lifelong history of developmental and personality abnormalities, differentiating between
the premorbid state and the active psychotic state can be difficult. It has also been suggested that the development of psychotic
conditions during childhood may have major adverse effects on development, a feature further complicating diagnostic assessment
(21 ).

Investigators have noted that social withdrawal, “shyness,” and disturbances in adaptive social behavior seem to be the first signs of
dysfunctional premorbid development. Eggers et al. suggested that these should be considered vulnerability factors, indicative of a
risk of psychotic illness (22 ). Recent work has also pointed to early language deficits and motor impairments as being significant for
very early-onset schizophrenia, in children younger than 12 years (23 ). However, a socially odd child is not usually schizophrenic. In
fact, most children who have hallucinations are not schizophrenic (24 ,25 and 26 ), because they lack the requisite persistence and
associated symptoms. Intellectual delays have long been considered as general risk factors for psychopathology and psychosis in
children (27 ). In fact, the estimated rates may be low, because most studies examining psychosis in children exclude patients with
mental retardation (28 ,29 ).

CLINICAL AND DEVELOPMENTAL CONSIDERATIONS

Part of "45 - Psychosis in Childhood and Its Management "

Developmental factors influence the detection, form, and context of psychotic symptoms in children. One problem of assessing
psychotic disorders in very young children compared with older children is that these symptoms in young children tend to be more
fluid and less complex. Isolated hallucinations can occur in acutely anxious but otherwise developmentally intact preschool children.
In older children, hallucinations may occur in the absence of other signs of psychosis, but they are usually associated with other
psychopathologic conditions, such as depression, severe anxiety, and posttraumatic stress disorder.

Often, there is an underestimation of the subtle differentiation among age-related cognitive preoccupations, pseudohallucinations,
and imaginative experiences. Further, it is often too difficult to tease out the physiognomic-animistic interpretations of the inner
and outer world on one hand and the first prominent psychotic phenomena such as delusions and hallucinations on the other.

It is critical to avoid rushing to a premature conclusion about unusual behaviors and beliefs in children. Such atypical mental
experiences in children can be recognized as prodromal or prepsychotic signs only after the manifestation of frank psychotic
symptoms. Odd beliefs and unusual behaviors deserve close observation, but they cannot be ascribed to psychosis without the
concomitant presence of a thought disorder.

For example, a young schizophrenic girl lived by the railroad tracks all her life. At age 11 years, about the time when her disorder
had its onset, she noted that the sound of the train whistle changed, and she began to wonder why. She came to believe that it had
a specific purpose and meaning—that it beckoned her. Until that time, such events were inconsequential and unimportant, but at
about age 11 years, she started to attach a different meaning to them. She was uncomfortable with these thoughts and realized that
it was not her usual pattern of thinking. Things around her started to have special meaning, her thoughts were “strange,” and she
was puzzled and bewildered.

This may be considered a predelusional phenomenon, idiosyncratic but not yet fixed. Over the next several years, she developed
ideas of reference, thought broadcasting, and thought insertion. She believed that the train whistle was sending special messages
from God to her. She no longer questioned these perceptions and believed them to be real. By age 14 years, she was diagnosed with
childhood-onset schizophrenia.

A formal thought disorder in a child is more ominous and requires careful psychiatric and neurologic evaluation. Distinguishing
between the formal thought disorder of schizophrenia and that of developmental disorders, personality disorders, and speech and
language disorders also presents diagnostic problems (30 ). Symptoms such as thought disorder have been noted to arise in persons
with pervasive developmental disorders, particularly those with good language skills, such as (often referred to as “high functioning”)
autistic persons and those with Asperger syndrome (31 ,32 ).

Although loose associations and incoherence are valid diagnostic signs of early-onset schizophrenia, these symptoms are also
sometimes seen in schizotypal children (33 ). The inclusion criteria of disorganized speech according to DSM-IV (34 ), rather than a
formal thought disorder, presents a particular challenge when assessing children, because disorganized speech is an inherent
component of many of the developmental disorders. Clearly, the assessment and ascertainment of delusions, hallucinations, and
thought disorder in linguistically impaired children are difficult and complicated.

Therefore, developmental disorders must be considered in the differential diagnosis of a child presenting with psychotic symptoms.
The use of comparable criteria across the age span facilitates analyses of progressive symptoms from
 P.616

childhood to adulthood. However, one of the difficulties in assessing psychotic disorders in very young children is to determine
whether nonspecific behavioral disturbances represent an incipient psychosis or are signs of autism or pervasive developmental
disorders (35 ,36 ).

Further, the conceptualization of psychoses in childhood as a neurodevelopmental disorder has drawn increasing attention,
especially as it relates to childhood-onset schizophrenia (37 ). Therefore, another alternative in the conceptualization of psychotic
episodes is a grouping of symptoms that are not part of the formal DSM or International Classification of Diseases (ICD) scheme. For
decades, clinicians recognized that a pattern of brief psychotic episodes, affective dysregulation, and poor social abilities occurs in
children. Early references on schizophrenia (17 ) and later writings (38 ,39 ) noted the diagnostic problem of children with poor
social development and psychosis. Now, the absence of a formal single diagnostic address for this syndrome has produced a wide
variety of terms applied to the same phenomena. The older literature suggested that such children may be considered to have
“borderline syndrome of childhood” (40 ), and then later “schizotypal disorder of childhood” was considered (38 ). In 1986, Cohen et
al. suggested the term multiplex developmental disorder and proposed that this condition was best understood as a developmental
deviation within the group of pervasive developmental disorders (41 ). Towbin and co-workers offered operational criteria and
preliminary validating evidence for the concept and criteria and used the term multiple complex developmental disorder (42 ).
Following Cohen et al., the view of Towbin and co-workers was that multiple complex developmental disorder was a higher-
functioning type of pervasive developmental spectrum disorder. Rather than pointing to one particular outcome, Towbin and
coworkers suggested that multiple complex developmental disorder was a nonspecific risk factor for a poor adaptation in adult life
but with a myriad of adult diagnostic outcomes such as schizophrenia, bipolar illness, or any of the more severe unstable personality
disorders. Further elaboration of these criteria have shown support for the concept and validated that children with pervasive
developmental disorder not otherwise specified and autism can be meaningfully separated from those with multiple complex
developmental disorder (43 ). Further exploration of the concept of multiple complex developmental disorder has received support
from neurophysiologic studies as well (44 ).

The National Institute of Mental Health (NIMH) project on early schizophrenia culled children for a study of clozapine. The most
common referrals were children whose symptoms closely resembled those of multiple complex developmental disorder. The NIMH
group suggested the term multidimensionally impaired (45 ,46 ) and offered criteria that were analogous to those described by
Towbin and co-workers. However, despite findings that many of these children met partial criteria for pervasive developmental
disorder not otherwise specified, the NIMH group preferred to consider the constellation a forme fruste of schizophrenia (46 ). Yet
longitudinal studies suggested that the constellation remains stable and does not progress to schizophrenia (46 ). Other work
reporting on similar children has used terms like such as “pervasive developmental disorder plus bipolar” (47 ) or “obsessive difficult
temperament” (48 ). As further exploration now points to “high rates of speech and language, motor, and social impairments in
patients with childhood-onset schizophrenia,” the association with pervasive developmental spectrum disorders is drawn even closer
for this very early-onset subgroup (23 ).

In addition to the developmental factors and disorders described earlier, the other differential diagnoses of childhood psychoses can
be classified as described in the following sections.

Functional Psychoses

Childhood-Onset Schizophrenia
Schizophrenic psychoses with onset before age 11 years are rare. The prevalence in this age group is about 0.01 to 0.05 per 1,000. In
addition, developmental status can affect the expression of the disorder. The earliest descriptions by DeSanctis (5 ), Bleuler (49 ),
and Kraeplin (6 ) reported the onset and occurrence during childhood and considered schizophrenic psychoses to be an early onset of
the same disease, which appeared to be on a continuum phenomenologically, that they observed in adolescents and adults.
Furthermore, it has been shown that schizophrenic psychoses can be diagnosed reliably in children using the same criteria as for
adults (20 ,36 ,50 ,51 ). Very few studies to date have dealt with the long-term outcome in childhood-onset schizophrenia. Most
studies have followed children for between 1 and 5 years (52 ). Because of methodologic difficulties, there is a striking absence of
data before the age of 11 years on the long-term course of psychosis. Asarnow emphasized the “crucial importance” of long-term
follow-up data for establishing the validity of psychotic symptoms manifested in early childhood (53 ). This is especially important
because children often describe “hearing voices,” especially in clinical populations. An astute clinician will delve into this symptom
in greater depth, to obtain a qualitative appreciation of these “voices.” Often, the child will describe this “voice” inside his head as
if hearing his own voice or that of an adult in his life. He most likely will not hear this voice through his ears and seems affectively
not to be too troubled by it. Conversely, a child experiencing true auditory hallucinations is frightened, puzzled, and unable to be
reassured. This differentiation is especially important because management of these youngsters often includes the use of
psychotropic medications, which, in and of themselves, require serious consideration because of their long-term adverse effects. If
the phenomenology of these so-called psychotic
 P.617

symptoms is not clarified, many youngsters with pseudohallucinations will be prescribed psychotropic medication needlessly. In
addition, they will wrongly be labeled with a psychotic disorder.

Premorbid developmental peculiarities have been reported in children with childhood-onset schizophrenia who have been followed
into their thirties. These peculiarities are primarily internalizing such as shyness, isolatory behaviors, lack of interest, awkwardness,
being fickle with peculiar facial expression, aggression, paranoia, anxious thoughts, and being mistrustful of others, along with
symptoms of depression. These signs have been reported to be much more common than externalizing, acting-out behaviors such as
temper tantrums, aggression, opposition, and hostility (22 ). From a developmental standpoint, the age of first manifestation of
nonpsychotic symptoms is younger than the age of onset of schizophrenic symptoms (53 ,54 ,55 and 56 ). However, the predictive
relevance in prepsychotic symptoms in children seems to be extremely uncertain because of the high variability of developmental
peculiarities.

The nature of the diagnostic subtypes varies markedly across the course of the illness. In patients with continuous predominantly
catatonic symptoms, the outcome is poor. Eggers et al. suggested that detailed case description helps to illuminate the
heterogeneous psychopathology of childhood-onset schizophrenia (22 ). These investigators found that various temporary premorbid
behavioral peculiarities were precursors of childhood-onset schizophrenia. Children with early-onset schizophrenic psychosis develop
a phenomenology of positive and negative psychotic symptoms that are similar to those seen in adult patients with schizophrenia,
and the course variability is perhaps even greater than in adult patients. Their findings contradicted the assumption that childhood-
onset schizophrenia is characterized only by negative symptoms, because a differentiation between premorbid and prodromal signs
proved to be arbitrary.

Since Kraeplin’s first description of dementia praecox in 1889, the onset and course of schizophrenia relied heavily on first
admission data and on the subsequent course of the disease. However, Hafner et al. argued that items taken from the preadmission
phase of the disease were often incorrectly used as premorbid characteristics (57 ). In an attempt systematically to account for the
age and gender distribution of the true onset and the symptoms and pattern of the early and later course, Hafner et al. developed
an Interview for the Retrospective Assessment of the Onset of Schizophrenia (IRAOS) (57 ). This instrument allows an objective,
reliable, and valid assessment of the symptoms, psychological impairments, demographic and social characteristics, and the
referring points in time of the early course of psychosis. Their findings suggested that the IRAOS provides information on the earliest
course of the disease and enables them to separate premorbid characteristics, possibly the most powerful predictors of the later
course and outcome, from contamination with symptoms and deficits belonging to the early phase of the disease. The influence of
certain life events on the early course is also made accessible to empiric research. Other instruments that have been used for
assessing psychotic symptoms in youngsters have been the Interview Schedule for Children (58 ), the Diagnostic Interview Schedule
for Children (59 ), the Schedule for Affective Disorders and Schizophrenia for School-Aged Children (60 ,61 ).

Several nonspecific and nondiagnostic neurobiological abnormalities have been reported in patients with schizophrenia. These
include deficits in smooth pursuit eye movements and autonomic responsivity (62 ,63 ). Neuroimaging findings include a progressive
increase in ventricular size and a fourfold greater decrease in cortical gray matter volume during adolescence, with the greatest
differences occurring in the frontal and temporal regions (64 ,65 ,66 and 67 ). Others findings reported in the literature are a
smaller total cerebral volume, correlated with negative symptoms (37 ), and frontal lobe dysfunction (68 ).

Schizophrenia with childhood onset is usually a severe and chronic disorder with a more guarded prognosis and poorer therapeutic
response to neuroleptic agents than schizophrenia with adolescent or adult onset. New research and data will help to clarify the
origin and pathogenesis of schizophrenia in children. Subsequently, development of more effective treatments and preventive
measures may reduce its severity.

Mood Disorders
Mood disorders such as major depression and acute mania can often be accompanied by psychotic symptoms. Over the past several
decades, the prevalence of mood disorders appears to have been increasing (69 ). Although information on the epidemiology of
psychotic depression in children is limited, Chambers et al. described the occurrence of psychotic depression in children (61 ). The
psychotic symptoms usually are mood congruent, but at times they can be quite like those seen in childhood schizophrenia
(20 ,70 ,71 and 72 ). This overlap in symptoms increases the likelihood of incorrect diagnosis, especially at the time of onset.
Sometimes, the negative symptoms of schizophrenia in children can be mistaken for those of depression. However, it has been
shown that children with schizophrenia have poorer premorbid adjustments, lower IQs, and more chronic dysfunction, when
compared with children who suffer from a depressive disorder (50 ). It is therefore prudent to make only a tentative diagnosis at the
outset that must be confirmed longitudinally. Careful follow-up of psychotic patients is needed to detect diagnostic errors. This
issue can be compounded, however, if the symptoms resolve with antipsychotic medications. It becomes unclear whether the child
improves because of treatment or spontaneous remission. Approximately one-half of adolescents with bipolar disorder may
 P.618

be originally diagnosed as having schizophrenia (20 ,70 ). Therefore, it is extremely important that longitudinal reassessment is
needed to ensure accuracy of the diagnosis. Despite an increased family history of depression in schizophrenic youth (20 ), family
psychiatric history can be an extremely helpful differentiating factors. However, the opposite is not true, that is, an increased
family history of schizophrenia in depressed or bipolar youngsters. Often, the rule of thumb is first to rule out mood disorder in a
child or adolescent before the diagnosis of schizophrenia is more strongly considered.

Even though there is an overlap of the quality of psychotic symptoms in children with mood disorders and childhood-onset
schizophrenia, often with careful examination, some of the mood-congruent symptoms can be ascertained. As clinicians, it is
important that we ascertain chronologically what came first, that is, a change in mood and then the onset of delusions or
hallucinations, or a disturbance in thought followed by a change in mood. For example, the child who first starts to have “strange
thoughts” and to hear voices over time becomes puzzled, fearful, distraught, and depressed. This is quite different from the child
who first starts to lose interest in activities, to feel irritable or depressed, to not want to play with friends, and who demonstrates
neurovegetative symptoms, such as a decrease in appetite, sleep disturbance, and lethargy. Subsequently, the child starts to think
he is a bad and evil person and then hears a voice that tells him he is a bad boy and that he should kill himself. The phenomenology
in this instance is quite different. However, it is not always this clear, and there is a high rate of misdiagnosis in both directions
(72 ,73 ).

Psychotic symptoms during a manic episode have been recognized for many years, although misdiagnoses of schizophrenia were, and
remain, relatively common (74 ). Bipolar disorder eventually develops in a minority of children initially hospitalized for major
depression (1 ). This is particularly so if the child has a positive family history of bipolar disorder, psychomotor retardation, rapid
onset of symptoms, mood-congruent psychotic symptoms, or pharmacologically induced mania or hypomania. The characteristics of
the delusions and hallucinations are often mood congruent (expansiveness, grandiosity, and euphoria). Therefore, a child
experiencing mania may have delusions of being “superman” with special powers, of being able to fly and leap from high places.
Conversely, the child may believe that he or she has special skills playing baseball, even though the child perhaps may have
problems with gross motor skills and is clumsy and uncoordinated. Similarly, the child may hear voices, the content of which are
mood congruent, with the altered state in mood (i.e., grandeur), and may believe that the voice is saying that he or she is superior
and can do anything.

Brief Reactive Psychosis

Occasionally, children and adolescents suddenly develop psychotic symptoms that can last from a few hours or days. The child
experiences these symptoms when under tremendous stress, such as after a death in the family, witnessed acts of violence or
destruction, or physical or sexual abuse. The acute psychotic symptoms often resolve quickly, with total recovery in a few days.
These youngsters may suddenly become disorganized, confused, agitated, or withdrawn. At times, their speech becomes nonsensical
and incomprehensible. They may also experience delusions and hallucinations. These, too, are usually short-lived.

Anxiety Disorders
Children who experience acute anxiety or who have a history of maltreatment, abuse or neglect report significantly higher rates of
psychotic symptoms when compared with controls (75 ). Several studies have documented psychotic-like symptoms in children with
posttraumatic stress disorder. In such instances, the psychotic symptoms actually represent intrusive thoughts or worries regarding
the traumatic event (73 ,76 ,77 ). Mental status examination usually reveals the lack of a formal thought disorder, and the
psychotic-like symptoms are more akin to derealization or depersonalization, as is often observed in traumatized children.
Furthermore, there is often a qualitative difference in the way children with anxiety disorders and those with childhood-onset
schizophrenia relate. The former have better-developed relationship and prosocial skills compared with the socially isolated,
awkward, and odd behaviors of a child with schizophrenia. An identifiable traumatic event, abuse, or neglect in the child’s history,
in and of itself, does not necessarily rule out a psychotic disorder, because children with both schizophrenia and mood disorders may
have had such experiences (73 ).

Organic Psychoses

Neurologic Conditions

Seizure Disorder
Children with seizure disorders can experience hallucinations as part of the seizure activity. Complex partial seizures, especially
those with a temporal focus, may be associated with interictal psychotic symptoms of delusions, hallucinations, and unusual
preoccupations. Caplan and co-workers described a formal thought disorder in children with partial complex seizures (78 ,79 ),
although their way of defining thought disorder makes it intertwine closely with language organization deficits. However, they did
emphasize that these epileptic children usually do not display negative symptoms such as those seen in schizophrenia. Hallucinations
in children with epilepsy typically are brief. Therefore, these children experience mainly positive symptoms, which
 P.619

are often short-lived. Caplan and co-workers also described a higher incidence of formal thought disorder in those children who have
lower IQs, earlier onset of the seizure disorder, and poor seizure control. They postulated that these symptoms may either reflect
the underlying neuropathology that produces the seizures or result from the “kindling phenomenon” as a secondary effect of the
seizure activity.

Deteriorative Neurologic Disorders

Psychotic symptoms have been described in children who have a deteriorative and degenerative neurologic disorders such as
subacute sclerosing panencephalitis (80 ). Other disorders include Wilson disease, lipid storage disorders, and Huntington chorea.
These are usually differentiated from childhood-onset schizophrenia by the presence of neurologic findings on physical examination
of the child, further corroborated by abnormal findings on laboratory testing. Children suffering from such neurologic deterioration
often have a gradual, persistent, but global decline in their neurologic condition.

Central Nervous System Lesions

These conditions include brain tumors, congenital malformations, and head trauma.

Metabolic and Hormonal Disturbances

Various metabolic and hormonal conditions can be responsible for psychotic symptoms in children. Endocrinopathies may include
disorders of the adrenal, thyroid, or parathyroid glands. Exogenous metabolic disturbances leading to psychotic symptoms can
include exposure to heavy metals.

Toxic Psychoses
Toxic psychosis or delirium usually occurs secondary to bacterial or viral infections, high fevers, and exogenous toxins including
medications, illicit drugs, alcohol, and poisonings. Unlike childhood schizophrenia or other psychotic disorders, in which impaired
thinking and communication are the most salient symptoms, toxic psychosis is more likely to cause vivid, disturbing visual or tactile
hallucinations and other perceptual problems. Auditory hallucinations can also occur, but their content is qualitatively different
from those experienced in childhood schizophrenia or mood disorders. These sensory experiences may be extremely frightening and
may be accompanied by agitation or by uncontrolled or even aggressive behaviors. Children and adolescents often describe the
experience as “losing their mind”—a frightening concept, and they can become disoriented, unable to orient to person or place, or
comprehend why they are behaving in an unusual manner. They may also experience fluctuating levels of alertness.

In children, infections (bacterial or viral) can cause encephalitis, meningitis, and human immunodeficiency virus–related syndromes,
which can result in delirious states. High fevers, regardless of origin, have been known to cause delirious states with perceptual
disturbances. In addition, chronic liver and kidney disease may cause delirious states associated with psychotic symptoms in children,
manifested by states of confusion, distortions in perceptions, and frank hallucinations.

The best example of medication-induced psychosis is that resulting from high doses of stimulants (the most commonly prescribed
group of medications in this age group). In young children, normal doses of common medications, such as over-the-counter
antihistamines and decongestants, can induce similar symptoms. Some of the other medications that can have a similar result are
steroids, which can cause not only a disturbance in mood (depression and manic symptoms), but also delirium. Children prescribed
anticholinergic drugs are also vulnerable to developing delirium, presenting with psychotic symptoms.

Other causes, especially in older children and adolescents, are alcohol intoxication, amphetamine-like drugs (“speed” and cocaine),
hallucinogenic drugs (LSD and psilocybin), solvents, and cannabis. Most children who develop drug-induced psychosis recover once
the drugs are out of their system.

The psychotic symptoms sometimes experienced by patients after anesthesia should be included in the category of toxic psychoses.
Although usually short-lived, this phenomenon is reported by patients to be a very frightening experience. Support, reassurance, and
ensuring safety at the time are usually sufficient in the management of patients after anesthesia.

MANAGEMENT AND TREATMENT

Part of "45 - Psychosis in Childhood and Its Management "

Assessment
Effective treatment requires knowledge of the psychotic disorders, diagnostic criteria, symptoms, and longitudinal course, in
addition to an understanding of the youngster’s developmental, social, educational, and psychological needs. Treatment strategies
therefore need to focus on the clinical symptoms and morbidity of the underlying disorder, while also addressing any comorbid
disorders or biopsychosocial stressors. The physician must prioritize symptoms and diagnoses, so a reasonable treatment plan
addresses multiple problems. A clinician examining a child for psychoses must first ascertain whether the child comprehends the
clinician’s question about delusions and hallucinations and whether the child endorses the psychotic symptoms only to please the
interviewer or to get attention. In addition, it is important to determine whether the child acts on the basis of the delusional or
hallucinatory perceptions—associated with an affective response of fear, dread, avoidance, or elation.

The assessment of the child with psychotic symptoms should include a careful, comprehensive, and thoughtful
 P.620

evaluation. The history is often obtained from multiple informants, and several sessions may be required to gain accurate
assessment of the child’s mental status. The assessment should include a detailed evaluation of the symptom presentation, course
of illness, and phenomenology. A developmental history of the child and a detailed family psychiatric history are invaluable
components of the evaluation and assessment. A positive family history, especially for an affective disorder or schizophrenia
because these disorders tend to run in families, often helps the clinician with the differential diagnosis in the child.

Once it is determined that the child is experiencing psychotic symptoms, it is important foremost to ascertain the cause of such
symptoms. This will, in large part, determine the management and treatment of the child presenting with psychosis. A thorough
physical examination is essential, and pertinent tests and procedures may be necessary, as clinically indicated. These may include
imaging studies, an electroencephalogram, toxicology screens, and renal and liver function tests. Some children may require
consultation with other pediatric specialists.

Psychological and projective testing are not indicated as a method of diagnosing specific disorders causing the psychosis. However,
they can be helpful for intellectual assessment and to determine developmental delays, because these deficits may influence the
presentation or interpretation of symptoms. Routine use of adaptive function measures is important for understanding actual
function in social, daily living, and communication domains. These can be quite helpful in planning and maintaining developmentally
relevant treatment goals. Similarly, speech and language evaluations are often helpful, especially with a child who appears to have
linguistic impairments on examination.

Treatment
If it is deemed that the cause is organic, then the first step is to diagnose and treat the underling cause of the psychotic symptoms.
This may include treating a partial complex seizure disorder, managing a metabolic imbalance, or treating an underlying infection or
reducing a fever. Conversely, if it is determined that there is no medical cause for the psychotic symptoms, then the next step is to
ascertain whether the psychosis is functional. If so, is it secondary to severe depression or acute mania with psychotic symptoms or
secondary to a schizophrenic illness?

Adequate treatment requires a combination of pharmacotherapy and various psychosocial interventions that target the child’s
specific difficulties. Some of this depends on the phase of the underlying illness (81 ):
Stage 1 (prodromal phase): The child may experience some period of deteriorating function, which may include social isolation,
idiosyncratic preoccupations and behaviors, and academic difficulties.
Stage 2 (acute phase): This is usually the time when the child comes to the attention of a mental health professional, when the
clinical picture is dominated by frank delusions and hallucinations and other positive symptoms such as a formal thought disorder
or strange and idiosyncratic behaviors.
Stage 3 (recovery phase): The symptoms usually begin to remit and dissipate. However, often there may still be the presence of
some psychotic symptoms, although they are less disturbing to the child. In this phase, the child may continue to experience
some levels of confusion, disorganization, or lability in mood.
Stage 4 (residual phase): The positive symptoms continue to subside, but the child continues to experience apathy, lack of
motivation, withdrawal, and restricted or flat affect.
Unfortunately, some children remain symptomatic and chronically impaired, despite what would be considered adequate treatment.
Usually, such impairment is characterized by persistent symptoms, which occur especially if the psychosis is secondary to a
schizophrenic illness, rather than the result of depression or mania.

Psychosocial interventions should include working with both the parents and the child. Interventions targeted at improving family
functioning, problem solving, communication skills, and relapse prevention have been shown to decrease relapse rates in adults
(82 ). Children may benefit from social skills training and may require specialized educational programs, academic adjustments, and
support at school. Ongoing illness teaching and medication education, are important to promote compliance with treatment and to
help in coping with the daily and sometimes long-term implications of the child’s illness. Every effort should be made for the child
to be maintained in the least restrictive setting, such as home. However, in some cases, the severity and chronicity of the
underlying illness may warrant long-term placement in a hospital or residential facility.

Pharmacotherapy is instituted in an attempt to treat the underlying cause of the psychosis, or for symptom control, in those
children who have psychotic symptoms secondary to a known origin. Informed consent from the parents or guardian should be
obtained before treatment with psychopharmacologic agents is instituted.

It is not in the purview of this chapter to discuss each medication in detail. For the treatment of major depression, the following
antidepressants have been used in children:
Tricycle antidepressants (nortriptyline, imipramine, desipramine)
Selective serotonergic reuptake inhibitors (fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram)
Nonselective serotonergic reuptake inhibitors (nefazodone, mirtazapine)
Monoamine oxidase inhibitors (phenelzine, tranylcypromine) (seldom used currently)
 P.621

Others: bupropion, venlafaxine

Mood stabilizers that have been used for the treatment of manic-depressive illness in children include the following:
Anticonvulsants (divalproex sodium, carbamazepine, gabapentin)
Lithium
Often, the use of antipsychotic medications in addition to the use of antidepressants or mood stabilizers is indicated in functional
psychosis.

If the suspicion is that of early-onset schizophrenia, then antipsychotics are first-line medications. Although children may
metabolize neuroleptics more rapidly than adolescents and adults, optimum doses for children are typically less than those required
in adolescents and adults.

First-line agents include traditional neuroleptic medications that block dopamine receptors or the atypical antipsychotic medications
that have a variety of effects including antagonism of serotonergic receptors. The atypical antipsychotic medications are reported to
be at least as effective for positive symptoms and may even be more helpful for negative symptoms. Further, there is some
suggestion that they have fewer adverse effects. Except for clozapine, the novel agents also appear to produce tardive dyskinesia.
Experience with novel antipsychotic agents is too scant to determine whether the risk of tardive dyskinesia is equal to or less than
with the older antipsychotics. Newer antipsychotic medications that have been used in children are risperidone and olanzapine.
They may be less sedating than the traditional neuroleptic agents such as haloperidol, fluphenazine, thioridazine, and
chlorpromazine. There have been some case reports in the literature of the use of clozapine for children and adolescents with
schizophrenia in whom normally adequate treatment with other traditional antipsychotic medications has failed.

For a child suffering from acute reactive psychosis, support and safety are the two primary considerations. If the child is extremely
stressed and acutely ill, hospitalization may be necessary to provide a safe and structured environment. Brief treatment with
antipsychotic mediations has often been effective for the alleviation of psychotic symptoms in some children. However, medications
will not eliminate the problem that originally caused the brief psychosis. Thus, psychotherapy is often helpful in helping the child
learn to cope with the emotional trauma that may have precipitated the episode.

Toxic psychosis requires immediate medical intervention to identify the cause and to provide appropriate treatment. Identifying the
cause may include laboratory tests such as serum electrolytes, liver function tests, toxicology screens, blood alcohol level, serum
levels of prescribed medications including theophylline, tricyclic antidepressants (nortriptyline, amitriptyline, imipramine,
desipramine), or mood stabilizers (valproic acid, lithium, carbamazepine). Neuroleptics, because of their common usage, comprise
another important group of medications that needs to be considered in the psychiatric population. The rare but possible
development of neuroleptic malignant syndrome, manifesting as a disturbance of sensorium, fever, rigidity, and high blood pressure,
should be considered. A history of treatment with neuroleptics and an elevated creatinine phosphokinase usually enable one to
determine this cause (83 ). Most children who develop drug-induced psychosis recover once the drugs are discontinued and out of
their system. The gravest danger occurs during the psychotic episode, when a child may cause serious harm to himself or herself or
to others, because judgment is impaired. Some children may need brief hospitalization until the cause is determined and the
psychotic symptoms dissipate. Except for the presence of neuroleptic malignant syndrome as the cause, brief treatment with
antipsychotic medications may be necessary to decrease the child’s agitation and to control the distorted perceptions.

In addition to the foregoing treatment strategies, other interventions and services may be needed to address either comorbid
conditions or associated sequelae of the underlying disorder causing the psychosis, such as substance abuse, depression, and suicidal
tendencies.

CONCLUSIONS
Part of "45 - Psychosis in Childhood and Its Management "

From the clinical perspective, the rapid change and development of childhood have immediate implications for diagnosis and
intervention. When one is treating children, it is important to maintain diagnostic fluidity and to tolerate the pressure of
uncertainty.

In the realm of childhood-onset psychopathology, we have a great deal to learn from the psychoses. The stability of a diagnostic
category over time is usually considered to be one measure of the construct validity of that category. One possibility is that lack of
stability of a diagnosis during childhood implies that it lacks validity. This is only important if one is trying to establish a unique
direct link with later-onset disorders and to apply the same terminology. However, another possibility is that some childhood
diagnoses are only risk factors for development of more enduring adult conditions, such as the relationship between conduct
disorder and antisocial personality disorder. Variability of normal and psychopathologic development and the heavy influence of
environmental features and familial functioning during childhood make it difficult to be certain about diagnoses in children.
Although categoric classification has its advantages and at times is necessary (34 ), dimensional perspectives can be important for
understanding these phenomena as well.

It is useful to recognize the close, reciprocal relationship between diagnostic classification and biological or genetic advances.
Advances in genetic and imaging studies should open the way to a different classification system that links
 P.622

symptoms, neural circuitry, and biological (genetic) markers more closely than any current system. We should not be too surprised
to discover that conditions once considered to be quite distinct are now closely linked, as has been found for Tourette syndrome,
obsessive-compulsive disorder, and attention-deficit/hyperactivity disorder (84 ). “Diagnostic stability” may refer to the stability of
these biological features, rather than the stability of clinical signs and symptoms. One could expect that developments in molecular
biology would shed light on the natural history, protective factors, and risk factors for a specific biological risk. However, these
developments will depend on increasingly reliable, reproducible diagnoses. The success of these strategies demands that probands
or cohorts have been reliably diagnosed according to the most valid criteria at the time. Thus, the diagnoses inform the biological
work, and the biological work, in turn, influences our classification and criteria. This process fosters a more accurate understanding
of the natural history, pathophysiology, and etiology of disorders and the relationships among disorders. It will inform us about
prenatal psychoneurohormonal factors that influence development, sexual differentiation, and maturation of the central nervous
system. In time, we stand to gain a much clearer understanding of the complex, diverse contributions to the development and
maintenance of psychotic disorders. We look forward to developing treatments that will offer more comfort and better functioning
to the patients and families afflicted with these chronic conditions. There is, in addition, a real prospect of finding protective
factors and preventive interventions that can avert the worst manifestations of these disorders.

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46
Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into
the Developing Brain

James C. Harris

James C. Harris: Departments of Psychiatry and Behavioral Sciences, Pediatrics, and Mental Hygiene, Johns Hopkins University
School Medicine, Baltimore, Maryland.

 HISTORICAL BACKGROUND
 DEFINITION AND CHARACTERIZATION
 PSYCHOPATHOLOGY AND BEHAVIORAL PHENOTYPES
 PREVALENCE
 BEHAVIORAL PHENOTYPES OF SPECIFIC NEURODEVELOPMENTAL DISORDERS
 ANIMAL MODELS: SIMULATIVE OR SUBSTITUTIVE
 CONCLUSION

HISTORICAL BACKGROUND
Part of "46 - Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain "

Increasing evidence indicates that specific neurodevelopmental disorders may be associated with particular patterns of behavior. A
description of behavior was included by Langdon Down in the first published description of a specific mental retardation syndrome,
Down syndrome (1 ). In his description, Down observed: “They have considerable powers of imitation, even bordering on being
mimics. Their humorousness and a lively sense of the ridiculous often colors their mimicry.” Later, he added: “Several patients who
have been under my care have been wont to convert their pillow cases into surplices (vestments) and to imitate, in tone and gesture,
the clergymen or chaplain which they have recently heard.” He also commented on personality traits, saying: “Another feature is
their great obstinacy—they can only be guided by consummate tact.” Although these stereotypes were not confirmed in subsequent
studies (2 ,3 ), the prospect of linking behavior and genetics was introduced in this first description of a neurogenetic disorder.
Subsequent early clinical descriptions, such as that of tuberous sclerosis complex by Critchley and Earl (4 ), identified peculiar, and
severe, behavioral problems in children and adults with that condition. Yet despite the early recognition of syndrome-specific
behavioral and psychiatric features, neurogenetic disorders were not empirically investigated for behavioral deficits until 1990s,
when new conceptual and methodologic procedures were introduced (5 ).

Two main reasons may explain this lack of interest after the early reports by Down and others. First, there was a general negative
reaction against eugenics and claims for genetic bases of personality (6 ). This negative reaction established a climate in which it
was not considered appropriate for academic investigators to emphasize the genetics of behavior. Second, there has been a major
emphasis on learning theory and its applications to the field of mental retardation, in which most genetic disorders are found.
Tremendous strides have been made in the education of even the most severely mentally retarded persons. Advances in academic
and social adaptive education, in conjunction with motor treatment, have placed greatest emphasis on how severely and multiply
handicapped people could attain greater degrees of independence and social integration. With the emphasis on normalization,
research into severe disorders in learning tended to be deemphasized. Moreover, the occurrence of associated psychiatric and
behavioral problems was interpreted more in terms of learning theory rather than in being unlearned behaviors associated with
behavioral phenotypes. The focus has been on addressing the potential of the individual person and the developmental possibilities.
Yet this focus could not continue to ignore reports from families and clinical observations of characteristic patterns of behavior and
stereotypes.

With the establishment of active and refined learning-based approaches and a better understanding of the interpretation of genetic
findings, reappraisal and revision of attitudes toward research with behavioral phenotypes have begun. O'Brien suggested three
reasons for this shift (7 ). First, research findings have been reliably reported with various syndromes. Second, there are continued
reports from family members as large family organizations have developed in the United States and other countries that describe
characteristic behavioral patterns and interpersonal responses. In meetings, parent groups frequently report similar behavior
problems and difficulties in management across syndromes. The interest in parent groups in improving the life of their children has
led to additional hypotheses and more refined observations on behavioral characteristics. Third, new techniques in genetics provide
new insights into
 P.626

the extent and mechanisms of the human genome as the basis of behavior. Advances in other aspects of neuroscience, including
neurophysiology and neuroanatomy, provide additional means of designating brain mechanisms that may be involved. With the
establishment of these new methods of evaluation and the identification of rating scales to measure behavioral phenotypes, there is
now an increased focus on behavioral phenotypes in developmental neuropsychiatry. Finally, comprehensive study of children with
different developmental disabilities may increase our appreciation of the relative contribution of genetic variables in the
pathogenesis of specific affective and behavioral disorders.

Nyhan introduced the term behavioral phenotype to describe outwardly observable behavior so characteristic of children with
genetic disorders that its presence suggests the underlying genetic condition (8 ). In speaking of compulsive self-injury in Lesch–
Nyhan disease (LND), a disorder that he initially described, Nyhan noted: “We feel that these children have a pattern of unusual
behavior that is unique to them. Stereotypical patterns of behavior occurring in syndromic fashion in sizable numbers of individuals
provide the possibility that there is a concrete explanation that is discoverable. In these children, there are so many anatomical
abnormalities, from changes in hair and bones to dermatoglyphics, that it is a reasonable hypothesis that their behaviors are
determined by an abnormal neuroanatomy that would be discoverable, possibly neurophysiologically, ultimately anatomically…
these children all seem self-programmed. These stereotypical patterns of unusual behavior could reflect the presence of structural
deficits in the central nervous system” (8 ).

Such observations have led to greater emphasis on assessment of behavior, and the recognition of behavioral phenotypes in some
disorders has led to closer scrutiny of known neurodevelopmental conditions. Initially, the focus was on documenting the patterns of
behavior because the study of brain and behavior requires the identification of welldefined syndromes for investigation. Now that
developments in the neurosciences provide a means to understand the biological bases of such behavioral patterns, the focus has
shifted to understanding the neurobiological mechanisms underlying characteristic behavioral patterns, including cognitive processes
and social interactions. Such patterns are reported in numerous syndromes arising from genetic or chromosomal abnormalities. Thus,
molecular analysis of the underlying genetic disorder has been initiated in several syndromes with the hope of revealing the
biological basis of the behavioral phenotype. However, because of the rarity of many of these syndromes and the complexity of their
genetic basis, establishing the validity of the association between syndrome and behavioral phenotype is difficult. Nevertheless,
Flint pointed out that evidence from animal studies with relevance to human behavioral phenotypes shows that the pathway from
genotype to phenotype may be accessible after careful delineation of each of the features of the behavioral phenotypes (9 ,10 ).
However, in regard to the study of cognition, he suggested that we require a greater integration of different levels of understanding
of cognition to exploit the genetic discoveries, “a rapprochement between molecular and systems neuroscience” (10 ).

Much of the research in behavioral genetics uses a “top-down” approach to the qualitative analysis of complex traits such as novelty
seeking, memory, personality traits, and intelligence (11 ). Linkage or association strategies are used to examine naturally occurring
alleles of candidate genes in a “wild-type population.” These alleles are usually functional polymorphisms rather than mutations and,
if they are quantitative trait loci, may be associated with individual differences in the trait in question. However, Tully suggested
that such genes may have minor effects on the phenotype of the individual because alleles with a more striking effect could reduced
fitness and would be selected against in evolution (12 ). Specifically, chromosomal deletions that may have cognitive and behavioral
consequences may be associated with monosomy (13 ). The loss of one copy of genes that are dose sensitive may be significant in
brain development. Such genes may play a fundamental role in development of the functional organization of the brain, but they
may not be as important for individual differences in the general population. Moreover, partial variants of disorders such as LND that
result in a range of enzyme levels may allow study of dose response to enzyme deficits.

This chapter uses a developmental perspective to provide a definition and characterization of behavioral phenotypes in
neurodevelopmental disorders, and it discusses etiologic factors, methods to understand underlying mechanisms, and natural history.
It addresses the question: What do behavioral phenotypes that occur in specific neurogenetic disorders teach us, and how may they
provide a portal to understand the developing brain? This question is considered by reviewing studies of neurogenetic disorders with
behavioral phenotypes: (a) LND, an X-linked disorder, that results from the absence of an enzyme, hypoxanthine-guanine
phosphoribosyltransferase (HPRT), that is involved in purine metabolism; (b) Prader–Willi syndrome (PWS) and Angelman syndrome
(AS), in which the parental origin of the genes involved (uniparental disomy or UPD) is an important factor in the cause; (c) fragile X
syndrome, a disorder caused by unstable trinucleotide repeat expansion that results in the absence of a gene that encodes an RNA-
binding protein thought to play a role in translational regulation of selective messenger RNA transcripts; and (d) Williams syndrome
(WMS), a contiguous gene disorder with an unusual cognitive phenotype in which language is preserved but the patient has severe
visual spatial disabilities. Each of these neurogenetic disorders provides a portal to understand neurodevelopment. Other nongenetic
disorders that are environmentally induced, such as fetal alcohol syndrome, are not discussed but also offer keys to understanding
the developing brain (23 ).

DEFINITION AND CHARACTERIZATION

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 P.627

The study of behavioral phenotypes emphasizes the discovery, among individuals with known chromosomal, genetic, or
neurodevelopmental disorders, of those mental and behavioral features causally related to the underlying condition. Examples are
the characteristic self-mutilation of fingers and lips in LND, the hyperphagia and compulsive behaviors in PWS, gaze aversion in
fragile X syndrome, and the superficial sociability, hyperlalia, and language disorder in WMS. When present, the behavior suggests
the syndrome. As Nyhan suggested, these are “syndromes of behavior” (14 ). Still, despite their behavioral presentations, not all
individuals with the disorder show the classic behavioral features, but the probability is greater that they will. The essential issue is
that the behavior suggests the diagnosis.

Efforts to define what is meant by a behavioral phenotype are continuing. Harris proposed that behavioral phenotypes are
stereotypic patterns of behavior that are reliably identified in groups of individuals with known neurodevelopmental disorders and
are “not learned” (15 ,16 ). They may be the consequence of neurodevelopmental abnormalities that are potentially discoverable.
This approach to definition is a phenonic approach that takes as its starting point observations of the behavior itself rather than
beginning with a discrete and genetically identifiable condition, such as Down syndrome. Using the phenomic approach, the
behavioral phenotype of Rett syndrome (17 ), with its characteristic hand and hand-to-mouth stereotypies, identified it as disorder
with a behavior phenotype many years before the genetic origin was recognized. Moreover, the phenomic approach does not
discount acquired disorders, such as fetal alcohol syndrome, as having behavioral phenotypes. The impact of alcohol on cellular
signaling is now well known, with its consequences of cell death, abnormal midline brain development, behavioral problems, and
learning disabilities (16 ,23 ).

Such considerations led Flint and Yule to propose the following definition that includes the characteristic types of behaviors: “The
behavioral phenotype is a characteristic pattern of motor, cognitive, linguistic, and social abnormalities that is consistently
associated with a biological disorder” (18 ). This does not mean that the behavior is present in all instances but that the probability
of its occurrence is increased. In the future, more may be learned about brain mechanisms by comparing persons with behavioral
involvement with others who have the same syndrome but without the behavioral features.

Although some investigators have sought to limit the study of behavioral phenotypes to known genetic disorders (11 ), knowledge of
the genetic disorder is only the first step. Links from gene to behavior are complicated in that one gene may lead to the encoding of
many, perhaps ten or more, different proteins; the number of genes and type of mutation determine complexity. For example, in
LND, the disorder of purine metabolism clearly leads to the overproduction of uric acid and renal stones, but the pathway to the
movement disorder and self-injury is not direct and may be mediated through effects on the arborization of dopamine neurons (19 ).
Moreover, there are variants of LND with different degrees of enzyme deficit, ranging up to 20%, that have clinical effects.

Thus, several caveats are necessary as we consider pathways from genes to behavior (11 ): (a) the behavioral descriptions, like other
physical features of neurodevelopmental disorders, have increased probability of occurring and do not occur in all cases; they are
not be fully expressed in all affected individuals; (b) the genetic background of the individual may affect the phenotypic expression;
(c) the possibility exists that environmental factors may modify expression; (d) the behavioral presentation may be modified by the
extent of mental retardation associated with the disorder; and (e) variability occurs in mouse models in which there may be species-
specific factors so mutant mouse models do not replicate the behavioral features. One must consider the genetic background, strain
differences, and the differences in the rodent physiology. In LND, the HPRT-deficient mouse has a uricase enzyme that breaks down
uric acid. Therefore, it is not a model for the hyperuricemic metabolic disorder, but it still may be a useful model to study
dopamine deficiency in the brain. Thus, aspects of the disorder may be modeled in transgenic mice or in other species.

In some animal models, links to specific pathophysiology have been established. The canine model of narcolepsy (20 ) is an
interesting example of an approach to a human clinical disorder. Mutations for canine, autosomal recessive, narcolepsy were
identified by linkage analysis in canine backcrosses, and homology was demonstrated between human chromosome 6 and canine
chromosome 12. Canine narcolepsy is caused by a disruption of a G-protein–coupled receptor, the hypocretin (orexin) receptor 2
gene (Hcrt2) in three canine breeds. However, human narcolepsy is not associated with frequent hypocretin gene mutation (20 ).
Nonetheless, most humans with narcolepsy have undetectable hypocretin 1 levels in cerebrospinal fluid.

PSYCHOPATHOLOGY AND BEHAVIORAL PHENOTYPES

Part of "46 - Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain "

Numerous neurogenetic disorders are associated with nonspecific behaviors that may be found in several syndromes. These include
attention problems, hyperactivity, impulsivity, self-injury, aggression, autistic-like behavior, and preservative behaviors. Such
presentations indicate vulnerability of the developing brain and perturbation of brain systems resulting in these clinical conditions.
However, because these behaviors occur across many syndromes, they lack specificity and do not qualify as specific behavioral
phenotypes.
 P.628

Still, these behavioral features should be included in the description of the disorders. For example, the relationship between
aggression and antisocial behavior has been suggested in monoamine oxidase A (MAOA) deficiency. Brunner et al. described an
association between abnormal behavior and MAOA deficiency in several males from a single large Dutch kindred (21 ). The affected
males differed from unaffected males in that they tested in the borderline range of mental retardation and demonstrated increased
impulsive behavior, that is, aggressive behavior, abnormal sexual behavior, and arson. Yet a specific psychiatric diagnosis was not
made in four affected males who were examined by psychiatrists. Because MAOA deficiency leads to increased 5-hydroxytryptamine
(5-HT) levels, the aggressive behavior in these persons may be an exception to studies linking low 5-HT with impulsive aggression.
Brunner et al. suggested that even if a possible association between MAOA deficiency and abnormal behavior is confirmed in other
kindreds, “the data do not support the hypothesis that MAOA constitutes an ‘aggression gene’.” These investigators noted that genes
are essentially simple and code for proteins, whereas behavior is complex; thus, a direct causal relationship between a single gene
and a specific behavior is highly unlikely. In MAOA deficiency, complexity is shown by the variability in the behavioral phenotype and
by the highly complex consequences of MAOA deficiency on neurotransmitter function. Thus, the full pathway from gene to complex
behavior must be considered; the concept of a gene that directly encodes behavior is simplistic (21 ). Still, a great deal may be
learned by considering such pathways in neurogenetic syndromes.

PREVALENCE
Part of "46 - Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain "

With increasing attention to neurogenetic disorders, the number of identifiable behavioral phenotypes is increasing. Careful
observations of behavior are necessary when considering intervention for neurogenetic disorders. Although standardized rating
scales and personality profiles have been developed to measure behavioral phenotypes (22 ,23 ), profiles pertinent to the specific
disorder are needed. Besides behavioral phenotypes, isolated special abilities that occur in genetically based syndromes require
assessment. These include special abilities in calculation and in music (24 ). These special abilities may potentially be related to the
proposed modular organization of the central nervous system.

BEHAVIORAL PHENOTYPES OF SPECIFIC NEURODEVELOPMENTAL

DISORDERS
Part of "46 - Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain "

The sections that follow discuss four syndromes in which behavioral phenotypes have been identified: LND, PWS/AS, fragile X
syndrome, and WMS. Characteristic behaviors are highlighted, findings on origin are discussed, and potential neurochemical and
neuroanatomic abnormalities are reviewed. Behavioral and pharmacologic therapies have had limited success in many of these
conditions, so better characterization of the individual condition is essential to establish treatment. Neuroanatomic studies, brain
imaging studies, and continuing investigations of neurotransmitter systems, endocrine rhythms, and sleep studies may provide
information that will be helpful in the future in treatment.

Lesch–Nyhan Disease
LND is a rare (1:380,000) sex-linked recessive disease caused by an inborn error of purine nucleotide metabolism. It is caused by an
almost complete deficiency of the enzyme HPRT, which is involved in the purine salvage (purine base recycling) pathway (25 ). Self-
injury is the major behavioral manifestation; this behavior was sufficiently characteristic that Nyhan introduced the term
“behavioral phenotype” as a descriptor (8 ). LND is of psychosocial and psychiatric importance because of the lifelong suffering
experienced by the involved child and his family, the uniqueness of the behavioral phenotype, and the resources needed for lifelong
patient supervision. Moreover, an understanding of the neurobiological basis of this disease may contribute to a better
understanding of brain mechanisms involved in self-injurious and compulsive behaviors.

Genetic and Metabolic Aspects

The HPRT-encoding gene is located on the X chromosome in the q26-q27 region and is made up of nine exons and eight introns
totaling 57 kilobases (kb). The HPRT gene is transcribed to produce a mRNA of 1.6 kb that contains a protein-encoding region of 654
nucleotides. More than 270 mutations throughout the coding regions have been identified (79 ). Techniques that provide information
on the three-dimensional structure of the HPRT protein make it possible to correlate structure and function of the enzyme (26 ).
Eads et al. reported the effects of single amino acid substitutions on the stability and activity of HPRT (26 ).

The gene involved in LND is on the X chromosome, so the disorder occurs almost entirely in males; occurrence in females is
extremely rare. The metabolic abnormality is the result of an abnormal gene product—a deficiency in the enzyme HPRT. This
enzyme is normally present in each cell in the body and is highest in the brain, especially in the basal ganglia. Its absence prevents
the normal metabolism of hypoxanthine and results in excessive uric acid production and manifestations of gout without specific
drug treatment (i.e., allopurinol). The full disease requires the virtual absence of the enzyme. Other syndromes with partial HPRT
deficiency are associated with gout without the neurologic and behavioral symptoms. Page and Nyhan reported that HPRT levels are
related to the extent of motor symptoms,
 P.629

the presence or absence of self-injury, and possibly the level of cognitive function (27 ). Hypoxanthine accumulates in the cerebral
spinal fluid, but uric acid does not because it is not produced in the brain and does not cross the blood–brain barrier.

Behavioral Phenotype
Self-injurious behavior usually is expressed as self-biting; however, other patterns of self-injurious behavior may emerge with time.
It is not uncommon for self-injury to progress to deliberate self-harm (19 ,28 ). Characteristically, the fingers, mouth, and buccal
mucosa are mutilated. The biting pattern is often asymmetric, so the patient may mutilate the left or right side of the body and
may become anxious if he perceives that this side of the body is threatened. Other associated maladaptive behaviors include head
or limb banging, eye poking, pulling of fingernails, and psychogenic vomiting (28 ).

Self-mutilation in LND is conceptualized as a compulsive behavior that the child tries to control but generally is unable to resist.
With increasing age, the affected child becomes more adept at finding ways to control his self-injury. He may enlist the help of
others to protect him against these impulses or may learn self-restraint.

A language pattern that consists of repeated ambivalent statements with anxiety and coprolalia (vulgar speech) is characteristic.
Moreover, the patient may be compulsively aggressive and may inflict injury on others through pinching, grabbing, or using verbal
forms of aggression. Frequently, he will apologize for this behavior immediately afterward and will say that the behavior was out of
his control.

Etiologic Factors
The cause of the neurologic and behavioral symptoms is not clearly established; however, abnormalities in dopamine function have
been demonstrated in three autopsied cases (29 ). The behavior is not caused by either hyperuricemia or by excess hypoxanthine
because LND partial variants whose HPRT levels are greater than 2 do have hyperuricemia but they do not self-injure. Moreover,
infants treated for hyperuricemia from birth whose uric acid level is normalized still develop self-injury despite having normal levels
of uric acid.

Wong and Harris et al. used positron emission tomography to investigate how dopamine dysfunction contributes to the self-injurious
behavior (30 ). These authors documented reductions in dopamine transporter density of 68% in putamen and 42% in caudate in six
patients with classic LNS and self-injurious behavior. To clarify the relationship between presynaptic dopamine transporter binding
in the striatum and self-injurious behavior further, Harris, Jinnah and Wong (30a ) studied seven patients with Lesch–Nyhan variants
(HPRT levels 1.8% to 20.0%) and two patients with HPRT levels less than 1.5%, all nine without self-injurious behavior (age range, 12
to 37 years). The extent of motor findings was documented on quantitated neurologic examination. Two patients with HPRT levels
less than 1.5% and two patients with HPRT levels of l.8% and 2.5% with severe movement disorder were not different in WIN 35,428
dopamine transporter binding in positron emission tomography imaging than the previously described classic patients with LND who
did injure themselves. The study of variant cases with motor symptoms but with no self-injurious behavior suggests that reductions
in dopamine receptor density are not a sufficient explanation of the self-injury. However, these authors found that HPRT level and
the extent of motor deficit were correlated with dopamine transporter binding in caudate and putamen in the nine cases. Dopamine
transporter binding was significantly correlated with HPRT levels in whole cells. Moreover, when the movement disorder was rated
on the Fahn-Marsden dystonia rating scale, putamen dopamine transporter density was significantly correlated with symptom
severity. These findings suggest that dopamine reduction is linked to the extent of the movement disorder, but it may not be a
sufficient explanation for self-injurious behavior, and other neurotransmitters need to be examined. Moreover, these variant
subjects with levels from 2% to 20% showed cognitive deficit profiles similar to those of classic LND (31 ).

Future investigation will need to take into account the existence of a variety of mutations in the HPRT gene structure. Why partial
HPRT deficiency does not lead to neurologic and behavioral symptoms remains unclear; perhaps neurotrophic factors are active with
minute amounts of the enzyme. It is advisable to study combined drug and behavioral treatment. An emphasis on parental training is
of particular importance for drug compliance and generalization of treatment effects. As in other inborn errors, continuous family
support is essential. Harris provides a description of a comprehensive treatment program for LND (19 ).

Prader–Willi Syndrome
PWS is a neurodevelopmental disorder characterized by obesity, short stature, cryptorchidism, mental retardation, hyperphagia,
learning disability, short stature, hypogonadism, hypotonia, small hands and feet, and dysmorphic facies. Patients have an increased
prevalence of daytime sleepiness, scoliosis, and other orthopedic abnormalities. Because of the obesity, heart failure and diabetes
may occur as complications. Although it is a rare disorder (1 in 10,000 to 15,000), its behavioral phenotype has assumed prominence
in genetics because of its relationship with AS, which has a different behavioral phenotype, although both disorders involve genomic
imprinting of the same region of chromosome 15.

Genetics
PWS may result from both chromosomal deletion and maternal UPD. In UPD, two copies of the maternal chromosome
 P.630

are inherited with no paternal contribution (32 ). Without the presence of the chromosome donated by the father, the normal
imprinting of the two maternally donated chromosomes leads to absence of gene expression in this interval. This results in a
functional abnormality that is essentially equivalent to the structural abnormality found in a deletion in the 15q11-q13 region that is
associated with the disorder. Moreover, in about 5% of cases, abnormalities in the mechanism of imprinting may occur when the
imprinting control center itself has a mutation.

Several genes are included in the most commonly deleted region in PWS. Some are paternally imprinted, and others are maternally
imprinted (33 ). Among these, ZNF 127, NDN, SNURF-SMRPN, IPW are paternally imprinted. Another gene, UBE3A (E6-AP ubiquitin
lipase), is maternally imprinted. Others genes in this region that are expressed from both maternal and paternal chromosomes
include three γ-aminobutyric acid (GABA) receptor subunits (GABRB3, GABRA5, GABRG3) (33 ). Because similar phenotypes result
from deletions and from imprinting in PWS, it is less likely that nonimprinted genes play a role in PWS or AS. Among these genes, a
specific gene for PWS has not been established, so several of these genes may contribute to the phenotype. For example, the SMRPN
gene is involved in protein slicing and is expressed throughout the brain; however, it is not thought that the PWS is the direct
outcome of this deficit. The NCD (necdin) gene does lead to failure to thrive in certain mouse strains, so it may be a factor; however,
those mice that survive do develop into apparently normal adults. Thus, the disorder is most likely linked to the loss of more than
one gene in this region. Conversely, the mutation of a single gene, UBE3A, has been found in cases of AS (34 ).

Behavioral Phenotype
The extent of cognitive impairment is variable in PWS. Some patients test in the normal range of intelligence, but most test in the
mild to moderate range of mental retardation. Others may test in the severe range of mental retardation. The behavioral phenotype
includes unusual food-related behavior (compulsive food seeking, hoarding, gorging), skin picking, irritability, anger, low frustration
tolerance, and stubbornness. Standardized methods of assessment have substantiated increased rates of depression, anxiety, and
compulsive behavior. Up to 50% of children and adults with PWS demonstrate behavioral disorders.

Compulsive eating is the most disabling of these behavioral manifestations and leads to obesity and the complications of severe
obesity, such as respiratory impairment and diabetes. The hyperphagia, which has been consistently found, has received the most
systematic behavioral evaluation. When not carefully supervised, patients may steal food and, in some instances, eat unpalatable
food, although this can be avoided with appropriate supervision. Holm and Pipes evaluated food-related behavior in the PWS (36 ).
They found that behavioral problems were most commonly related to food and included food stealing, foraging for food, gorging,
and indiscriminate eating with little food selectivity. No special circumstances that resulted in food stealing or gorging were
identified.

Besides the food-related compulsions, emotional lability with temper tantrums, stubbornness, negativism, skin picking and
scratching, and non–food-related obsessions have been examined. A questionnaire survey involving 369 cases identified compulsive
and impulsive aggressive behavior (37 ). These authors used the Overt Aggression Scale, the Yale-Brown Obsessive-Compulsive
Disorder Scale, a clinical global rating, and DSM-III-R criteria to diagnose self-stimulation and self-injury, compulsive behavior, and
obsessive behaviors. These investigators found that skin picking was the most common form of self-injury, observed in 19.6% of this
sample. Other types of self-injury with lower frequency were nose picking, nail biting, lip biting, and hair pulling. The second
behavioral problem area was compulsive behavior; food hoarding was the most severe manifestation and occurred in 17.7%. Other
compulsive behaviors included counting, symmetric arrangements of objects, checking, and hand washing, but they were less
common. Obsessive thinking was far less characteristic, with only 1.4% rated in the severe range on an item dealing with concerns
about contamination. State et al. reviewed the evidence in regard to compulsive behaviors in PWS and the relationship with
obsessive compulsive disorder (38 ). Behavioral problems identified in the preschool years persist throughout the school years and
continue into adolescence and adulthood.

Etiologic Factors
Investigators have proposed that the genetic abnormality in PWS leads to hypothalamic dysfunction that results in aspects of the
clinical phenotype, such as dysregulation of feeding, delay in sexual development, sleep disorder, and abnormality of
thermoregulation. In support of hypothalamic dysfunction, Swaab et al., in a postmortem study, found reduction in oxytocin cells in
certain regions of the hypothalamus (35 ). However, other brain regions and neuropeptides may be involved in PWS. Because the loci
of GABA subunits is in the area around the 15q11-13 region, GABA has been measured in PWS, and abnormalities have been reported
in plasma levels in some patients.

To clarify the mechanism leading to the behavioral phenotype further, differences between deletion and maternal UPD causes have
been assessed (39 ). Similar studies have been completed in AS (40 ). Differences in intellectual functioning in PWS with a paternal
15q11-q13 deletion versus maternal UPD of chromosome 15 were evaluated using measures of intelligence and academic
achievement in 38 patients with PWS (24 with deletion and 14 with UPD).
 P.631

The patients with UPD had significantly higher verbal IQ scores than those with deletion (p < .01). The magnitude of the difference
in verbal IQ was 9.1 points (69.9 versus 60.8 for UPD and deletion PWS patients, respectively). Only 17% of subjects with the 15q11-
q13 deletion had a verbal IQ greater than or equal to 70, whereas 50% of those with UPD had a verbal IQ greater than or equal to 70.
Performance IQ scores did not differ between the two PWS genetic subtype groups. This report documents the difference between
verbal and performance IQ score patterns among patients with PWS of the deletion versus the UPD subtype. Comprehensive
treatment of behavioral problems in PWS is described by Holm et al. (41 ).

Angelman Syndrome
In contrast to PWS, investigators have shown that one gene in the deleted region can lead to AS (34 ). AS is a neurologic disorder
with a heterogeneous genetic origin. It most frequently results from a de novo interstitial deletion in the 15q11-q13 region, but it is
also caused by paternal UPD or an imprinting mutation. The remaining 20% to 30% of patients with AS exhibit biparental inheritance
and a normal pattern of allelic methylation in the 15q11-q13 region. In this biparental inheritance group, mutations in the UBE3A
gene have been shown to be a cause of AS. Moncla et al. described the phenotypic expression in 14 patients with AS involving eight
UBE3A mutations (34 ). These were made up of 11 familial cases from five families and three sporadic cases. Some subtle
differences from the typical phenotype of AS were noted. Consistent features were psychomotor delay, a happy disposition, a
hyperexcitable personality, EEG abnormalities, and mental retardation with severe speech impairment. The other main features of
AS—ataxia, epilepsy, and microcephaly—were either milder or absent in various combinations among these cases. Moreover,
myoclonus of cortical origin was commonly observed with severe myoclonic seizures. Most of these patients were overweight. This
study showed that ataxia, myoclonus, EEG abnormalities, speech impairment, characteristic behavioral phenotype, and abnormal
head circumference are attributable to a deficiency in the maternally inherited UBE3A allele. Finally, analysis of mutation
transmission showed an unexpectedly high rate of somatic mosaicism in normal carriers. These clinical findings have important
consequences for genetic counseling in AS.

Fragile X Syndrome
The fragile X syndrome is characterized by mental retardation, behavioral characteristics, and the physical findings of a long face
with large, protruding ears and macroorchidism (42 ). Fragile X syndrome is the most common known cause of inherited mental
retardation, and it may also result in learning disabilities and social deficits in those who do not test in the mentally retarded range.
After the identification of the fragile X mental retardation (FMR1) gene, the cytogenetic marker (a fragile site at Xq27.3) was
replaced by molecular diagnosis. Recognition of this gene has broadened our understanding of the spectrum of the fragile X
syndrome.

Genetics
Fragile X syndrome is caused by massive expansion of CGG triplet repeats located in the 5′-untranslated region of the FMR1. The
cloning of the FMR1 gene led to the characterization of its protein product FMRP. The full mutation is associated with a process of
methylation; the addition of methyl groups along the “backbone of the DNA helix” (42 ). In patients with fragile X syndrome, the
expanded CGG triplet repeats are hypermethylated, and the expression of the FMR1 gene is repressed, which leads to the absence
of FMR1 protein (FMRP) and subsequent mental retardation. The encoded protein is a ribosome-associated, RNA-binding protein
thought to play a role in translational regulation of selective messenger RNA transcripts. FMRP is an RNA-binding protein that
shuttles between the nucleus and cytoplasm. This protein has been implicated in protein translation because it is found associated
with polyribosomes and the rough endoplasmic reticulum (43 ). A similar mechanism is proposed for FMR2, which encodes a large
protein of 1,311 amino acids and is a member of a gene family encoding proline-serine–rich proteins that have properties of nuclear
transcription factors (44 ).

The fragile X syndrome was one of the first examples of a “novel” class of disorders caused by a trinucleotide repeat expansion in
the X chromosome. In the genetically normal population, the CGG repeat varies from six to 54 units. Affected subjects have
expanded CGG repeats (more than 200) in the first exon of the FMR1 gene (the full mutation). Phenotypically normal carriers of the
fragile X syndrome have a repeat in the 43 to 200 range (the premutation). The process of methylation silences transcription so a
fully methylated full mutation results in no FMR1 protein’s being produced. The absence of FMR1 protein results in fragile X
syndrome. Two additional disorders result in a fragile site at Xq27.3; there are FRAXE, which is usually associated with a milder form
of mental retardation, and FRAXF, which is not consistently associated with mental retardation. These two mutations also have CGG
repeat expansions and are distal to the FMR1 site. The transcriptional silencing of the FMR2 gene also has been implicated in FRAXE
mental retardation. FRAXE individuals have been shown to exhibit learning deficits, including speech delay and reading and writing
problems.

The frequency of the premutation and mutation may be variable in different populations because of founder effects (42 ). Thus, the
prevalence in an English study was 1 in 2,200, and in an Australian study it was 1 in 4,000, but it
 P.632

was higher in Finland, where it is proposed that the initial settlers included one or more fragile X carriers.

Behavioral Phenotype
There is a substantial degree of genetic and phenotypic heterogeneity in the physical, cognitive, and behavioral phenotype. The
behavioral phenotype has been the subject of considerable study and includes mental retardation and learning disabilities, language
impairment, hand flapping, gaze aversion, perseveration, and neuropsychiatric disturbance, principally attention-
deficit/hyperactivity disorder and pervasive developmental disorder–like symptoms. These patients are more interested in social
interactions than those with autistic disorder; the avoidance of social contact may be secondary to hyperarousal or increased
sensitivity to stimuli associated with social situations. The behavioral phenotype may be more helpful than the physical phenotype in
diagnosis because most prepubertal patients do not have macroorchidism or the characteristic long face.

Attentional difficulty and concentration problems are commonly associated, and hyperactivity may be a presenting symptom in
nonretarded boys with fragile X syndrome. Self-injury, most commonly hand biting and scratching, may be elicited by excitement
and by frustration. Female patients with fragile X syndrome may be unaffected, although abnormalities in social interaction,
thought process, and affect regulation have been reported in carriers. Both schizotypal features and depression have also been
found in carriers.

Most girls with the full mutation show shyness and social anxiety. In women with the full mutation, the social anxiety is associated
with social awkwardness and schizotypal features. Anxiety disorders, avoidance disorder, and mood disorder symptoms are common
(42 ).

Gaze Aversion
Gaze aversion is a striking feature of affected males with fragile X syndrome. There is consistency in gaze aversion over repeated
trials in the same individual; nearly all male patients with fragile X syndrome who are more than 8 or 9 years old avert their gaze on
greeting another person. Their unusual greeting is characterized by both head and gaze aversion along with an appropriate
recognition of the social partner (45 ). This greeting response is qualitatively different from gaze aversion that is described in
autistic patients. Those with Down syndrome and nonspecific mental retardation do not show this behavioral pattern on greeting.
The idiosyncratic gaze behavior in fragile X syndrome may disrupt social interactions. Despite their apparent social anxiety and
aversion to eye contact, male patients with fragile X syndrome are otherwise socially responsive and can be affectionate.

Speech and Language

Speech and language in fragile X syndrome is generally delayed, even thought the IQ may be in the normal range. Deficits in both
receptive and expressive language include dysfluency, production of incomplete sentences, echolalia, palilalia (reiteration of the
speaker’s own words and phrases in a perseverative manner), verbal perseveration, and poor fluency in conversation. Compulsive
utterances and shifts in speech pitch are common, and auditory processing and memory deficits are present.

Etiology
The FMR1 protein is expressed most abundantly in neurons and testes with the localization primarily in the cytoplasm. High
concentrations of FRM1 mRNA have been found at the synapse in rat brains, especially in areas involved in synaptogenesis in the
hippocampus, cerebral cortex, and cerebellum (46 ). Hinton et al. found thin and immature dendritic branches with small synapses
in neuroanatomic studies of the neocortex in three male patients with fragile X syndrome (47 ). The expression of the FMR2 protein
also has been characterized. To characterize the expression of the FMR2 protein, polyclonal antibodies were raised against two
regions of the human FMR2 protein and were used in immunofluorescence experiments on cryosections of mouse brain. The FMR2
protein is localized in neurons of the neocortex, Purkinje cells of the cerebellum, and the granule cell layer of the hippocampus.
FMR2 staining is shown to co-localize with the nuclear stain 4,6-diamidino-2-phenylindole (DAPI) and confirms that FMR2 is a nuclear
protein. The localization of FMR1 and FMR2 protein to the mammalian hippocampus and other brain structures involved with
cognitive function is consistent with the learning deficits seen in patients with fragile X syndrome. Comprehensive treatment of
fragile X syndrome is described by Hagerman and Cronister (48 ).

Williams (Williams–Beuren) Syndrome

WMS is a rare (1 in 25,000), genetically based neurodevelopmental disorder associated with a characteristic physical, linguistic,
cognitive, and behavioral phenotype. This syndrome provides a unique opportunity to study personality development, linguistic
functioning, and visuospatial development. The syndrome is characterized by congenital facial and cardiovascular anomalies
(supravalvular aortic stenosis and peripheral pulmonary stenosis), failure to thrive, and mental retardation that may be
accompanied by transient idiopathic infantile hypercalcemia (49 ). Adolescents with WMS have expressive language abilities that are
better than expected for their mental age. Because of their hyperverbal speech, the investigation of WMS allows the study of the
dissociability of components of language and other cognitive brain systems. In mentally retarded patients with WMS, linguistic
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abilities may be selectively spared, unlike language learning disability occurring in normally intelligent children (50 ).

In WMS, a deletion of 1.5 Mb on one copy of chromosome 7 results in the specific physical, cognitive, and behavioral features.
Molecular dissection of the WMS phenotype may lead to identification of genes important in human cognition and behavior.

Genetics
WMS is caused by a chromosomal deletion at 7q11.23. A contiguous gene deletion disorder, it results from hemizygous deletion of
about 20 genes (51 ). This chromosomal region is highly repetitive, and the deletion arises from recombination between misaligned
repeat sequences flanking the WMS region. The deletion breakpoints cluster within the repeats, so most patients with WMS have
similar, although not identical, deletions of 1.5 Mb.

The first deleted gene identified in the critical region was that for elastin (ELN). Studies of patients having deletions or point
mutations confined to this gene showed that hemizygosity for ELN causes supravalvular aortic stenosis but not the other typical
features of WMS.

Several other genes have now been identified that are deleted in most patients with WMS. These include the following: LIMK1,
which codes for a protein tyrosine kinase expressed in the developing brain; that for syntaxin 1A (STX1A), which encodes a
component of the synaptic apparatus; RFC2, which codes for a subunit of the replication factor C complex involved in DNA
replication; and FZD3, homologous to the Drosophila tissue-polarity gene, “frizzled” (51 ).

Cognitive Phenotype
Bellugi et al. proposed that “the cognitive hallmark of WMS is dissociation between language and face processing (relative strengths)
and spatial cognition (profound impairment)” (50 ). The WMS phenotype demonstrates specific dissociations in the higher cognitive
functions. These investigators proposed that general cognitive deficits are present but linguistic abilities are spared. They found
extreme spatial cognitive deficits with intact face processing. Of special interest is the social phenotype in WMS: an overly friendly,
engaging personality and excessive sociability with strangers (52 ). WMS subjects show an unusual positive response in their social
judgments of unfamiliar persons.

Howlin et al. investigated cognitive, linguistic, and academic assessments in a representative sample of 62 adults with WMS (average
age of the group was 26 years; mean full-scale IQ was 61) (53 ). Less difference was found in verbal and performance IQ and
between receptive and expressive language skills in the adults than that found in children. Still, subtest scores documented an
almost identical cognitive profile to that found in children. Reading, spelling, arithmetic, and social adaptation remained at a low
level, with functioning around a 6- to 8-year age equivalent. The consistency in intellectual abilities in both child and adult studies
of patients with WMS supports the notion of a syndrome specific pattern of cognitive, linguistic, and adaptive functioning.

The use of adult neuropsychological models to explain developmental disorders of genetic origin such as WMS has been challenged
(54 ,55 ). It is assumed that uneven cognitive profiles found in childhood or adulthood in WMS characterize infant starting states and
that modules underlying these abilities start out either intact or impaired. However, findings from two experiments with infants
with WMS (selected for study based on claims of innate modularity) suggest a within-syndrome double dissociation: for numerosity
judgments, WMS subjects do well in infancy but poorly in adulthood, whereas for language, WMS subjects show poor performance in
infancy but do well in adulthood. The theoretic and clinical implications of these findings in WMS emphasize the importance of an
developmental approach to neurogenetic disorders. Karmiloff-Smith et al. previously proposed that in WMS, language follows a
different path to normal acquisition and may turn out to be more like second language learning (56 ).

Finally, Tager-Flusberg et al. tested the hypothesis that the WMS phenotype involves sparing abilities involved in the domain of
understanding other minds (mentalizing or theory of mind) (57 ). They compared a group of mentally retarded adults with WMS to an
age-, IQ-, and language-matched group of adults with PWS, and a group of age-matched normal adults, on a task that tests
mentalizing ability. The task involved identifying the correct labels to match photographs of complex mental state expression
focused on the eye region of the face. The adults with WMS performed significantly better than the adults with PWS on this task,
and about half the group performed in the same range as the normal adults. Such findings provide support for the proposal that
mentalizing is a distinct cognitive domain. The authors proposed that this sparing of cognitive capacity could be “linked to the
relative sparing of limbic-cerebellar neural substrate in WMS, which is also connected to cortico-frontal regions that are known to be
involved in understanding complex mental states.”

Linking Genes and Cognition

An approach to studying cognition is to carry out genetic and psychometric testing of patients who have small deletions within the
WMS critical region. LIMK1 and STX1A are good candidate genes to investigate cognitive or behavioral aspects of WMS. The gene for
LIMK1 was implicated as a cause of the visuospatial characteristics of WMS (58 ); however, other investigators were unable to
substantiate this association in three further cases (59 ). The genes for STX1A
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and FZD9 were proposed as involved, based on brain-specific gene expression in the developing (FZD9) or adult (STX1A) central
nervous system. However, when these genes were underexpressed by 50%, as is expected in WMS, Korenberg et al. reported that
deletion of these genes was not associated with significant effects on overall cognition (51 ). However, these authors did propose
that genes responsible for mental retardation and other features of the disorder are “located in the region telometric to RFC2
through GTF21 at the telometric border of the deletion.” Moreover, mild cognitive deficits reported in a subject deleted for elastin
and LIMK1 genes (59 ) were consistent with findings in those with deletion of genes in the WMSTF through LIMK1 region having mild
cognitive deficits. Thus, studies of patients with rare and atypical deletions may be informative in identifying candidate genes to
understand the cognitive deficit.

Linking Anatomic and Behavioral Changes

WMS is associated with specific neuromorphologic and neurophysiologic findings. There is proportional sparing of frontal, limbic, and
neocerebellar structures on magnetic resonance imaging (60 ). Abnormal functional organization of the neural systems that underlie
language processing is revealed through studies using event-related potentials (61 ). Event-related potential studies suggest
abnormal cerebral specialization for spared cognitive functions in WMS. The lack of uniformity in the cognitive, neuromorphologic,
and neurophysiologic domains of WMS makes it a compelling model for elucidating the relationships among cognition, the brain, and,
ultimately, the genes.

Another approach is to investigate anatomic changes in brain regions in WMS that may the result of gene deletions. In WMS,
Galaburda and Bulligi found that the overall shape of the brain is not consistently abnormal (62 ), although in some cases abnormal
brain shape is apparent. The most consistent anatomic finding is abnormal length of the central sulcus producing an unusual
configuration of the dorsal central region. This includes the distal portion of the superior-parietal lobule and dorsal frontal gyrus.
These regions may be linked to abnormal behavior in patients with WMS. Cytoarchitecture of WMS forebrain appears mostly normal,
although subtle dysplastic changes are noted. Abnormal neuronal size of cortical neurons was suggested in one region and may be
linked to increased subcortical connectivity. Elastin does not stain in the cerebellum, whereas Lim kinase does stain in cortical
neurons.

Thus, in WMS, the link of neuroanatomy and behavior seems to fit a dorsal ventral dichotomy and not a frontal-caudal, left-right, or
cortical subcortical dichotomy. Gallaburda and Bellugi proposed that the dorsal portions of the hemispheres, the frontal and
parietal-occipital regions, may be involved (62 ). They noted that some language functions are preserved that are linked to ventral
systems. Face recognition, also a ventral function, is preserved despite severe visuospatial dysfunction, a dorsal function. Anatomic
findings also suggest possible involvement of the visually linked lateral nucleus of the amygdala. Galaburda and Bellugi speculated
that this could be related to the lack of appropriate fear in WMS of new and unfamiliar faces, perhaps also threatening ones.
Moreover, because this region may receive auditory projections, WMS subjects may not be sensitive to threatening voice and speech.
Further work is needed at architechtonic and histologic levels to confirm sparing of ventral regions. To understand the linking of
genes with neuroanatomy, it is necessary to find more genes with brain developmental effects. Of particular interest in this regard
is the proposal that the region deleted in WMS may be a hotspot in mammalian brain evolution (51 ). Hagerman outlined a
comprehensive approach to treatment of WMS (63 ).

ANIMAL MODELS: SIMULATIVE OR SUBSTITUTIVE

Part of "46 - Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain "

Animal models may be used to elucidate critical brain mechanisms involved in disorders with behavioral phenotypes. Early animal
models focused on the impact of traumatic events during the developmental period, as exemplified by the social isolation and
chronic stress (learned helpless) models of depression (64 ). These animal models generally simulated rather than substituted for the
disorder. Animal models have used pharmacologic challenges to study neurochemical mechanisms linked to aberrant behavior or
have introduced transgenic mice as substitutive models of conditions with behavioral phenotypes. Examples of these models include
pemoline models of stereotyped self-biting behavior in the rat (65 ), SNAP mutant mouse model of attention-deficit/hyperactivity
disorder (66 ), and transgenic and knockout mouse models for fragile X syndrome (67 ,68 ), MAOA deficiency (69 ), and LND (70 ,71
and 72 ). These models may contribute to the understanding of psychopathology. However, despite genetic replication of a disorder
in the mouse, the behavior may not be replicated, so even these animal models often simulate aspects of the condition and are not
fully substitutive.

Molecular genetic techniques combined with techniques to manipulate the developing mouse embryo make it feasible to produce
such genetic animal models. Embryonic stem cells are isolated from a pregnant mouse with identifiable coat color that acts as a
donor. The embryonic stem cells are grown in cell culture and then are genetically modified with the insertion of genetic material
or through mutation of endogenous genes. Modified embryonic stem cells are microinjected into a blastula that is isolated from
another mouse that ordinarily has a different coat color. The blastula is then reimplanted into a female host mouse and develops in
utero. The inserted stem cells are incorporated into the
 P.635

developing fetuses, and progeny that contain genetically altered cells are chimeras that can be identified by their mosaic coat
colors. As adults, these chimeras, in which genetically modified cells have been involved in the establishment of the germ cell line,
may then transmit the altered gene to their own offspring. It takes several generations to produce an affected animal, by using
these embryonic stem cell techniques that depend on whether the needed phenotype can be produced in the heterozygous,
homozygous, or hemizygous condition.

To illustrate these animal models, we may contrast animal models of LND based on the use of the neurotoxin 6-hydroxydopamine (6-
OHDA), transgenic mouse models of LND, calcium channel blocker models of self-injury, and the mutant mouse model of fragile X
syndrome.

Lesch–Nyhan Disease 6-OHDA (Rat Model)

After the demonstration of dopamine deficiency in striatum in autopsies of brain in three human patients with LND (35 ), Breese et
al. administered 6-OHDA to neonatal and adult rats to test the effects of dopamine depletion in an animal model (73 ,74 ). These
authors demonstrated that the age at which neural function is disrupted is an important factor in the type of motor and behavioral
symptoms observed after a neural insult to basal ganglia structures. They documented a relationship between dopaminergic
supersensitivity and self-injurious behavior. Rats treated with 6-OHDA in the neonatal period demonstrated self-biting with
mutilation when they were challenged as adults with L-DOPA or a D1 dopamine agonist, but no such self-injurious behavior was
found in the adult rats treated with 6-OHDA. Because of the self-biting, the neonatal 6-OHDA–treated rat was proposed as a model
for LND, and dopamine deficiency was linked to self-injury. In these studies, rats that were not HPRT deficient were given injections
of 6-OHDA at 5 days of age to denervate basal ganglia regions. These brain regions developed supersensitive dopamine receptors.
Self-biting was documented in the lesioned animals when they were challenged as adults with a dopamine agonist; however,
untreated adult rats did not show this behavior.

HPRT-Deficient Mouse
For LND, molecular techniques were used to produce two HPRT-deficient strains of mice. One strain was produced by retroviral
interruption of the human HPRT gene in the embryonic stem cells (72 ). Another model was produced through the selection of
embryonic stem cells for spontaneous mutations in the HPRT gene (70 ,71 ). In both instances, the mouse strains produced had
nondetectable levels of HPRT. However, neither strain showed the spontaneous behavioral abnormalities or neurologic presentation
seen in patients with LND. Tests of both cognitive functions and motor functions were intact in these animals. Similar findings were
documented in a double knockout that is HPRT/APRT deficient (75 ).

This HPRT-deficient transgenic mouse model of LND (70 ) may still contribute to our understanding of LND. Reductions in dopamine
of 40% or more (76 ) have been documented (77 ) in these animals. Because of questions about strain differences, Jinnah et al.
studied the caudate nucleus in five HPRT-deficient strains of mice and made comparisons to littermate controls (77 ). Reductions of
dopamine and also of the dopamine transporter of 35% to 40% were found in these animals. These results indicate an abnormality in
the dopamine system despite apparently normal spontaneous behavior.

The absence of behavioral changes in the HPRT-deficient mice was unexpected. Originally, it was thought that uricase, which is
present in rodents but is not present in primates, may act in a protective manner to lessen behavioral manifestations because uric
acid, which normally builds up in the blood in LND, and hypoxanthine, which accumulates in cerebrospinal fluid, would not do so in
mice because of the presence of uricase. This explanation is consistent with the inability of treatment with allopurinol, a xanthine
oxidase inhibitor that prevents the accumulation of uric acid, to improve the behavior disorder in patients with LND. However, the
mice were still found to have reduced dopamine in brain (76 ). Thus, it is the consequence of the HPRT deficits on dopamine, and
possibly other neurotransmitter systems, that leads to the behavior in humans. This mouse differs from the Breese rat model in that
dopamine depletion is complete in the Breese rat model but only 40% to 50% reduced in the HPRT-deficient mouse. Dopamine
depletion is substantially greater in human subjects (76 ) than in the mice; therefore, this difference may account for the
differences in behavior.

Bay K 8644 Model of Self-Injurious Behavior

Another approach to study self-injury is the calcium channel blockers model proposed by Jinnah et al. (78 ). The L-type calcium
channel agonist (+/-) Bay K 8644 causes motor abnormalities in adult mice. These authors showed that administration of this drug
could also cause the self-injurious biting, particularly when it was given to young mice. Self-biting was provoked by injecting small
quantities of (+/-) Bay K 8644 directly into the lateral ventricle of the brain, a finding indicating a central effect of the drug. Similar
behaviors can be elicited by administration of another L-type calcium channel agonist, FPL 64176. The self-biting elicited by (+/-)
Bay K 8644 can be inhibited by pretreating the mice with dihydropyridine L-type calcium channel antagonists such as nifedipine,
nimodipine, or nitrendipine. Moreover, self-biting is not inhibited by nondihydropyridine
 P.636

antagonists including diltiazem, flunarizine, or verapamil. The known actions of (+/-)Bay K 8644 as an L-type calcium channel
agonist, the reproduction of similar behavior with another L-type calcium channel agonist, and the protection from this behavior
that results from certain L-type calcium channel antagonists implicate calcium channels, and their possible association with
neurotransmitter deficits, in the mediation of the self-biting behavior (78 ).

Fragile X Mouse Model

The mutant mouse model of fragile X syndrome demonstrates another use of an animal model for a neurogenetic disorder.
Transgenic fragile X knockout mice were developed to provide an animal model to study the physiologic function of the fragile X
gene (FMR1) and to understand better the clinical phenotype caused by the absence of the fragile X protein.

The fragile X mouse model demonstrates macroorchidism and cognitive, affective, and behavioral features similar to the human
condition (79 ). In the Morris water maze test, the Fmr1 knockout mice learned to find the hidden platform nearly as well as the
control animals, but they showed impaired performance after the position of the platform was modified. The fragile X knockout
mouse exhibited subtle deficits in spatial learning but normal early-phase long-term potentiation.

Jin and Warren expanded these studies by examination of late-phase hippocampal long-term potentiation, the protein synthesis–
dependent form of long-term potentiation, in the Fmrl knockout mice (43 ). Initially, they found that late-phase long-term
potentiation was normal and proposed that either absence of fragile X mental retardation protein has no influence on long-term
potentiation or that any such influence is too subtle to be demonstrated by this technique. Moreover, when they examined spatial
learning in this knockout mouse using the hippocampus-dependent Morris water maze, near-normal performance was observed.
However, because the knockout mouse strain they used differed from that used in the earlier investigations that did show learning
deficits, their studies were repeated using the same mouse knockout line that showed the deficit. Now significant, but subtle,
increased swim latencies on the Morris maze test in reversal trials were found to be in agreement with the earlier studies. Thus,
strain differences among mouse strains influence the behavior in the Fmrl knockout phenotype. Because the finding were subtle,
these authors chose to investigate a paradigm less dependent on hippocampal function, one using the conditional fear paradigm. In
this paradigm, the knockout animals showed significantly less freezing behavior than their wild-type littermate with two types of
stimuli, contextual and conditional fear stimuli. These researchers concluded that that amygdala dysfunction may also be involved
in fragile X syndrome.

These examples from LND and fragile X syndrome illustrate how animal models may contribute to our understanding of behavioral
phenotypes: self-biting in LND and learning and fear responses in fragile X syndrome.

CONCLUSION
Part of "46 - Behavioral Phenotypes of Neurodevelopmental Disorders: Portals into the Developing Brain "

The study of behavioral phenotypes in neurodevelopmental disorders demonstrates the complexity in mapping pathways from genes
to cognition and complex behavioral phenotypes. Behavioral phenotypes occur in disorders with mendelian inheritance (LND) and
nonmendelian inheritance (PWS/AS, FRX). An investigation of these syndromes demonstrates that recognition of the involved gene is
only the first step. Identification of the involved protein and of its expression in brain is critical. To clarify the mechanism, the use
of animal models, neuroanatomic study, brain imaging techniques, systems neuroscience, and detailed descriptions of behavior are
needed. The study of partial variants of the disorder (LND, WMS), comparison of deletion versus UPD (PWS, AS), and the study of
atypical subjects who exhibit some but not all features of the disorder (WMS) are essential in understanding developmental
pathways. Moreover, a neurodevelopmental model is essential because brain modularity of function cannot be assumed. Animal
models must be carefully chosen because genetic background may influence the expression of the disorder. Such models may be
important in simulating aspects of the disorder, but they may not substitute for the human condition. Flint proposed that success in
the study of behavioral phenotypes requires a screen for regions of monosomy, the use of a sophisticated battery of
neuropsychological and behavioral tests to describe the phenotype, a transcript map to identify quickly the genes that are likely to
affected by the deletion, and a way to of deciding which genes are dosage sensitive (5 ). These are challenges that lie ahead as we
continue to investigate behavioral phenotypes as portals to understanding the developing brain.

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71. Williamson DJ, Hooper ML, Melton DW. Mouse models of hypoxanthine phosphoribosyltransferase deficiency. J Inherit
Metab Dis 1992;15:665–673.

72. Kuehn MR, Bradley A, Robertson EJ, et al. A potential animal model for Lesch–Nyhan syndrome through introduction of
HPRT mutations into mice. Nature 1987;326:295–298.

73. Breese GR, Baumeister AA, McCown TJ. Behavioral differences between neonatal and adult-6-hydroxydopamine treated
rats to dopamine gonists: relevance to neurological symptoms in clinical synromes with reduced brain dopamine. J Pharmacol
Exp Ther 1984;231:343–354.

74. Breese GR, Criswell E, Duncan GE. A dopamine deficiency model of Lesch–Nyhan disease: the neonatal-6-OHDA–lesioned rat.
Brain Res Bull 1990;25:477–484.

75. Engle SJ, Womer DE, Davies PM, et al. HPRT-APRT-deficient mice are not a model for Lesch–Nyhan syndrome. Hum Mol
Genet 1996;5:1607–1610.

76. Jinnah HA, Gage FH, Friedmann T. Animal models of Lesch–Nyhan syndrome. Brain Res Bull 1990;25:467–475.

77. Jinnah HA, Jones MD, Wojcik BE, et al. Influence of age and strain on striatal dopamine loss in a genetic mouse model of
Lesch–Nyhan disease. J Neurochem 1999;72:225–229.

78. Jinnah HA, Yitta S, Drew T, et al. Calcium channel activation and self-biting in mice. Proc Natl Acad Sci 1999;96:15228–
15232.

79. Kooy RF, D’Hooge R, Reyniers E. Transgenic mouse model for the fragile X syndrome. Am J Med Genet 1996;64:241–245.

SUGGESTED READINGS
Harris J. Behavioral phenotypes. In: Harris J, ed. Assessment, diagnosis and treatment of the developmental disorders. New
York: Oxford University Press, 1998:251–376.

O'Brien G, Yule W, eds. Behavioural phenotypes. Cambridge: Cambridge University Press, 1995.
 P.639

Section VI
Schizophrenia and related disorders

Daniel R. Weinberger

Schizophrenia and related disorders - Introduction

The schizophrenia section of the Fifth Generation reflects both a subtle shift in emphasis in schizophrenia research and a
refinement of earlier trends. Molecular and systems neuroscience and genetics are the basic biological sciences of psychiatry, and
their increasingly important role in research is reflected in the schizophrenia section. Schizophrenia research is inexorably on a path
from clinical phenomenology to cellular and molecular pathogenesis. Although this complex disease will never be reducible to one
gene or one signal-processing pathway, the molecular origins of risk and of manifest psychopathology are being elucidated. The main
topics covered in the Fourth Generation volume, including treatment of acute psychosis, mechanisms of antipsychotic drug action,
basic models of pathophysiology, application of brain imaging technologies, and efforts to characterize symptoms in terms of
failures of specific neuronal systems, are represented here, but the presentations are more scientifically mature. Consistent with
the completion of the reference sequence for the human genome and the increasing sophistication of approaches to complex
genetic disorders such as schizophrenia, clinical and molecular genetics are now reviewed as a new chapter in this section. Because
of the introduction of more effective and much better tolerated antipsychotic drugs, the economics of treating schizophrenia has
changed, and this topic also is now covered as a new chapter. Finally, a new chapter is devoted to the issue of developing
informative animal models of schizophrenia. The traditional view that a disorder of human perception, cognition, and comportment
could not be modeled in the experimental animal has yielded in the face of heuristic models of neurochemical, physiological, and
molecular aspects of schizophrenia. These models promise to open new pathways for drug discovery and to test specific hypotheses
about etiology and pathophysiology.

Neuroimaging has been a popular tool for asking specific questions about brain anatomy and function that may help to unravel the
pathophysiology of the illness. There is no doubt, after more than 2 decades of neuroimaging research in schizophrenia, that the
schizophrenic brain is abnormal in a variety of experimental domains. The details of the abnormalities are still to be fully clarified,
but the level of analysis has been meaningfully elevated. New technologies in brain imaging have allowed testing of much more
sophisticated hypotheses. Magnetic resonance imaging has replaced positron emission tomography as the primary functional mapping
tool, and new approaches to in vivo chemistry with nuclear medicine resonance spectroscopy and to anatomic tract tracing with
diffusion tensor imaging have revealed abnormalities not approachable with earlier methods. Positron emission tomography has
become primarily a neurochemical assay technique. Positron emission tomography imaging of dopamine systems has refined our
understanding of the role of dopamine in the pathophysiology of a symptomatic episode and of the mechanism of an antipsychotic
response.

Progress in schizophrenia research has followed discoveries in the basic neurosciences. This is illustrated in several chapters that
address the development and plasticity of the prefrontal cortex and the importance of intrinsic prefrontal circuitry and
prefrontocentric distributed networks to the pathophysiology of schizophrenia. The study of postmortem
 P.640

tissues has been revived by the availability of cellular and molecular assay tools that permit surveys of gene and protein expression
at an industrial scale. Numerous provocative molecular findings have emerged from these preliminary forays into the cell biology of
the illness. Although the mystery of this devastating illness is still unsolved, the clues that have emerged in the past 5 years and the
opportunities for discovery that now exist make it very likely that much more of schizophrenia will be understood at the cellular
level in the next 5 years.
 P.641

47
Schizophrenia: Course Over the Lifetime
Philip D. Harvey

Michael Davidson

Philip D. Harvey: Mount Sinai School of Medicine, New York, New York.

Michael Davidson: Sheba Medical Center, University of Tel Aviv, Tel Aviv, Israel.

Among the lifelong remitting and relapsing illnesses, the course of schizophrenia is among the most widely debated. At the core of
the debate are the following questions:

1. What is the best way to investigate the course of schizophrenia?

2. What are the manifestations preceding and shortly after the first psychotic episode?
3. How do the manifestations of the illness, both clinical and biological, change over the life span, especially during later
life and senescence?
4. Should schizophrenia be conceptualized as the response of a stable encephalopathy to different stages of the life cycle
(1 ), as a progressive, degenerating disease (2 ), or as a hybrid of the two concepts (3 )?

This chapter attempts to provide a critical assessment of these questions in light of the latest empiric data and current
conceptualization of this disease.

The results of the major studies on the course of the illness over 20 to 40 years of follow-up are consistent in reporting a chronic,
generally persistent course of illness for 50% to 70% of the patients who receive an initial diagnosis of schizophrenia (4 ,5 ,6 ,7 ,8
and 9 ). However, a more careful examination of the reports reveals marked heterogeneity in course both between and within
cohorts (10 ,11 and 12 ). The reason for this heterogeneity may be that different studies have examined widely diverse samples of
subjects and may also be related to the different definitions of what constitutes a good outcome. These definitions range from
disease-free for the majority of life to simply not floridly psychotic at the time of last assessment (13 ). Very few of these studies
included elderly patients in their samples or accounted for attrition, and even fewer examined longitudinal biological changes. This
is unfortunate because accurate information on the course of schizophrenia is essential to plan the delivery of care, to evaluate
treatment effectiveness, to provide information to newly diagnosed patients and their families, and to advance schizophrenia
research. The paucity of data on the course of schizophrenia is mostly the result of limitations inherent in studying a relatively low-
incidence illness of unknown origin and pathophysiology, with an insidious onset and a course affected by a multitude of personal
and social factors.

 WHAT IS THE BEST WAY TO INVESTIGATE THE COURSE OF SCHIZOPHRENIA?

 WHAT ARE THE MANIFESTATIONS PRECEDING AND SHORTLY FOLLOWING THE ONSET OF THE FIRST PSYCHOTIC EPISODE?
 HOW DO THE ILLNESS’ MANIFESTATIONS CHANGE DURING LATER LIFE AND SENESCENCE?
 SCHIZOPHRENIA: STABLE ENCEPHALOPATHY OR PROGRESSIVE DISEASE WITH CORRESPONDING LIFELONG BIOLOGICAL
CHANGES?
 CONCLUSIONS
 DISCLOSURE

WHAT IS THE BEST WAY TO INVESTIGATE THE COURSE OF

SCHIZOPHRENIA?
Part of "47 - Schizophrenia: Course Over the Lifetime "

Study Design
The ideal way to determine the course of schizophrenia is to follow a randomly sampled birth cohort throughout the entire age of
risk for schizophrenia and then continue to follow the incident cases, and appropriately selected controls, through the entire life
span. A related but less informative strategy is to follow-up apparently healthy persons hypothesized to be at high-risk of
schizophrenia such as first-degree relatives of affected persons (14 ,15 ,16 and 17 ). An alternative strategy is the prospective
follow-up of patients from the first time they seek help for psychosis (18 ,19 ,20 ,21 ,22 ,23 ,24 ,25 ,26 ,27 ,28 and 29 ).
Unfortunately, the birth-cohort strategy is impractical because schizophrenia is a very low-incidence disease (.87%) (30 ), the age of
risk spans over more than 4 decades of life, and the age of risk appears different for males and females. Thus, following a birth
cohort of 10,000 individuals for 40 years, starting at age 5 years, would detect approximately 90 cases of schizophrenia (not
accounting for attrition), a number that is insufficient to make any statement regarding the course of a heterogeneous syndrome
such as schizophrenia. Similarly, the high-risk strategy is limited in scope because it excludes most future patients with
schizophrenia who do not have affected first-degree relatives, in addition to the problems of investing in a very large research effort
for a
 P.642

relatively small number of persons who will convert into cases.

Therefore, the most often employed strategy to map out the course of schizophrenia has been to start the follow-up only after a
diagnosis of psychosis is established (first psychotic episode cohorts). However, these are selected cohorts in that they include only
persons who seek help (often in an academic center) and consent to participate in research (31 ). Furthermore, this strategy does
not provide prospectively collected information on events preceding the first psychotic episode. Moreover, it is conceivable that
some of the patients recruited for the first-episode studies have been chronically ill for several years but undiagnosed (32 ,33 ). This,
in turn, could explain the discrepancies between studies finding differences between first-episode patients and patients hospitalized
on a long-term basis and other studies that do not find such differences (34 ).

Regardless of the study design, all prospectively followed schizophrenic cohorts will be characterized by high attrition. There are
many reasons for attrition: lack of insight, disappointment with the care received, recovery accompanied by the wish to forget and
conceal the experience of illness, and being too sick to maintain contact. Whether following-up 100% of the cohort would find the
outcome to be better, worse, or the same (and in which aspect) remains unclear. Even if the debate on the best way to follow
patients to describe the course of the illness could be settled, and the attrition rate could be reduced (both of which are unlikely),
it still is not clear what defines a case of schizophrenia and therefore who should be followed to elucidate the long-term course of
illness.

What Is a Case of Schizophrenia?

The debate on the nosologic boundaries of schizophrenia is as old as the term itself, and the last hundred years of research have
done little to settle this debate (35 ,36 ,37 ,38 and 39 ). On the contrary, the pendulum has swung back and forth between a
discrete nuclear definition of schizophrenia based mostly on psychotic features and severely impaired functioning and a continuum
that includes questionable psychotic manifestations, schizophrenia spectrum personality disorders, and moderate to severe
deviations from normality based on psychometric indicators (32 ,40 ).

Despite the contribution of the modern diagnostic classifications to the definition of schizophrenia, the abandonment of the
continuum-based concept and adoption of the dichotomous model have not occurred, largely because the continuum model has
received considerable pathophysiologic support. On the contrary, the schizophrenia-nonschizophrenia distinction is a matter of
operational convenience brought about by treatment and economic developments emerging since the 1960s. The emergence in the
1950s of antipsychotic drug treatments that ameliorated psychosis while producing severe adverse effects called on the medical
community to distinguish between patients who should and who should not be treated with these medications. Throughout the 1980s,
as the accounting between providers of health care and health insurance organizations was becoming more thoughtful, the latter
began to demand definitions of which patients were entitled to reimbursement and which were not. Finally, clinical investigators
into the biology of schizophrenia also supported a model clearly distinguishing between schizophrenia and nonschizophrenia as more
amenable to research. Needless to say, the course of illness of a cohort of schizophrenic patients depends on the definition of the
cases enrolled in the cohort (41 ). Hence, until objective biological markers can be combined with phenomenologic criteria to define
a case, the question of the course of “exactly what illness?” will continue to be raised.

It would also be reasonable to assume that regardless of the degree of the cohort’s heterogeneity, part of the variability in the
course of illness is determined by the interaction between the affected individual and a wide array of societal, familial, and
personal interactive influences (42 ,43 ,44 ,45 ,46 ,47 and 48 ). A few of these influences can be captured by careful collection of
demographic and treatment information. However, the effects of changes that occur over many decades, such as changes in health
care delivery, changes in the public perception of severe mental illness, and the interactions between these changes and aging, may
not be amenable to survey and quantification. For example, how will deinstitutionalization, reduction of stigma, intensive
community care, managed care, novel neuroleptics, open international borders and resultant migration, and the influence of
advocacy groups affect the definition and course of schizophrenia?

In summary, the inherent limitations of studying birth and high-risk cohorts, coupled with the observation that many of the dynamic
changes occur over a time span of 3 to 5 years before and immediately after the first diagnosed episode of psychosis, have been the
impetus for the proliferation of first-episode studies in the 1990s. These studies can provide some useful information about
schizophrenia, particularly because most patients experiencing schizophrenic symptoms in Western societies are likely to be
diagnosed and treated at least once by mental health professionals.

WHAT ARE THE MANIFESTATIONS PRECEDING AND SHORTLY

FOLLOWING THE ONSET OF THE FIRST PSYCHOTIC EPISODE?
Part of "47 - Schizophrenia: Course Over the Lifetime "

Premorbid Phase
The observation that that some schizophrenic patients have premorbid abnormalities dates back to Bleuler (49 ). History taken on
the first contact with a mental health professional
 P.643

often reveals subtle or flagrant motor, cognitive, emotional, and behavioral deviations during childhood, social withdrawal and
mood and personality changes during adolescence, and attenuated psychotic symptoms several months to several years before the
first treatment contact and the diagnosis of psychosis (51 ,52 ,53 ,54 ,55 ,56 ,57 ,58 ,59 ,60 ,61 and 62 ). The period immediately
preceding the onset of psychosis, during which behavior and functioning deteriorate from a stable, premorbid level of functioning,
as well as the behavioral changes that identify it, is referred to as the prodrome. However, the factors that precipitate the
transition from prodrome to the first incident of help seeking and the resultant diagnosis are not necessarily distinctly related to the
illness itself.

Factors such as the educational level of patients and their families, socioeconomic status, and availability of health care may all
determine when the first contact occurs (63 ,64 ,65 ,66 ,67 and 68 ). Moreover, events such as the sudden unavailability of a
caregiver able to maintain a highly symptomatic patient in the community or any change in the threshold of abnormal behavior
tolerated by the community can precipitate treatment contact, hospitalization, and diagnosis. Hence, the presence of the
premorbid manifestation, the onset of the prodrome, the emergence of the symptoms that define an episode of the illness, and
ascertainment of the full syndrome of illness including formal diagnosis do not necessarily coincide and are not always clearly
distinct points in time (31 ). Methods employed to investigate the phenomena preceding the first contact for help and the diagnosis
of schizophrenia are the high-risk method, the birth-cohort method, and the historical prospective (or follow-back) method.

The high-risk studies that followed-up children and siblings of patients affected by schizophrenia into adulthood demonstrated that
these relatives were more likely than the general population to be affected by emotional and behavioral abnormalities and abnormal
psychophysiologic reactions (69 ,70 ,71 ,72 ,73 ,74 ,75 ,76 ,77 ,78 ,79 ,80 and 81 ). For instance, one study compared cognitive and
behavioral assessments of twin pairs healthy at the time of testing and discordant for psychoses later on with twin pairs who both
remained healthy. The healthy twin from the ill pair performed better than the ill co-twin but worse than the average of the twins
from the healthy pair (82 ) (Fig. 47.1 ). Thus, abnormalities were found to be associated both with schizophrenia and with being a
nonpsychotic identical twin of a schizophrenic patient. Even though the increased risk can be demonstrated in targeted populations,
this strategy has not been completely successful in defining the premorbid aspects of schizophrenia. This is because most persons
who belong to the high-risk groups represent a small, atypical subgroup of patients with schizophrenia and because of the relatively
small number, approximately 10% to 15% at most (30 ), of high-risk persons who eventually develop schizophrenia.

FIGURE 47.1. Intellectual functioning in members of twin pairs concordant and discordant for schizophrenia.

National health authorities have conducted follow-up studies of persons born in a geographically defined area over a specified
period (birth cohort) to study protective and risk factors for healthy development and disease. Among the most publicized and
complete studies are two British studies: the Medical Research Council National Survey of Development, covering all births during
the week of 3 to 9 March 1946, and the National Child Development Study, covering all births during 3 to 9 March 1958 (83 ,84 ).
Persons born during these 2 weeks were interviewed and assessed, together with their parents, several times during early childhood
and adolescence. Developmental and scholastic achievement data collected on these cohorts were later linked to the data in a
registry containing diagnoses of patients discharged from psychiatric hospitals. An overview of these studies indicates that, as a
group, persons with future schizophrenia cases had delayed developmental milestones, speech and behavioral difficulties, and lower
IQ scores compared with noncases (individuals who did not appear in the psychiatric registry). Although future cases were
overrepresented in the lowest third of the IQ scores, these future cases had scores that were distributed over the entire range. The
decline in IQ was not limited to a particular test, and the magnitude of decline ranged between 0.25 and 0.75 standard deviations
(SD). Thus, the level of performance seen was not necessarily even outside the average range of IQ scores (defined as IQs between
90 and 110, which is 0.67 SD above or below the average score of 100).

Follow-back or historical prospective studies examine the archival premorbid histories of individuals who are already diagnosed as
suffering from schizophrenia. They can be based on the linkage of databases containing routine psychometric tests administered by
educational or military authorities to large numbers of healthy adolescents with national psychiatric registries. This strategy takes
advantage of large-scale, readily available data enabling the testing of hypotheses with high statistical power. The disadvantage of
the strategy is that, like birth-cohort studies, the data contained in the archival assessments are not aimed at the detection
 P.644

of schizophrenia or its premorbid manifestations, which may be responsible for the low predictive specificity found in many of these
studies. Several follow-back studies have produced results very similar to the birth-cohort studies, findings confirming, both
quantitatively and qualitatively, the cognitive and behavioral abnormalities of future schizophrenic patients (85 ).

For instance, one study based on a national population of adolescents called by the nonselective Israeli Draft Board revealed that
apparently healthy persons who several years later developed schizophrenia had lower mean group scores than their healthy
classmates by about 1 SD on items reflecting social adjustment and IQ (53 ). The differences derived from a “shift to the left” of the
future patients, one that was clearly more pronounced on social adjustment than on IQ.

Despite the consistency between the studies’ results, their interpretation remains uncertain. The premorbid signs of the illness are
widely variable, and a single “typical prodrome” cannot be identified. For example, for some persons, the premorbid manifestations
consist of shyness detectable in elementary school, many years before the manifestation of psychosis. For others, the premorbid
manifestations consist of IQ scores 0.67 SD lower than expected, detected in adolescence, or in nonpsychotic paranoid thoughts
manifested several years before the first psychotic episode in persons with unimpaired IQ. Yet for others, the premorbid
manifestations consist of withdrawn behavior and depressed mood preceding psychosis only by a few months. Furthermore, for some
patients, the prodrome is manifested as a crescendo of progressive, continuous deterioration during childhood and adolescence and
for others as the barely detectable presence of a few minor cognitive abnormalities. Finally, it is possible that some of the
variability in the quality and time of manifestations of premorbid manifestations reflects limitations of the study designs, which are
often cross-sectional assessments (34 ). It is conceivable that a true prospective follow-up study, specifically designed to detect
signs of premorbid schizophrenia and conducted from birth through age of risk, would reveal that the same person who manifests
mild delay in developmental milestones as a toddler (56 ), shyness and learning difficulties in elementary school (50 ,52 ), restricted
peer interaction as a teenager (86 ), and depressed mood and unusual thoughts in adolescence (87 ) would have psychosis in early
adulthood (30 ). Alternatively, a particular premorbid manifestation could lead to a particular subtype of schizophrenia (86 ). It is
uncertain whether these various premorbid or prodromal manifestations, which differ in quality, severity, and time of onset, bear
the same relation to the first psychotic exacerbation or to the course of the schizophrenic illness.

Despite these uncertainties and even though with current psychometric tools, premorbid abnormalities are detected only in a few
persons with future schizophrenia, their presence has opened up both conceptual and practical lines of investigation. Conceptually,
it would be interesting to explain the pathophysiologic relationship between the premorbid symptoms and the manifestation of the
illness. Practically, it would be helpful if the prodrome could be developed into a reliable predictor of future illness, based on which
a secondary prevention strategy could be implemented.

Because the clinical manifestation of schizophrenia could represent an accumulation of genetic and environmental risk factors (or
lack of environmental protective factors), the premorbid abnormalities, particularly the early-life ones, could be conceptualized as
markers of vulnerability. This is consistent with a “multiple-hit” hypothesis by which, in addition to the genetic and environmental
factors that have led to the premorbid manifestations, an environmental insult or a gene expressed later in life may be necessary to
develop the full syndrome of schizophrenia. A corollary hypothesis would suggest that, depending on the additional, later insults,
the same early-life manifestations (e.g., avoidant personality traits) could remain stable through life with no pathologic implication,
could evolve into milder mental disorders such as a schizophrenia spectrum personality disorder, or could lead to schizophrenia. If
indeed the phenotype of schizophrenia reflects the consequences of an accumulation of genetic and environmental risk factors,
studying the course of the disease from birth through the end of the age of risk may be required to identify specific etiologic
patterns. Alternatively, it is possible that a subgroup of these persons who manifest certain premorbid abnormalities may be
inevitably destined to manifest schizophrenia in the future, and for these, and only these persons, the prodromal manifestations are
obligatory precursors of the illness.

Is Secondary Prevention a Realistic Goal?

From a practical point of view, it would be tempting to use the occurrence of the premorbid and prodromal manifestations of the
illness to identify persons at imminent risk of developing schizophrenia and to intervene before the onset of the first psychotic
episode, in an attempt to delay or ameliorate it (47 ,57 ,88 ,89 ,90 ,91 ,92 and 93 ). It would be reasonable to hypothesize that any
intervention that would delay or attenuate the first psychotic episode would have a major impact on the long-term outcome of the
illness. This idea draws support from studies indicating that patients with shorter duration of untreated psychosis have more rapid
symptomatic remission and may incur less deterioration in the long run (94 ,95 and 96 ).

However, the relatively low specificity of the premorbid symptoms such as subtle cognitive deficits, poor social adjustment, changes
in personality, and depressed mood has given rise to concerns that an excessive number of persons could be exposed unnecessarily
to the stigma of a provisional diagnosis of severe mental illness. Although it is possible to improve the specificity of prediction, for
example, by
 P.645

targeting only persons at very high risk (e.g., first-degree relatives of schizophrenic patients who also manifest putatively prodromal
symptoms), this strategy would exclude the 90% of future patients who do not have an affected relative. Furthermore, even if the
prediction could be improved, it is not certain that effective prevention exists. Antipsychotic drugs proven to reduce symptoms and
to prevent exacerbation in patients who already experienced psychosis may or may not be effective in delaying the onset of
psychosis.

Moreover, the notion that psychosis exerts a toxic effect on the brain and that longer duration of untreated psychosis should result
in worse outcome has been challenged (20 ,97 ). It has been argued that (a) duration of untreated psychosis cannot be accurately
assessed, (b) the delay in requesting and obtaining treatment is not the cause of a worse outcome but the result of an insidious-
onset illness that is a more severe form, and (c) long duration of persistent untreated psychosis and persistence of psychosis despite
treatment both reflect the same psychosis-severity phenomena without proving a causal relationship between the two. Finally, the
proof that the duration of untreated psychosis correlates with the more relevant indices of outcome such as quality of life or overall
illness outcome is still equivocal.

For all these reasons, the question of treating persons who are not yet floridly psychotic has stirred public debate beyond the
professional community. Yet because of the potential benefits of secondary prevention on one hand and the risks and ethical
implications associated with it on the other, it is essential to search for rational strategies to assess the risk-to-benefit ratio.
Examining such ratios in an area where preventive measurements are already an accepted reality would be such a strategy. For
example, even though after remission from the first psychotic episode, only 60% of drug-free patients have an exacerbation of their
illness within the first year, 100% of patients are routinely treated with neuroleptics. Hence 40% are exposed to the adverse effects
of neuroleptics, although they are not likely to experience a worsening of their symptoms. Similarly, seven families of schizophrenic
patients must go through the effort, expense, and potential adverse effects of intensive family therapy for 1 year, to prevent
relapse on the part of one of seven recently discharged patients with schizophrenia (98 ).

The dilemma of preventive treatment is not limited to psychiatry. For instance, approximately 70 elderly patients with moderate
hypertension must be treated with antihypertensive drugs for 5 years to save one life, and 100 men with no evidence of coronary
heart disease must be treated with aspirin for 5 years to prevent one heart attack (99 ). In a study using the number needed to treat
method, which is the number of persons who need to receive treatment to prevent one bad outcome, it was calculated that one
must administer antipsychotics to 35 adolescents with paranoid or schizotypal personality disorder for 1 to 3 years to delay
hospitalization for schizophrenia by 6 months to 1 year in a single patient. This calculation assumes that approximately 5% of these
adolescents will convert to schizophrenia, and it also assumes a 60% treatment success rate in delaying conversion, which is the
same rate by which neuroleptics can induce extended remission in first-episode patients (100 ).

The early detection and treatment strategy is supported by preliminary results from a community clinic where youths with
prodromal symptoms were treated with open-label neuroleptics plus supportive measures or supportive measures alone (101 ,102 ).
The results indicated that more members of the neuroleptic-treated group were symptom-free for a longer period than similar
youths given only supportive therapy or those who refused to enroll in the trial. In a different study, nonpsychotic, first-degree
relatives of patients complaining mostly of cognitive deficit also were found to benefit from neuroleptic treatment (103 ). In
summary, although there is much interest in the events leading to the first psychotic episode and a strong appeal for secondary
prevention, the information currently available is still tentative. In contrast, much information and a few solid practical implications
regarding the first episode of psychosis are known.

First Episode of Psychosis

Most studies of patients followed for 2 to 5 years after the first episode of illness have provided highly informative data regarding
the early course of positive (3 ,34 ,105 ) and negative (19 ,104 ,106 ,107 and 108 ) symptoms, cognitive functioning
(109 ,110 ,111 ,112 ,113 ,114 ,115 and 116 ), functional status (117 ,118 and 119 ), and response to treatment (95 ). Furthermore,
some of the studies have described radiologic changes in the brain after the first episode (120 ,121 and 122 ).

Often, the appearance or worsening of psychotic symptoms constitutes the trigger for the first contact with a mental health
professional and subsequent diagnosis and hospitalization. Hence, it is no wonder that what is described as the first episode of
schizophrenia is dominated by the presence of positive symptoms, mostly fully formed delusions and hallucinations. Almost 90% of
first-episode patients treated with neuroleptics experience a rapid, albeit transient, remission of their psychotic symptoms. Despite
the good initial response to treatment, relapse with reoccurrence of psychotic symptoms is common. Predominance of negative
symptoms and hebephrenic, catatonic presentations are not part of the characteristic presentation of the first episode. Occasionally,
however, negative symptoms of insidious onset are present on the first episode, and the response of these symptoms to treatment is
very limited. Cognitive deficits are common and relatively severe at the time of the first episode. Performance on most cognitive
tests is approximately 1 SD below age- and education-adjusted expectations, with more that 50% of the first-episode patients
performing even worse (123 ). The impairment affects almost
 P.646

all aspects of cognition; however, specific areas of impairment are distributed unevenly. For example, deficits in memory,
abstraction, and attention are more severe than deficits in verbal or perceptual skills (124 ). This impairment, measured on
remission from the first episode, goes beyond the one-third to two-thirds SD deficit that characterizes the premorbid cognitive
performance of the schizophrenic patients, and it raises the question whether it reflects a progressively deteriorating process. In a
cross-sectional comparison of Raven Progressive Matrixes scores (a valid measure of IQ), it was found that apparently healthy
adolescents closer to their first hospitalization for psychosis performed more poorly than adolescents who were tested several years
before their first exacerbation, but better than patients whose disease had already exacerbated (125 ) (Fig. 47.2 ). Furthermore,
cognitive performance appears to be slightly worse in patients with chronic disease (114 ) in comparison with first-episode patients,
a finding providing indirect evidence of further cognitive deterioration beyond the first episode (110 ,111 ). In contrast to psychotic
symptoms, cognitive functions are less responsive to the neuroleptic treatment administered for schizophrenia (126 ).

FIGURE 47.2. Scores on the Ravens Progressive Matrices as a function of time until first admission for schizophrenia.

In contrast to the evidence from studies of conventional antipsychotic treatments that suggest little improvement in cognition with
treatment, two separate studies demonstrated modest longitudinal improvements in certain areas of cognitive functioning
(111 ,127 ). These findings suggest diversity in the course of cognitive deficit even early in the illness, although they also indicate
that there is no consistent pattern of specific dimensions of improvement. Furthermore, even though an improvement in cognition
was seen in these studies, no research to date has demonstrated that many first-episode patients show evidence of normalization in
their cognitive functioning. Thus, although evidence of worsening in cognitive functioning associated with duration of illness was
collected from the study of patients with a longer duration of illness (124 ), patients with multiple psychotic episodes (116 ), and
elderly patients with continuous psychosis (reviewed later), there is still marked heterogeneity of recovery of cognitive functioning
immediately after the first episode.

Despite the good remission of psychosis achieved by most first-episode patients (95 ), and even though negative symptoms and
cognitive impairments are not very severe at this stage of the illness, most patients are already affected by persistent social and
vocational decline in the first psychotic episode. For instance, in a study reported by Ho and colleagues (128 ), more than half of a
sample of first-episode patients with schizophrenia were found to be supported by public funds within 12 months of their first
episode of illness, and fewer than 25% of them had a job or went to school. Despite evidence of improvement in cognition on the
part of some patients at the time of the first episode, continuing cognitive and functional deficit is the rule.

Taken together, the premorbid and first-episode studies indicate that many of the manifestations of schizophrenia, including
psychosis, are present many months to few years before the formal diagnosis, and most, but not all, patients respond well to
treatment in terms of their positive symptoms and have a better course of illness in several different domains for the first year or 2
of illness than later. Occupational and cognitive deficits are clearly disproportionate compared with the severity of psychotic
symptoms in most cases, despite evidence of improvement on the part of some patients. However, these results may be biased,
because most first-episode studies enroll patients who (a) were sufficiently sick to need hospitalization, but (b) became sufficiently
well to be able and willing to consent to be followed-up after discharge, yet (c) are not sufficiently recovered to be completely out
of the treatment network. More important, most first-episode studies last less than 5 years because of attrition, funding, or other
factors.

HOW DO THE ILLNESS’ MANIFESTATIONS CHANGE DURING LATER LIFE

AND SENESCENCE?
Part of "47 - Schizophrenia: Course Over the Lifetime "

Middle Course of Schizophrenia

Until the early 1990s, the characteristics of schizophrenia in patients older than 55 years were largely the subject of speculation. As
of 1993, it was estimated that less than 5% of all of the research ever performed on patients with schizophrenia had included any
patients older than 55 years (129 ). It was “common knowledge” that by age 55 to 60 years the illness has run its course, psychotic
symptoms had burned out, and most patients did not need or did not benefit from medications. Since the early 1990s, however, a
substantial amount of research on this topic has been completed, with this area one of the fastest developing aspects of research on
schizophrenia. This research has considered all the topic areas covered by studies on younger patients
 P.647

and has yielded a considerable amount of information that has helped to refine thinking about schizophrenia in general.

One of the sources of the common knowledge that the course of schizophrenia was established into old age was the consistent
findings of symptomatic, cognitive, and functional stability on the part of patients after their first few episodes. Although many
patients experience multiple psychotic episodes through middle age and many patients experience continuous psychotic symptoms,
there was little evidence of change in cognitive or functional status on the part of these patients. Most research on the course of
functional status suggests that the impairments noted at the time of the first episode are rarely reduced. Estimates of the
proportion of patients with schizophrenia who are employed are in the range of about 40%, with most patients employed in
noncompetitive, sheltered settings (130 ). Likewise, independent living is the exception for patients with schizophrenia. There is
also no significant evidence that functional status in patients with schizophrenia changes markedly over time or is altered by
treatment with older antipsychotic medications (131 ). This large body of data raises issues of importance when older patients are
studied, including whether changes seen in later life are part of the natural course of the illness or whether they are the result of
additional comorbidities.

Cognitive and Functional Deficits in Older Patients

It has been consistently reported, however, that many patients older than 65 years who have a lifelong course of schizophrenia,
especially those with a history of long-term institutional care, have marked deficits in cognitive and functional status (132 ,133 and
134 ). Similar findings have been reported at different research sites in the United States and in the United Kingdom (135 ). Because
of the lack of data regarding the lifetime course of functional and cognitive deficits in schizophrenia, it is not clear whether the
presence of severe deficits in functioning seen in these elderly institutionalized patients with schizophrenia is the result of
deterioration in their cognitive and functional status or is a lifelong feature of their adjustment. There are multiple potential
methodologic issues associated with the study of older patients, particularly patients with a history of long-term institutional stay.
Among these issues are the difficulty in identifying the patients’ “true premorbid status,” long-term treatment with antipsychotic
medications, extremely invasive somatic treatments, and institutionalization and demoralization, potentially leading to poor
motivation to cooperate with testing. There is no question, as would be expected from studies of younger patients, that chronically
institutionalized patients have low levels of premorbid functioning, in domains of educational, social, occupational, and
independent living skills (132 ,133 and 134 ). However, the functional history of these institutionalized patients is inconsistent with
the idea that their current, grossly impaired status could possibly be their lifelong level of functioning.

Many of these questions are being addressed by a longitudinal cohort study carried out by the Mt. Sinai School of Medicine group
since the late 1980s, as well as other investigators who have become increasingly interested in this population. A study of the
baseline characteristics of the Mt. Sinai sample demonstrated that these elderly patients manifested moderate to severe negative
and positive schizophrenic symptoms not dissimilar to the symptoms present in younger institutionalized patients (133 ). Many of
these patients had cognitive and social performance compatible with dementia (136 ) that could not be accounted for by somatic
treatment, lengthy institutionalization, poor motivation and education, or comorbidity. For example, in the original publication on
this population (133 ), it was demonstrated that psychosurgery, insulin coma, electroconvulsive therapy, and the severity of
negative symptoms were not the factors accounting for cognitive deficits. Relevant to the issue of motivational deficits, in a
subsample of the patients from that study (137 ), the average level of education was found to be more than 11 years, and their
reading performance was higher than the tenth grade level. In contrast to these indicators of educational achievement, the current
average Mini-Mental Status Examination (MMSE) score was 20 (consistent with moderate dementia). Thus, some elderly
institutionalized patients with schizophrenia appear to manifest decline in their functioning relative to premorbid functioning.

Studies of the cognitive performance of elderly schizophrenic patients have identified “double dissociation” performance profiles
that discriminate them from patients with clearly identified dementia (138 and 139 ), and a profile of differential deficits has been
identified. Differential deficits cannot be caused by a single constant factor, such as failing to provide adequate effort when
assessed. These data suggest that studies of very poor-outcome long-stay patients, although clearly reflecting the most seriously ill
subset of the population, are not hugely biased by the obvious factors associated with long institutional stay.

Longitudinal Course of Cognition and Functional Status in Late-Life Schizophrenia:

Patients with Chronic Illness
As noted earlier, some elderly institutionalized patients with schizophrenia appear to manifest decline in their functioning well past
premorbid levels at some time in the course of their illness. The time course, prevalence, and correlates of this decline are as yet
undiscovered. There is surprisingly little longitudinal research on cognitive functioning and functional skill deficits in schizophrenia.
One metaanalysis suggested that indicators of cognitive performance were
 P.648

largely stable over time in 15 follow-up studies of patients with schizophrenia (140 ). The total sample size in all these previous
studies was only 639, and 225 of those patients were chronically institutionalized patients studied by the Mt. Sinai group in a short-
term (1- to 2-year) follow-up study (141 ). In contrast, in two separate published longitudinal studies of the course of cognitive and
functional status in elderly poor-outcome patients with schizophrenia (142 ,143 ), the Mt. Sinai group found that about 15% of these
patients per year showed evidence of cognitive and functional worsening. The second study also demonstrated statistically
significant cognitive and functional decline over an average of 2.5 years in 57 geriatric schizophrenic patients who entered the study
as chronically hospitalized but were reassessed after discharge to nursing home care (143 ). These data suggest that some proportion
of elderly patients with schizophrenia with a history of long-term institutional care experience a notable decline in their functioning
over a relatively brief follow-up period. These data suggest the possibility of some adverse effect of aging after a lifetime of poor
functional outcome and extensive cognitive deficits.

The Mt. Sinai group completed a larger follow-up study based on 1,102 patients. Some of these patients were unavailable for later
study at each of the subsequent reassessments, because they had died or were discharged to nursing home care, where follow-up
could not be performed. The primary analyses examined the development of new-onset severe cognitive and functional impairment.
Patients were divided on the basis of their baseline Clinical Dementia Rating (CDR) (148 ) score, such that patients with baseline
scores of 1.0 or less were considered less impaired. Worsening in cognitive and functional status was defined as having a CDR score
at a subsequent follow-up of 2.0 or greater. At baseline, there were 456 patients with CDR scores of 1.0 or less, whose average
MMSE score was 20.8.

The actuarial life-table method, with discrete interval procedures, was used to assess the cumulative risk of cognitive and functional
decline over the three intervals between assessments, while considering subject attrition. The cumulative “survival” (i.e., no
worsening in cognitive and functional impairment) was then calculated, and survival curves were constructed. At the first follow-up
time beginning at 15 months, 17% of patients met criteria for worsening (corrected), and at the second follow-up time beginning at
30 months, 20.4% of the remaining patients met criteria for worsening. At the third follow-up time beginning at 48 months, 25.3% of
the remaining patients manifested worsening of their cognitive and functional deficits. Thus, over the entire follow-up period, a
corrected rate of cognitive decline of 51% was noted.

The influences of potential risk factors on rates of cognitive and functional decline over the entire follow-up period for the initially
higher-functioning patients were examined. The Wilcoxon statistic was used to measure the difference in survival curves as a
function of risk factor status. Gender was unassociated with risk for cognitive and functional decline, as were neuroleptic treatment
status, age, and age at first psychiatric admission. In contrast, three risk factors were associated with increases in risk for cognitive
and functional decline. Patients with more severe positive (Wilcoxon statistic [1df] = 4.28; p < .05) and negative (Wilcoxon statistic
[1df] = 17.03; p < .0001) symptoms were found to be at higher risk for decline. In addition, patients with more education were less
likely to experience a cognitive and functional decline than were patients with lower levels of formal education (Wilcoxon statistic
[1df] = 8.65; p < .01).

In the final analysis, presented in Fig. 47.3 , the influences the risk factors previously demonstrated as significant predictors of risk
for decline were examined for their influence on the rate of cognitive and functional decline over the entire follow-up period. First,
patients were divided at the median level for both baseline severity of symptoms and education levels and were assigned to one of
four groups
 P.649

on the basis of their status for symptom severity and education level. The Wilcoxon statistic was then used to measure the
difference in survival curves as a function of combined risk factor status. Patients below the median level for education and above
the median for severity of symptoms were at the highest risk of cognitive and functional decline. This group’s level of risk was
significantly greater than those with similarly high levels of positive symptoms but higher levels of education (Wilcoxon statistic [1df]
= 8.49; p < .001) and those with lower levels of positive symptoms and higher levels of education (Wilcoxon statistic [1df] = 15.31; p
< .001). Similarly, a significant interaction was seen between the influences of negative symptom severity and education level on
risk rates for cognitive and functional decline.

FIGURE 47.3. Effects of combined symptom (positive and negative) severity and educational level factors on survival from
cognitive and functional decline over a 60-month follow-up period.

Certain potential limitations that must be addressed in the interpretation of these data. No control for institutionalization as a
direct risk factor was used in this study. Despite the difficulty in identification of such as a group, there is no other direct way to
index institutionalization effects. Similarly, these data do not control specifically for the development of subtle new-onset medical
conditions. This is a less difficult question to address in later research. Finally, as noted earlier, multiple additional factors,
including subtle environmental changes, may interact with the easily measured risk factors examined in this study.

Longitudinal Course of Cognition and Functional Status in Late-Life Schizophrenia:

Better-Outcome Patients
Although the studies just reviewed indicate that some proportion of poor-outcome patients experience cognitive and functional
decline, there is no evidence to date of cognitive decline in patients with a history of better lifetime functional outcome. Cross-
sectional comparisons of older and younger better-outcome patients conducted by the University of California at San Diego (UCSD)
group found little evidence of relatively poorer cognitive performance on the part of older patients (138 ,144 and 145 ). It is
impossible to determine, of course, from cross-sectional data that these older better-outcome patients, with minimal evidence of
previous decline in their cognitive and functional status, would never experience a decline at a later date. Furthermore, the
proportion of patients in the UCSD samples older than 65 years was only about 15%, a finding suggesting that if the risk of cognitive
and functional decline increases with age, these patients may only be entering the period of increased risk. Finally, few of these
patients had a history of symptom severity consistent with extended periods of treatment-refractory psychosis, and very few would
have met the criteria for kraeplinian status previously demonstrated to be associated with very poor lifetime functional outcome
(146 and 147 ). These data suggest the need to determine whether long-term institutionalization or the patient characteristics that
cause institutionalization are the operant factors in the cognitive decline seen in worse-outcome patients. One of the best possible
strategies could be to perform a longitudinal comparison of outpatients with and without a prior history of long-term institutional
stay, to separate patient characteristics from current environmental factors.

Persistent Symptoms Revisited: Duration of Continuous Psychosis

The data from follow-up studies of poor-outcome patients suggest that persistent schizophrenic symptoms, combined with evidence
of premorbid educational underachievement, are associated with marked increases in risk for functional decline over relatively short
follow-up periods. These data again raise the issue of persistent symptoms as a risk factor for the later course of illness and also
suggest that evidence of lifelong intellectual compromise may increase this risk. These data may help to address some of the
differences in findings between previous studies of ambulatory patients and these extremely impaired, continuously refractory
patients. First, these institutionalized patients have persistent symptoms that have kept them hospitalized for decades and clearly
distinguish them from ambulatory samples. As previously demonstrated, functional deficits and negative symptoms do not interfere
with discharge to nursing home care in this population, whereas persistent positive symptoms do (149 ,150 ). Second, these patients
are all older than 65 years. In previous longitudinal studies, even institutionalized patients younger than 65 years old had essentially
no risk of cognitive and functional decline over a 6-year follow-up period (151 ). It would not be a surprising finding that ambulatory
patients in this age range who have never been institutionalized would not have elevations in their risk for decline either.

These data may provide a heuristic for understanding the variance in outcome, measured by cognitive performance and ratings of
functional status, in older patients with schizophrenia. In institutionalized patients with similar periods of institutional stay, MMSE
scores range from 0 to 30, and functional limitations range from moderate deficits in social skills to incontinence and complete
dependence on others for feeding and bathing. In addition, better-outcome patients clearly have indications of higher levels of
premorbid and current cognitive functioning. These data suggest that the interaction of reduced levels of educational attainment,
often referred to as a marker of cognitive reserve (152 ), and particularly persistent symptoms of illness, may predict functional
decline. The previous suggestion that education attainment is an indicator of a cognitive risk-protective factor for dementia (153 )
appears relevant to schizophrenia. Thus, patients with schizophrenia in late life who have severe and persistent psychotic symptoms,
as well as reduced levels of educational attainment, appear to have a much greater risk of worsening in functional status than
 P.650

patients whose positive symptoms are less treatment refractory and whose cognitive reserve may be greater.

The length of time that some of these patients have experienced continuous psychotic symptoms, despite conventional antipsychotic
treatment, is staggering. Some of these patients have been treated since the 1950s with conventional medications, with little relief
of their symptoms. The duration of untreated psychosis seen in typical samples of first-episode patients with schizophrenia pales in
comparison with these histories of continuous psychosis. This duration of continuous psychosis is much more similar to that typically
seen at the time of the initial introduction of antipsychotic medication in the 1950s. At that time, long duration of untreated
psychosis was found to be associated with risk for greater functional deficit after initiation of antipsychotic treatment than for
patients whose symptoms were treated sooner after the development of illness (96 ). Much later research will need to address the
issues of the impact of continuous psychotic symptoms, in terms influence on the course of illness and whether continuous psychosis
despite treatment has the same impact on development as lengthy periods of untreated psychosis at the outset of the illness.

SCHIZOPHRENIA: STABLE ENCEPHALOPATHY OR PROGRESSIVE DISEASE

WITH CORRESPONDING LIFELONG BIOLOGICAL CHANGES?
Part of "47 - Schizophrenia: Course Over the Lifetime "

A most controversial aspect of schizophrenia is whether the few biological and many phenomenologic abnormalities reported are
consistent with a degenerative, progressively deteriorating course of the illness (154 ,155 ,156 ,157 and 158 ) or a static course for
accounted by an early (developmental) insult (1 ,159 ,160 and 161 ). The neurodevelomental models suggest that a perinatal
neuronal insult disrupts normal neural maturation and results in disruption of neuronal circuits and thus abnormal neuronal function.
It is further postulated that the clinical manifestation of symptoms is triggered by interaction between the initial defect with
neuronal maturation processes such as neuronal migration, glial proliferation, and synaptic pruning. This maturation process, in turn,
accounts for the gap between the hypothesized early-life insult and later clinical manifestation.

The neurodevelopment concept has prevailed mostly because schizophrenia lacks specific biochemical and histologic changes (gliosis,
cellular debris, or amyloid deposits) closely paralleling behavioral abnormalities that define progressive degenerative disorders.
Furthermore, because Alzheimer disease has been seen as the prototype of a progressive neurodegenerative disorder, the absence
of fast and relentless worsening of illness has been taken as evidence against a degenerative hypothesis in schizophrenia. However,
an overview of the data regarding the course and the biology of schizophrenia reveals no sufficient evidence to settle this debate,
and the same behavioral evidence can be interpreted to support either of the two hypotheses. For example, subtle cognitive,
behavioral, and motor deviation from norms are present in childhood, are amplified in adolescence, and exacerbate shortly before
and after the first psychotic episode. This can be interpreted a classic interaction between an early defect and brain maturation or
as the behavioral consequence of a slowly progressive degenerative brain process. In addition, lack of consistent worsening of
psychosis across episodes argues for the static hypothesis, whereas progressively poorer antipsychotic response after each additional
episode could be interpreted as evidence of a slowly progressive degenerative process.

Similarly, biological findings, mostly structural neuroimaging studies, have produced results compatible with both hypotheses
(120 ,121 ,162 ,163 ). Some investigators reported no evidence of progressive brain disease, in either the domains of overall cerebral
size (i.e., cortical atrophy) or the size of the cerebral ventricles (i.e., ventricular enlargement) (164 ,165 ). However, some cross-
sectional and longitudinal studies have produced different results. There are several limitations, of course, in using neuroimaging to
make direct inferences about changes in the brain, particularly in reference to whether these changes are degenerative.

Brain Structure Immediately after the First Episode

One of the interesting recent topics in the area of the course of schizophrenia is that of changes in brain structure after the first
episode. Research by DeLisi and colleagues suggested that some patients recovering from the first episode of the illness have
evidence of progression in the size of their cerebral ventricles (166 ,167 ). This progressive ventricular enlargement is consistent
with that seen in patients with more chronic illness, both during adolescence for childhood-onset patients (168 ) and during middle
age for poor-outcome patients with a more typical age of onset (169 ). These changes are modest, but they are also detected in
relatively short follow-up periods. Because the patients in the studies by DeLisi et al. experienced an increase in the ventricular size
of about 3.5% in 5 years, the amount of change that would be expected over a lifetime, if this change were continuous, could be
substantial.

Changes in Brain Function in Patients with Established Illness

Changes in cerebral structure have also been noted in patients with an established illness. In a 5- year prospective study (169 )
comparing middle-aged patients with schizophrenia who varied in their lifetime functional outcome from chronic “kraeplinian”
patients with community dwellers, the kraeplinian patients demonstrated progressive ventricular enlargement. These data,
consistent with those of
 P.651

the first-episode and childhood-onset studies, suggest that the cerebral change is dynamic over the life span in patients with
schizophrenia. In addition, two separate sets of cross-sectional studies, examining p300 prolongation, suggested that older patients
have longer latencies (170 ,171 ). The finding of prolonged p300 latency can be associated with the presence of neurodegenerative
diseases (172 ). Finally, cross-sectional studies have also found that older patients show relatively greater atrophic changes in the
size of the olfactory bulb (173 ), and this has been found to correspond to a concurrent deterioration in olfactory sensitivity (174 ).
Because olfactory deficits are also detected with consistency in patients with degenerative conditions, these data are consistent
with the p300 data just reviewed. The data to date on the processes of dynamic cortical change in schizophrenia are hardly
conclusive. There are, however, multiple, albeit indirect, suggestions that the idea that brain structure and function in
schizophrenia are immutably stable over the life span in all patients is open to question. These are important issues that will shape
future research in this area.

Is Integration Possible?
In an attempt to account for the phenomenology, course, and epidemiology of schizophrenia, McGlashan and Hoffman postulated
that reduced synaptic connectedness resulting from early, developmental disturbance of synaptogenesis or faulty synaptic pruning is
at the root of schizophrenia (3 ). More specifically, an innately sparse synaptic substrate combined with normal pruning in childhood
and adolescence or a normal substrate combined with abnormally accelerated pruning could theoretically reach a critical threshold
at which deficient synaptic connectivity manifests as abnormal perceptions and ideas. Because the model incorporates both static
and dynamic components, it can account for some of the apparently competing postulations of schizophrenia pathophysiology.
Caution is required, however, because integrative models of the development of schizophrenia have been foiled in the past by the
remarkable heterogeneity of the illness.

CONCLUSIONS
Part of "47 - Schizophrenia: Course Over the Lifetime "

From the time schizophrenia was defined, it has been viewed as a chronic, lifelong disease. Yet its specific manifestations along the
life cycle have been poorly described, mostly because of inherent methodologic difficulties. Improved medical record keeping and
the realization that understanding the course of illness is essential to the understanding the pathophysiology of the illness have been
behind the modern long-term follow-up studies. Determining, for example, the earliest manifestations of the illness or the trigger
and the length of the window of deterioration has implications for preventive and palliative treatment. Even if otherwise possible,
the identification of clinical correlates of progressive cortical atrophy by computed tomography, magnetic resonance imaging, or the
biological meaning of spectroscopic abnormalities in synaptic connectivity requires detailed description of the illness course. As
more research is focused on these issues, important information about the nature of schizophrenia itself will result.

DISCLOSURE
Part of "47 - Schizophrenia: Course Over the Lifetime "

Dr. Harvey has received research support from Janssen and Lilly and has served as a consultant or on a speakers bureau for the
following companies: Pfizer, Janssen, Lilly, HMR, BMS, and Astra-Zeneca.

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48
Neurocognitive Functioning in Patients with Schizophrenia: An
Overview

Terry E. Goldberg

Michael F. Green

Terry E. Goldberg: Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of Health, Bethesda,
Maryland.

Michael F. Green: Department of Psychiatry and Biobehavioral Sciences, University of California School of Medicine, Los Angeles,
California.

Increasingly, neurocognitive paradigms are used to study patients with schizophrenia. With such paradigms, the cognitive
abnormalities in schizophrenia are characterized by means of experimental and clinical tests. These techniques have indicated that
some types of cognitive impairment are not only reliably present in schizophrenia, but are also central and enduring features of the
disease. This chapter, a revision of the one published in 1995, focuses on certain recent advances in characterizing the precise
nature of cognitive impairments in schizophrenia, on understanding the implications of these for treatment given the course and
relationship to outcome of these variables, and on novel applications of neurocognitive approaches to the genetics of schizophrenia.
Cognitive abnormalities were noted by early investigators of schizophrenia. In the original clinical descriptions of schizophrenia
made by Kraepelin (64 ), he commented, “Mental efficiency is always diminished to a considerable degree. The patients are
distracted, inattentive… they cannot keep the thought in mind.” Some years later, Shakow (95 ) began a series of studies in which
he examined abnormalities in patients’ reaction time in response to different types of readiness information and imperative stimuli.
Hunt and Cofer (54 ) noted the intellectual quotient (IQ) of schizophrenic patients to be lower than that of normal controls.
However, the increasing influence of psychodynamic theory tended to minimize the significance of the cognitive deficits of
schizophrenia. It was thought that the deficits displayed on formal psychologic testing were secondary to impaired motivation or
cooperation, gross breakdowns in reality testing, or disordered thought processes.

This view changed rapidly with the advent of in vivo techniques of brain imaging. First, it became evident that the lateral cerebral
ventricles of patients with schizophrenia are larger than those of controls on computed tomography (96 ). Second, functional brain
imaging suggested that the frontal lobe blood flow or metabolism of schizophrenic patients is decreased. Moreover, it was shown
that one type of cognitive impairment, poor performance on the Wisconsin Card Sorting Test (WCST), is directly linked to impaired
activation of the prefrontal cortex in regional cerebral blood flow (106 ). It was within this context that a series of studies in which
broad neuropsychological test batteries were used demonstrated that patients with chronic schizophrenia could not be reliably
discriminated from heterogeneous brain-damaged populations (69 ). These findings led to a reinterpretation of the original
neuropsychological studies; it was increasingly realized that patients with schizophrenia perform in the range typically found in
brain-damaged populations because schizophrenia involves structural and functional abnormalities of the brain that are, in some
sense, primary, and compromise to a differential degree frontal lobe and temporal lobe function. From this perspective,
schizophrenia is viewed as a disease of cortex in which information processing dysfunction is an obligatory concomitant.

We examine certain crucial, conceptually driven issues that derive from this view: What is the course of global cognitive impairment
in schizophrenia? What is the character of neurocognitive impairments in schizophrenia? What is the relevance of traits like
neurocognitive impairment to linkage or association studies in which the goal is to discover susceptibility genes relevant to the
etiology of schizophrenia? We conclude this chapter by noting that neurocognitive impairments may be of prognostic significance in
schizophrenia because of the importance of such functions in providing orientation to and encoding relevant environmental
information, remembering new information, propitiously
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retrieving old information, and working on line with old and new information to make responses or decisions. In this account,
cognitive impairments in schizophrenia can be considered target symptoms that must be corrected.

 COURSE
 COGNITIVE IMPAIRMENTS
 NEUROCOGNITION AS AN INTERMEDIATE PHENOTYPE
 NEUROCOGNITIVE DEFICITS AND FUNCTIONAL OUTCOME IN SCHIZOPHRENIA
 EFFECTS OF MEDICATIONS ON NEUROCOGNITIVE DEFICITS
 CONCLUSIONS

COURSE
Part of "48 - Neurocognitive Functioning in Patients with Schizophrenia: An Overview "

Several contrasting views of the course of cognitive function in schizophrenia are extant. One view suggests that cognitive deficits
become progressively worse throughout the long duration of the illness. After an insidious onset, patients’ intellectual functions
become weaker and social skills become coarser (73 ). A second view suggests that cognitive deficits, once they arise, remain
relatively stable; this view is thus consistent with the notion of a static encephalopathy.

It is clear that once the clinical manifestations of the illness become overt, a sharp decline in cognitive ability takes place in many
patients. In longitudinal studies spanning the premorbid and morbid periods, Schwartzman and Douglas (92 ) found a significant
decrement in the performance of schizophrenic patients tested on an army intelligence examination (standard deviation of nearly
0.5), whereas the score of controls improved. (The patients were similar to controls in the premorbid period.) In the study of
Weickert et al. (105 ), about 50% of a large series of treatment-refractory patients exhibited a large decline in IQ (> 10 points) from
estimated premorbid levels, although a minority of patients had marked cognitive limitations from early on (see ref. 89 ). This is not
say that subtle premorbid deficits do not exist in the majority of patients. Recent population-based studies have demonstrated
attenuations in intelligence measures in schizophrenic patients-to-be (13 ,16 ), in addition to delays in the attainment of some early
developmental milestones (58 ).

Studies of patients during their first episode of schizophrenia substantiate the view that marked cognitive abnormalities are present
at the very onset of the illness. In several studies (8 ,36 ), the neuropsychological profile of first-episode schizophrenic patients was
remarkably similar to that of patients with chronic schizophrenia and did not show a decline at 1- to 2-year follow-up. Both groups
of patients performed poorly on a wide range of tests, including tests assessing memory, executive functioning, and attentional
abilities.

A number of cross-sectional studies searched for evidence of decline during the chronic phases of the illness. Davidson et al. (14 )
reported a decline of two to three points per decade in a global measure of cognitive functioning, the Mini-Mental State Examination,
across the range of 25 to 95 years. (To place this in perspective, the decline in patients with Alzheimer disease is 1.5 points per
year.) Consistent with these results, Harvey et al. (49 ) recently found that elderly patients in this cohort display a marked decline
on a clinical global rating of functioning. However, when patients were followed longitudinally on a variety of cognitive measures for
1- to 2-year periods, little change was evident. As Harvey noted, the changes were not continuous, nor did they occur in the sample
as a whole. It is possible the effects observed were secondary to the interaction between compromised cognitive reserve in
schizophrenia and normal aging; it is also possible that high doses of neuroleptics and long-term institutionalization had a significant
effect on daily living skills and some cognitive functions.

In contradistinction to these findings, Goldstein and Zubin (41 ) found no differences in performance on the complex cognitive tasks
of the Halstead Reitan Battery between large samples of younger and older patients with chronic schizophrenia. Heaton et al. (51 )
demonstrated that a large sample of older schizophrenic outpatients did not manifest deterioration in performance above and
beyond that of normal aging. Hyde et al. (55 ) used a cross-sectional approach in which successive cohorts of schizophrenic patients
were assessed. The study design allowed comparison over an extremely wide range of duration of illness (patients ranged in age
from 18 to 70 years). In addition, each cohort was matched on a measure of premorbid intellectual ability, and patients with
confounding neurologic or systemic diseases were excluded. No significant differences between age cohorts were noted on tests
known to be sensitive to progressive dementias: the Mini-Mental State Examination, Dementia Rating Scale, verbal list learning, and
semantic fluency. Thus, over five decades of illness, no progression was noted. A synthesis of these results suggests that in the
modal patient, a sharp decline in cognitive ability, including general intellectual efficiency, occurs around the time of the onset of
clinical symptoms (± 3 to 5 years), which is followed by an arrest in deterioration and a long period of impaired but stable cognitive
function.

This view of the natural history of schizophrenia is consistent with a neurodevelopmental perspective (107 ) in that a prenatal lesion
remains silent for years before manifesting itself in overt symptomatology and cognitive impairment. Contrary to some
interpretations, Kraepelin (64 ) held to this account, stating, “As a rule, if no essential improvement intervenes in at most two or
three years after the appearance of the more striking morbid phenomena, a state of weak mindedness will be developed which
usually changes slowly and insignificantly.” At the very least, the set of findings suggests that cognitive impairment in schizophrenia
is an enduring feature of the disorder.

COGNITIVE IMPAIRMENTS
Part of "48 - Neurocognitive Functioning in Patients with Schizophrenia: An Overview "

It is possible that nearly every cognitive function of a schizophrenic patient is impaired, and to an equivalent degree (1 ,2 ).
However, we examine three functions in detail because (a) evidence has been found of differential impairments,
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especially in the cognitive domains related to frontal system executive and attentional systems and medial temporal memory
systems; (b) these measures are important in regard to outcome (see below); (c) ongoing and systematic experimental work
indicates that these cognitive functions can be mapped onto neural systems in a principled manner in normal and schizophrenic
persons (30 ); and (d) such measures are useful in intermediate phenotyping.

Attention
Early descriptions of the clinical phenomenology of schizophrenia emphasized impairment of volitional attention. This clinical
observation has been amply supported by many years of experimental study with the use of a wide variety of tasks. Recent models
have sharpened the lines between selective attention, shifting attention, and biasing for and encoding relevant target information.
We investigate some of these functions by examining three tasks: the Continuous Performance Test (CPT), the Covert Visual
Orienting test, and the Stroop Test.

The classic test of selective attention is the Stroop color–word task, in which a word (e.g., red) can be printed in incongruent colors
(e.g., green). Depending on instructions, the task is either to name the actual word or name the ink color in which the word is
written. The attentional task requires the subject to focus selectively on one dimension of the stimulus and ignore or inhibit
contextually inappropriate response tendencies. Normal subjects are slowed when they have to name a color of ink that is
incongruent with the word because they have to inhibit their overlearned tendency of reading the word (see ref. 68 for review).
Schizophrenic patients may have differential problems on this task in reaction time or accuracy, a finding that has been taken to
suggest that they have disproportionate difficulty in inhibiting overlearned tendencies (of reading the word), and may be susceptible
to failure in conditions of cognitive conflict more generally, because they are unable to use the contextual information
appropriately (e.g., by focusing) (22 ,83 ).

Another type of task requires covert shifts of attentional resources in response to task instructions or cues, but this time in
anticipation of a target in a particular location. It was pioneered by Posner and Dehaene (84 ). In this paradigm, participants view a
central fixation point flanked by two small squares, within which a target is to appear. Participants are to respond as quickly as
possible to the target. The reliability with which a preceding cue predicts the location of the target is manipulated and thus
provides a measure of two components of selective attention: engagement (the benefit of a valid cue as evidenced by a fast
response) and disengagement (the cost of focusing on an invalid cue followed by orientation and response to the actual location of
the target). Although qualitative problems have been reported in patients in this domain (e.g., hemifield-dependent RT effects or a
disproportionately slow response to invalid cues), several other studies have not found differences beyond general slowing (35 ).

A test of “sustained” attention, the CPT, has been used to demonstrate consistently that patients with schizophrenia “miss” targets
(77 ). This task involves monitoring a random series of numbers or letters that are represented continuously, often at a rate of
approximately one per second. Participants are asked to detect a target event by pressing a response button and to avoid
responding to foils or distracting stimuli.

In an important study of the CPT, Servan-Schreiber et al. (94 ) showed that when the delay interval between the cue and the
stimulus to which a response is to be made was increased to 5 seconds, patients were disproportionately inaccurate in their
responding. It is possible that the use of rather long delays changes the basic nature to one of delayed response. Thus, in a recent
study, Elvevaag et al. (26 ) were unable to replicate these findings of delay-induced impairment. Indeed, of the numerous errors
made by the patients with schizophrenia, disproportionately more were omission errors at short delay intervals and low target
probabilities, a finding taken to suggest a specific problem in rapidly encoding and acting on the imperative stimulus (i.e.,
constructing a representation of the stimuli to be attended to) under certain unengaging situations (i.e., when few responses are
required) or in biasing perceptual representations for target recognition, presumably by an executive system. Based on work on a
very different paradigm involving short-term memory in the auditory system, Javitt et al. (56 ) also proposed that precision or
efficacy of encoding is impaired in schizophrenic patients.

Memory
Memory impairment is often the most striking feature of neurocognitive impairment in schizophrenia. Newer work has sought to
determine if patients with schizophrenia have qualitative abnormalities in specific stages of mnemonic processing. Toward this end,
Elvevaag and colleagues conducted an encoding study in which subjects had to state whether the letter a was present in a word
(shallow level) or make a decision as to whether the word represented a living thing or not (deep level). Much previous work has
demonstrated that words are recalled better when they are encoded deeply. Preliminary results indicated that although patients’
performance was worse than that of controls, they showed the same benefit of deep encoding (B. Elvevaag and T. E. Goldberg, 2001,
unpublished observations). Although Kareken et al. (60 ) noted that failures in strategy-driven semantic encoding on this task
contributed to impaired performance, their measures were indirect. Elvevaag and colleagues (24 ) also examined schizophrenic
patients’ susceptibility to “false recognition.” They found that patients not only did patients make fewer false-positive errors by
incorrectly recognizing semantic lures when poor general memory
 P.660

(e.g., impaired recall or recognition) was covaried, but they also again were not differentially impaired (24 ). Consistent with this
finding, another study found that susceptibility to interference effects in patients with schizophrenia in so-called AB-ABr paradigms
(in which an initial list of paired associates is presented, followed by representation after the items have been “shuffled”) is not a
differential problem, but rather one that is confounded by general memory problems (B. Elvevaag and T. E. Goldberg, unpublished
observations). Together, these findings demonstrate that patients with schizophrenia respond in a systematic and lawful manner to
a variety of manipulations that target specific mnemonic encoding and orthogonalization (e.g., resistance to interference) processes.
Thus, patients may have subtle impairments in different mnemonic processing stations that additively or interactively produce
effects of large magnitude.

Moreover, the memory problem in schizophrenia does not appear to be one of binding (the ability to learn associations between
various items and distinguish those items from other items that may be similar). This has implications for those who premise
aberrant consciousness based on so-called binding abnormalities (12 ).

Working Memory
Patients with schizophrenia often seem unable to maintain some form of volitional control over the maintenance and manipulation
of even basic information. They appear to have difficulty in formulating plans, initiating them, and flexibly changing a strategy once
it is no longer effective; they also have difficulty in using feedback efficiently. Moreover, patients sometimes have problems when
interrupted; they appear to forget what they were doing after only short periods of interference. One construct that attempts to
capture these types of processing failures is working memory, which can involve not only the storage of information over brief
delays, but the simultaneous storage and processing of information in a capacity-limited store or computational workspace. These
types of behavior have been investigated in various laboratory-based neurocognitive tasks, including the Brown–Peterson test, digit
span, WCST, Intradimensional/Extradimensional Set Shifting Test, and various delayed-response tasks.

Patients with schizophrenia have difficulty on the Brown–Peterson test, in which words have to be remembered over short delays
during which covert rehearsal is prevented, presumably because of a compromised executive component. Patients are differentially
sensitive to longer delays and larger memory sets (38a ). However, patients perform abnormally even on basic short-term verbal
working memory tasks, including digit span (100 )

Several investigators demonstrated that schizophrenic patients exhibit deficits on the WCST, which demands set shifting, response
to feedback, and abstraction (28 ). Patients seem to have difficulty abstracting concepts, and they also make perseverate responses
to incorrect responses. Shallice et al. (95a ) stressed the consistency of executive deficits in their detailed analyses of single cases,
as most patients in their series displayed difficulties in generating rules for the WCST or solving puzzles of the Tower of Hanoi type.

Strong evidence indicates that the WCST may involve the working memory system. For instance, Sullivan et al. (101 ) found that
WCST perseveration is strongly associated with other tests that are thought to require working memory, including self-ordered
pointing (in which a subject monitors his or her own series of responses). Gold et al. (34 ) found the WCST to be highly correlated
with a letter–number span task that involves information maintenance and manipulation over short delays. Statistical differences
between normal and schizophrenic subjects on the WCST were eliminated when letter-number span performance was covaried,
which suggests that both tasks are performed in a similar multimodal or all-purpose cognitive workspace

Much recent work has focused on a task requiring both intradimensional and extradimensional set shifting, in effect a componential
version of the WCST. In intradimensional shifts, subjects are required to change their response set to an alternative design within a
category (e.g., a new exemplar of a line design) while an irrelevant dimension (e.g., shape) introduced earlier continues to be
ignored. In a later stage, an extradimensional shift is demanded as new exemplars are introduced, but subjects are now required to
respond to the previously irrelevant dimension (e.g., shapes rather than lines). Subjects make decisions based on feedback after
each trial. Patients with chronic schizophrenia display markedly impaired attentional set shifting on the
intradimensional/extradimensional task. They demonstrated a significantly higher rate of attrition at the intradimensional shift
stage in comparison with patients with frontal lobe lesions, and they were similarly impaired in comparison with patients with
frontal lobe lesions at the extradimensional shift stage (79 ). Patients with chronic disease also showed impairments in regard to
Tower tasks, spatial memory span, and spatial working memory tasks. Thus, patients with schizophrenia showed an overall deficit in
executive function, often greater than that observed in patients with frontal lobe lesions (80 ).

Several studies have indicated that an impairment of working memory is present in schizophrenia, even in patients who are
relatively intellectually intact. For instance, Pantelis et al. (79 ) found that although patients with a high IQ performed better than
patients with low IQ on the intradimensional/extradimensional task, their performance was still remarkably abnormal, especially in
the extradimensional shifts. Elliot et al. (21 ) were able to confirm these results, even in patients with preserved intellectual
function (i.e., IQs > 100). Weickert et al. (105 ) used a different methodology to reach similar conclusions. They found that nearly
all patients—irrespective of whether they exhibited
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developmentally compromised intellectual function, normal premorbid intellectual function that declined significantly (the modal
subgroup in this study), or preserved intellectual function (i.e., both current and putative premorbid IQ was normal)—displayed
deficits in comparison with a normal control group on the WCST measure of perseveration. These results indicate that working
memory may represent a core deficit in schizophrenia.

Another set of studies also argues for a deficit in working memory’s “visual scratchpad.” They are particularly important because
failure on this class of delayed response tasks is often taken to be the signature of abnormalities in dorsolateral prefrontal cortex,
an area uniquely positioned and designed to exert control over a wide variety of information processing. Using an ocular motor-
delayed response paradigm developed by Goldman-Rakic (40 ) for use in primates, Park et al. (82 ) found that patients with
schizophrenia have grave difficulties maintaining information for location over a brief delay in which they have to perform an
interference task. Fleming et al. (29 ) replicated the findings of this study by using short-term memory for visual patterns. Because
patients in these studies also were impaired on control tasks that did not have delays, encoding problems may have contributed to
overall level of performance.

NEUROCOGNITION AS AN INTERMEDIATE PHENOTYPE

Part of "48 - Neurocognitive Functioning in Patients with Schizophrenia: An Overview "

Although schizophrenia is a heritable condition, linkage studies in which diagnosis is used as a phenotype have been disappointing,
as few significant or replicable chromosomal loci have been identified (20 ). Using psychiatric diagnosis as the major phenotype may
be a major confound. One possible reason is that people may not inherit schizophrenia per se, but rather a variety of information-
processing deficits from which schizophrenia emerges. In other words, although impairment in any given cognitive process may exact
only a small cost in social and vocational functioning, a constellation of impairments may be disabling and result in the emergence
of psychosis. Thus, understanding the genetic architecture of individual processes may well be critical for understanding the
genetics of “schizophrenia.” This account is consistent with a polygenic model of schizophrenia, which implies that the genetic
complexity of schizophrenia qua schizophrenia can be reduced by determining affected status based on neurobiological or
neurocognitive dimensions; the genetic architecture of these dimensions is simpler than that of schizophrenia but segregates both
illness and family risk for illness. This approach involves identifying abnormalities that (a) are quantitative, stable, and enduring; (b)
have a pathophysiology that involves neural systems implicated in the disorder; and (c) have a clear effect on outcome. Certain
cognitive functions may meet these criteria.

A spate of work has examined early stimulus processing and cognition in relatives of patients with schizophrenia. This so-called high-
risk approach has several strengths; for example, abnormalities cannot be attributed to florid psychopathology, cooperation, and
medication. It can also be used to identify cognitive processes that may serve as intermediate phenotypes. Several recent studies, in
addition to many older ones (65 ), have produced strong evidence that relatives of patients have subtle impairments in select
cognitive functions. In a study of Cannon et al. (6 ), siblings showed deficit profiles intermediate between those of patients and
controls in verbal memory, abstraction, attention, and language. Faraone et al. (27 ) examined neurocognitive performance in 35
relatives (sibs and children) and 72 normal controls and found deficits in abstraction, attention, and verbal memory in relatives.
Classification analysis was highly significant. Studies with other paradigms, including backward masking, delayed response, and
verbal working memory, also revealed differences between sibling and controls (10 ,46 ,82 ). In an important study, Cassens et al.
(7 ) showed that a variety of tasks demanding frontal lobe processing, including complex verbal working memory, semantic encoding,
and source monitoring, are not only heritable but are impaired in a stepwise genetically-at-risk fashion in the monozygotic and
dizygotic co-twins of schizophrenic persons.

However, simply examining group differences does not directly address issues of familiality/heritability. A newer approach uses
computations of relative risk (RR) (88 ) in necessarily large samples of controls, sibling, and index cases. One type of RR is based on
comparisons of concordance rates for impairment on a given trait within sibships with the rate of impairment in the general
population. The statistic indicates whether a given quantitative trait is familial and by inference heritable. It is important for
predicting the strength of genetic effects on a given phenotype.

In an earlier study in which Goldberg et al. (39 ) examined monozygotic twins discordant for schizophrenia, they found subtle
attenuations of performance in otherwise-well co-twins when they were compared with normal twins on neurocognitive measures
indexing working memory, speed of information processing, and episodic memory. The concordance for these traits was thus higher
than the concordance for illness. Based on these results, Egan and colleagues (19a ) have used a variety of paradigms to assess
specific cognitive functions in a sample of schizophrenic index cases, their well siblings, and healthy controls. Specific tests were
selected because they reliably measure impairments in schizophrenic patients, are stable, and, in many cases, are known to be
heritable. These criteria are obviously of key importance in determining if a person is impaired because of genetic or environmental
factors, or simply because of measurement error.

They first assessed RR of the CPT, given that prior work from other groups had suggested that this type of test might be sensitive to
certain cognitive impairments in relatives of
 P.662

patients (19 ). In the study of Chen et al. (9 ), who reported an extremely high RR in a Chinese cohort when degraded and
nondegraded versions of a CPT were used, seemingly minor features of the methodology, including a sample in which siblings and
parents with attendant age differences and a low educational level were combined and psychotic relatives were included, might
have led to artifactual inflation of risk computations. Egan et al. (19a ) examined 147 patients with schizophrenia, 193 of their
siblings, and 47 controls. They did not include parents, and the educational of the groups was high and equivalent in siblings and
controls (above grade 13). The IQ of index cases was 94, for their siblings it was 107, and for controls it was 108. The percentage of
siblings carrying the schizophrenic spectrum diagnosis was relatively low—under 5%. In a version of the CPT that had flanking
distracters, they found that 50% of patients, 24% of siblings, and 18% of controls performed one standard deviation below the control
mean when d′ was used as a dependent measure. The RR for this phenotype was 2.1. This finding suggested that the cognitive
demands that this test imposes are under genetic control, the alleles that control this type of information process may be
overrepresented in some families of schizophrenic patients, and that this finding is not redundant with diagnosis. However, it was
not clear whether CPT impairment is a disease-modifying variable or a susceptibility trait, given that the sibling group as a whole did
not differ from controls. In contrast, examination with a test of continuous working memory (the so-called n-back task, which
demands rapid encoding of stimuli, temporal coding, interference by a restricted set of stimuli, and maintenance) revealed that at
“2-back” the RR was above 7.5 and that the sibling group as a whole was significantly impaired in comparison with normal controls,
which suggests that the genetic structure that underlies impaired performance may also confer liability (37 ).

Impairments in several other domains of cognition have also been examined. To assess the suitability of cognitive function for use as
a phenotype in genetic studies, Egan et al. estimated RR (19a ) in the aforementioned cohort of siblings. They hypothesized that the
RR of cognitive dysfunction would be moderate and that different subgroups of families would demonstrate different patterns of
impairment. A set of instruments measuring these constructs included IQ, set shifting and working memory, memory, speed, and
fluency. RR was estimated by using cutoff scores of one and two standard deviations below the control mean. Patients performed
markedly worse than controls on all tests except a measure of premorbid intelligence. The entire sibling group showed impaired
performance on the WCST, letter fluency, and Trails B. Siblings of patients with impaired performance also showed deficits on the
CVLT, Wechsler Memory Scale-Revised (WMS-R), and Trails A. When one standard deviation was used as the cutoff, the RR of siblings
was elevated on the Trails B (RR, 3.8). Trends (p = .01 to .05) toward an increased RR were also seen with the California Verbal
Learning Test (CVLT), WCST, letter fluency, memory for stories, and Wide Range Achievement Test (WRAT) (RR, 1.7 to 2.8). When
two standard deviations was used as the cutoff, the RRs were generally higher, ranging from 4.3 to more than 13. Correlations
between tests of different cognitive functions were weak, which suggests they measure relatively independent processes; factor
analysis confirmed this. Multiple regression analysis also demonstrated that impairment on one test did not predict impairment on
another test in the sibling group. Thus, cognitive dysfunction along several dimensions is familial and probably genetic. The use of
cognitive phenotypes may reduce clinical and genetic heterogeneity and improve the power of genetic studies of schizophrenia.

NEUROCOGNITIVE DEFICITS AND FUNCTIONAL OUTCOME IN

SCHIZOPHRENIA
Part of "48 - Neurocognitive Functioning in Patients with Schizophrenia: An Overview "

By any standard, schizophrenia is a remarkably disabling illness. Among young adults in developed countries, it ranks near the top of
causes of disability in both men and women (75 ). There is now increasing support for the idea that key aspects of disability, such as
reductions in social competence and the capacity for independent living and vocational success, are the result of neurocognitive
compromise.

Although the neurocognitive deficits of schizophrenia have been long recognized, their functional consequences have only recently
been appreciated. Throughout most of the twentieth century, studies of the neurocognition of schizophrenia focused rather
narrowly on attempts to define and characterize the deficits. However, initial forays to study the implications of these deficits for
daily living suggested that neurocognitive deficits may be critical for functional outcome (50 ). Starting in the early 1990s, a large
number of studies examined the associations between rather specific neurocognitive measures and functional outcome in
schizophrenia. This being said, individual studies were underpowered with small sample sizes and were mainly atheoretic. To make
inferences even more difficult, there was little overlap in either the neurocognitive or the functional outcome measures.
Nonetheless, some conclusions from this literature can be drawn.

The literature generally supports the conclusion that neurocognitive deficits are related to functional outcome in schizophrenia
(42 ,45 ), including skill acquisition in psychosocial rehabilitation programs, laboratory assessments of social problem-solving ability
or analogue measures of instrumental skills, and broader aspects of behavior in community outcome and activities of daily living.

Indeed, using intrapair differences in twins concordant for schizophrenia, Goldberg et al. (38 ) observed that virtually
 P.663

all the variance on the Global Assessment Scale could be accounted for by differences in the performance of four neuropsychological
variables: IQ, memory for stories, fluency, and card sorting. In this design, the experience of illness, institutionalization, medication,
psychotic symptomatology, and, of course, genome is shared. Although in one sense the design “stacks the deck” because of its
artificiality, it does illustrate the importance of neurocognition in predicting level of functioning. This is not to say that symptoms
do not have an impact on social and vocational outcome; they do, at least in the short term. What is important to note is that
cognitive impairment may also contribute in a unique manner to outcome. These results suggest that patients’ deficits in learning
new information, rapidly completing tasks, purposefully recalling old information, and generating novel plans or hypotheses may
have an impact on their capacity to perform a job efficiently, take part in social transactions, and make decisions.
It is not clear which neurocognitive measures are the most useful predictors and correlates of functional outcome. Despite this lack
of consensus, studies on the relationships between neurocognition and functional outcome have frequently included assessments for
one or more of the neurocognitive constructs listed in Table 48.1 .

TABLE 48.1. NEUROCOGNITIVE CONSTRUCTS

EXAMINED IN STUDIES OF FUNCTIONAL OUTCOME

This literature includes a substantial number of replicated findings (Fig. 48.1 ). A tally of replications by themselves is not entirely
useful because they do not indicate how many times an association was sought, nor the strength of the relationships. Metaanalysis is
more useful for examining the strengths of associations across studies. Table 48.2 shows the results of metaanalyses of four key
neurocognitive constructs collapsed across the three outcome domains. In this type of analysis, the combined sample sizes are large
and the relationships between neurocognition and functional outcome are highly significant. The metaanalyses demonstrate that
these four neurocognitive constructs are significantly related to functional outcome and that the effect sizes for these relationships
are generally in the medium range.

FIGURE 48.1. Neurocognitive constructs and

functional outcome. Two levels of replication are
represented. A heavy arrow indicates that at least
four studies found a significant relationship
between the neurocognitive construct and the
outcome domain; a thin arrow indicates that
significant relationships were uncovered in two or
three studies.

TABLE 48.2. METAANALYSES:

NEUROCOGNITIVE PREDICTION OF
FUNCTIONAL OUTCOME

Most of the studies in this area have used rather specific measures of neurocognition, and it is the results of these
 P.664

studies that are reflected in Fig. 48.1 and Table 48.2 . Although effect sizes for the individual constructs are mainly in the medium
range, they can become quite large when global or composite measures of neurocognition are used instead of individual measures
(48 ,104 ). Such composite measures indicate that neurocognition can explain between 20% and 60% of the variance in outcome.

How do these relationships compare with those for clinical symptoms? In general, psychotic symptoms (hallucinations and delusions)
fare rather poorly as predictors and correlates of functional outcome (43 ). Negative symptoms are more highly correlated with
functional outcome, but across studies, the relationships are neither stronger nor more consistent than those for neurocognitive
deficits (17 ,48 ,104 ). Little is known about disorganized symptoms, which often constitute a separate syndromal dimension that
includes formal thought disorder, although recent studies suggest that this type of symptom may be related to functional outcome
(76 ,86 ).

The relative contributions of symptoms and neurocognition to functional outcome have only rarely been tested with appropriate
statistical analyses, including multiple regression (48 ,71 ). These studies do, however, support the idea that the neurocognitive
contributions to outcome are stronger than those of symptoms. In one study (104 ), sophisticated path analyses were used to test
the associations among positive symptoms, negative symptoms, cognition, and activities of daily living in two separate samples of
schizophrenic patients. A global measure of cognition had strong relationships with activities of daily living (48% and 42% of the
variance in the activities of daily activities for the two samples). Various causal models were tested in which certain pathways were
omitted. The pathway from cognitive impairment to functional outcome was necessary in the model; the fit was poor when it was
omitted. To the extent that symptoms were correlated with functional outcome, the relationships seem to be indirect. In other
words, although negative symptoms covary to at least a modest extent with neurocognition (17 ,104 ) and their relationship to
function appears to be mediated through this overlap, in toto the results suggest that cognitive impairment, rather than symptoms,
most strongly influences functional outcome.

Just as the neurocognitive deficits in schizophrenia are not fully specific to schizophrenia, the correlations with functional outcome
are unlikely to be specific to schizophrenia. Based on the role of neurocognitive deficits in other disorders, one would not one
expect them to be. The functional consequences of neurocognitive deficits have been observed in a variety of neurologic conditions,
including head injury, Alzheimer disease, multiple sclerosis, Parkinson disease, and AIDS encephalopathy (51 ,87 ,103 ). In fact,
neurocognitive deficits have been associated with activities of daily living even in a nonclinical sample of elderly persons (74 ).

The work so far has been aimed at determining whether neurocognition is related to functional outcome. At this time, it can be
concluded that it is related, and the effect sizes are generally medium for individual constructs and generally large for composite
measures. However, rather little is known about how neurocognition is related to functional outcome. It is likely that some cognitive
domains have direct, causal relationships, although others may be related to functional outcome through mediators, such as social
cognition or the application of knowledge and reasoning to problem solving.

EFFECTS OF MEDICATIONS ON NEUROCOGNITIVE DEFICITS

Part of "48 - Neurocognitive Functioning in Patients with Schizophrenia: An Overview "

One of the most surprising aspects of conventional antipsychotic medications is that although they usually have a profound impact
on psychotic symptoms, their effects on neurocognitive
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deficits tend to be negligible (7 ,11 ,98 ). Occasionally, treatment with conventional antipsychotic medications has led to
improvement in basic perceptual or attentional processes (3 ,98 ). However, it can be concluded that changes in neurocognition, if
they occur, are small compared with the changes in psychotic symptoms. In terms of disability, this presents a rather unfortunate
mismatch in which the domain of illness most affected by conventional medications is not the domain most closely linked to
functional outcome.

Conventional antipsychotic medications probably do not directly impair neurocognitive abilities, but they can do so indirectly when
they involve the simultaneous administration of anticholinergic medications. Anticholinergic medications given for extrapyramidal
side effects compromise certain neurocognitive abilities. Although the range of effects of anticholinergic medications is not well
characterized, they may disrupt aspects of secondary verbal memory that rely on rehearsal strategies (18 ). Other aspects of
memory, including immediate or working memory, appear to be less affected (4 ,38 ), and the effects on other neurocognitive
abilities, such as visual processing, are relatively unknown.

The situation with newer atypical antipsychotic agents appears to be more promising. Initial interest in the neurocognitive effects of
new antipsychotic medications was stimulated by a series of (mainly open-label) studies of clozapine (4 ,38 ,47 ,53 ,67 ). The results
of these studies were surprising in two respects: First, in most of the studies, clozapine treatment resulted in improvement in verbal
fluency (i.e., the ability to generate words that begin with a certain letter or belong to a certain semantic category) and possibly
psychomotor speed. Second, the initiation of clozapine treatment in some studies appeared to have at least short-term detrimental
effects on visual memory and possibly verbal working memory (38 ,47 ,53 ).

A large number of studies are emerging for recently approved antipsychotic medications: risperidone, olanzapine, and quetiapine
(31 ,85 ,90 ,99 ). These studies (again, mostly open-label) have generally shown that they have benefits for neurocognition in
comparison with conventional antipsychotic medications. Indications of short-term detrimental effects, similar to those seen in
some clozapine studies, have so far not been reported for the other newer antipsychotic medications. A rather comprehensive
review (72 ) and a metaanalysis (61 ) of the existing literature have both provided a basis for optimism about the beneficial
neurocognitive effects of newer medications. The metaanalysis of Keefe et al. (61 ) showed significant effects for the new
generation of medications in comparison with conventional agents across a range of neurocognitive areas, including attention,
executive functions, and verbal fluency.

The emerging optimism in this area should be tempered by the fact that the lion’s share of the studies have been “open-label,” with
the associated risks of experimental bias that can accompany such studies. In many of these studies, a single group was assessed at
baseline while on a conventional medication and then assessed again after being switched to an atypical medication. Inferences
from these types of studies are necessarily tentative because no control is made for repeated testings and possible practice effects.
A small number of parallel group blinded studies are emerging for clozapine (4 ,108 ), risperidone (44 ,62 ), and olanzapine (85 ).
These studies offer more convincing support for the proposal that new medications convey neurocognitive benefits in comparison
with conventional medications.

Even more than clinical trial studies of symptom reduction, studies of neurocognitive effects raise questions about alternative
explanations for treatment effects. For the most part, studies have not been designed or analyzed in a way that allows one to rule
out indirect effects. For example, if a newer antipsychotic medication has a better clinical effect than a conventional medication, it
may improve neurocognition as an indirect benefit of greater symptom reduction. This explanation, although plausible, seems
unlikely. Several studies have noted that changes in neurocognition appear to be independent of any changes in symptoms
(38 ,44 ,47 ,67 ,62 ). An alternative explanation is that the neurocognitive benefits of newer medications are mediated by a reduced
need for anticholinergic medications. Although this may turn out be true in some instances, the differential use of anticholinergic
medications did not explain the effects of risperidone on immediate and secondary verbal memory in one project (44 ,92 ). The
beneficial effects of newer medications on neurocognition may be mediated by lower rates of extrapyramidal symptoms. It is not
known whether such effects will be seen in comparisons with very low doses of conventional medications, when side effects are
minimal. Moreover, although a number of mechanisms have been proposed (5-hydroxytryptamine subtype 2A antagonism, indirect
glutamate release, dopamine D4 antagonism), all are problematic, and simple reduction of D2 blockade or transient D2 blockade
remains a viable explanation of “atypicality” (59 ). Thus, it is possible that the administration of conventional neuroleptics in
inappropriately high doses resulted in a lack of improvement, although dose-reduction studies do not support this explanation
(93 ,97 ). The pharmacologic basis for cognitive enhancement remains obscure. In any event, a single neurotransmitter effect seems
unlikely to account for the effects, which probably involve a constellation of actions at serotonergic, adrenergic, cholinergic, and
dopaminergic receptors (72 ). However, it is important to recognize that such actions probably are initiators of changes that
ultimately effect gene expression for major excitatory and inhibitory transmitters and their receptors and neuroplasticity.

Although these alternative explanations require serious consideration, it remains possible that the neurocognitive effects of the new
generation of antipsychotic medications
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are a direct result of the medications themselves (rather than indirect effects of a reduction of clinical symptoms or anticholinergic
medications). If so, the effects are serendipitous. These medications were not developed or initially evaluated with neurocognition
in mind. The possible role of adjunctive pharmacology specifically for neurocognitive deficits is now receiving serious consideration.
A key challenge for directing studies of adjunctive nosotropic medications in schizophrenia is deciding which neurotransmitter
systems are most critical in the pathophysiology of neurocognitive deficits in schizophrenia. One that has been implicated is the
glutamate system (78 ). Based on the cognitive and behavioral effects of antagonists of the N-methyl-D-aspartate (NMDA) subtype of
glutamate receptor, such as phencyclidine and ketamine, it has been suggested that schizophrenia may involve hypofunction of this
receptor (66 ,70 ). Because the NMDA receptor is modulated by glycine, glycinergic agents can provide a useful means for
manipulating glutamate function. Glycine itself has been utilized by Javitt et al. (56 ) with some effect on negative symptoms. The
administration of D-cycloserine, a partial glycine agonist, resulted in a benefit in choice reaction time when it was utilized in
conjunction with conventional antipsychotic medications (32 ), but not when added to clozapine (33 ). A full glycine agonist, D-
serine, demonstrated some success in improving WCST performance (102 ). If these studies of adjunctive agents result in reliable
improvement in neurocognition in schizophrenia, it may become routine for schizophrenic patients to receive a medication for each
of the major domains of illness, clinical symptoms and neurocognitive deficits.

CONCLUSIONS
Part of "48 - Neurocognitive Functioning in Patients with Schizophrenia: An Overview "
In the lay imagination, schizophrenic patients experience problems in living because they are divided against themselves, out of
touch with reality, and disorganized. The view of scientists, once not altogether different, has changed. Not only have the symptoms
been defined and codified, but the neurobiological underpinnings of the disorder have begun to be described. Emerging also is a
view in which cognitive impairments may be a relatively central feature of the disorder. Cognitive impairments are involved in the
genetic etiology of schizophrenia. They seem enduring in that they are present for much of the clinical history and are associated
with outcome. Cognitive impairments also may have a relatively well-delineated profile in which executive, memory, and
attentional deficits are prominent. This account carries with it implications for treatment, in that cognitive impairments should be
considered target symptoms in the same way as hallucinations, delusions, and anergia.

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49
Molecular and Population Genetics of Schizophrenia

Ming T. Tsuang

Michael J. Owen

Ming T. Tsuang: Department of Psychiatry, Harvard Medical School; Department of Epidemiology, Harvard School of Public Health;
Harvard Institute of Psychiatric Epidemiology and Genetics, Boston, Massachusetts.

Michael J. Owen: Department of Psychological Medicine, University of Wales College of Medicine, Cardiff, Wales.

For much of this century, we have believed that genes play a role in the etiology of schizophrenia, but we have been frustrated in
the search for specific mutations by diagnostic dilemmas and technologic shortcomings. Slowly, however, we have made substantive
strides in the diagnosis and treatment of schizophrenia, and considerable progress toward an explication of its underlying
neurobiology. In this chapter, we focus on the epidemiologic and genetic work that underlies much of the current clinical progress
and the promises, perhaps to be fulfilled in the not-too-distant future, to facilitate the treatment, and even prevention, of this
devastating disorder. We begin with updates on the epidemiology of schizophrenia and related disorders, followed by a review of its
molecular genetic bases. We then consider research strategies that promise to explicate the genetic etiology of schizophrenia
further in the next few years.

 EPIDEMIOLOGY OF SCHIZOPHRENIA
 GENETIC EPIDEMIOLOGY OF SCHIZOPHRENIA
 EPIDEMIOLOGY OF SPECTRUM DISORDERS
 MODE OF INHERITANCE
 MOLECULAR GENETICS: LINKAGE STUDIES
 CANDIDATE GENE ASSOCIATION STUDIES
 ANTICIPATION AND TRINUCLEOTIDE REPEATS
 HIGH RATES OF SCHIZOPHRENIA IN ADULTS WITH VELOCARDIOFACIAL SYNDROME
 FUTURE DIRECTIONS
 CONCLUSIONS
 ACKNOWLEDGMENTS

EPIDEMIOLOGY OF SCHIZOPHRENIA
Part of "49 - Molecular and Population Genetics of Schizophrenia "

Psychiatric epidemiology studies the distribution of disorders in well-defined populations. Its methodology emphasizes the use of
representative samples with reliable and valid diagnoses, and a specified time of onset (1 ,2 ), to identify risk factors that explain
why some populations are more vulnerable to psychiatric illness than others. Epidemiologic methods are also critical to an
understanding of how frequently a disorder occurs, a concept often expressed in terms of prevalence, incidence, and lifetime risk.
We consider the risk for schizophrenia by focusing on these measures.

Prevalence Rate
The prevalence rate of a disorder is the number of people in whom the disorder is diagnosed divided by the total number of persons
examined in the population under study. The computed rate depends on several factors: the definition of the disorder, the total
number of individuals examined in the population, and the procedure used to choose whom to examine. Ideally, the sample used to
compute prevalence should be representative of the population as a whole. The prevalence rate is usually expressed as the number
of cases per thousand people surveyed within a year, which is called the 1-year prevalence per thousand. Studies of schizophrenia
from around the world usually report these rates to range from a low of 0.6/1,000 to a high of 17/1,000 (3 ,4 ,5 and 6 ). Lifetime
prevalence rates usually range from 0.9/1,000 to 3.8/1,000 (7 ,8 ,9 and 10 ), depending on the diagnostic criteria and methods of
ascertainment.

The prevalence rates for schizophrenia are relatively constant between countries. Whether we consider East versus West, developed
countries versus less developed countries, or other classifications, the 1-year prevalence of schizophrenia is approximately 0.5% and
the lifetime prevalence is approximately 1.0%. In other words, schizophrenia is found in approximately one-half of one percent of
the population at any point in time.

Incidence Rate
Another way of reporting the rate of schizophrenia in a population is to estimate the number of new cases that appear in the
population during a specified period of time; this is called the incidence rate. Prevalence rates (as discussed above) include both
new and old cases because once schizophrenia has emerged, it usually demonstrates a chronic, unremitting course. In other words,
once patients are classified as schizophrenic, they usually remain schizophrenic. Like prevalence rates, incidence rates vary
according to a number of variables, including the standards of diagnosis. In addition, incidence studies of schizophrenia entail the
difficulty
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of how to define onset. Unlike the onset of some disorders (e.g., stroke or head injury), that of schizophrenia is often insidious and
confused easily with other problems. For example, early symptoms like social withdrawal or unusual thinking may be ignored or
mistaken for indications of depression or substance abuse. Because the time of onset is difficult to determine, incidence rates are
usually based on a patient’s first visit to psychiatric services for schizophrenic symptoms. Thus, misdiagnosed and untreated cases
can affect the accuracy of incidence rates significantly.

The incidence rate is usually expressed as the number of new cases in a given period per 100,000 population. For schizophrenia,
incidence rates range from a low of 0.10 to a high of 0.70 (11 ,12 ). As was the case with the prevalence figures, the incidence of
schizophrenia is generally stable over time and across geographic areas (13 ).

Lifetime Risk
Most persons with schizophrenia first become ill between 20 and 39 years of age. We call this the high-risk period for schizophrenia.
Men tend to be younger at the time of onset than women (14 ,15 and 16 ), although schizophrenia develops in men and women at
approximately equal rates (2 ). Because of the variability of age at onset, prevalence and incidence rates vary according to the age
and sex composition of the population studied. The age distribution is particularly important when the probability or risk that
schizophrenia will develop in a person during his or her lifetime is estimated (i.e., the lifetime risk). To estimate lifetime risk, the
age distribution of the population surveyed should be taken into account (17 ).

The lifetime risk for schizophrenia ranges from 0.3% to 3.7%, depending on the definition of schizophrenia and the method of survey
used (11 ,12 ,18 ,19 ). The World Health Organization study shows a narrow range of lifetime risks in 10 countries around the world
(0.5% to 1.7%) (13 ), although it is higher in some genetically isolated populations in Palau, Micronesia, and areas of Finland (20 ,21 ).
Taken together, studies of the lifetime risk for schizophrenia in the general population suggest it is around 1%. In other words, a
schizophrenic disorder will develop in approximately one in every hundred people at some time in their life.

Risk Factors
Schizophrenia occurs around the world and in all cultures. International differences in rates of the disorder are usually attributed to
diagnostic differences rather than to differences in true rates of illness. The use of broad, ambiguous diagnostic criteria before the
late 1970s was an important factor underlying artificial differences in rates of mental disorders recorded in different geographic
locales. In the 1960s, for example, the hospital incidence of schizophrenia in the United States significantly exceeded that of Great
Britain (28.0/100,000 vs. 17.9/100,000 population), whereas the incidence of mood disorders in British hospitals exceeded that in
the United States (36/100,000 vs. 7/100,000). These differences disappeared, however, when identical methods of diagnosis and
assessment were used (22 ). Similarly, in the World Health Organization study, differences in incidence among 10 countries
diminished when narrow, standardized diagnostic criteria were utilized (13 ,23 ).

A variety of risk factors have received attention in schizophrenia. These include, among others, a family history of the disorder, low
socioeconomic status, complications during pregnancy and childbirth, sex, and fetal viral infection (18 ,24 ,25 ,26 ,27 and 28 ). A
family history of the disorder and a negative relationship to social class are especially strong and frequently replicated risk factors.
The familial basis of schizophrenia is considered further in the section on the genetic epidemiology of schizophrenia. It should be
emphasized that although the focus of this chapter is the genetics of schizophrenia, many of the risk factors for the illness cited
above are environmental, a fact that underscores the combination of genetic and environmental factors underlying the disorder.
This point is stressed further in the discussion of twin studies, below; given an identical twin with schizophrenia, the risk that the
disorder will develop in the other twin (who has identical genes) is far less than 100%.

GENETIC EPIDEMIOLOGY OF SCHIZOPHRENIA

Part of "49 - Molecular and Population Genetics of Schizophrenia "

Family Studies
There is little question that schizophrenia (and related disorders) runs in families. In a review of 40 European studies, selected for
similarities in diagnostic and ascertainment procedures and performed between 1920 and 1987, the lifetime risks for schizophrenia
in relatives of schizophrenic patients were as follows: parents, 6.0%; siblings, 9.0%; offspring of one parent with schizophrenia,
13.0%; offspring of two parents with schizophrenia, 46%; and identical twin of a patient with schizophrenia, 46% (18 ,19 ). Note that
the risk to offspring exceeds the risk to parents. Because the biological relationship is the same (i.e., first-degree relatives), the risk
to offspring of patients should be identical to the risk to parents of patients. This would be true under any genetic model. The
difference occurs because, by definition, parents have reproduced, and the presence of schizophrenia has an adverse affect on the
probability of doing so. The risks to second-degree relatives ranged from 6.0% for half-siblings to 2.0% for uncles and aunts. First
cousins, a type of third-degree relative, had an average risk of 2.0%. Consistent with a genetic etiology, these figures show that as
the degree of biological/genetic relatedness to a schizophrenic patient increases, so does the risk for schizophrenia.

Although recent studies have used more rigorous research methods and narrower, criterion-based definitions of schizophrenia,
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they have essentially confirmed the familial basis of schizophrenia and provided evidence consistent with the genetic hypothesis. In
their family study of the Roscommon area in Western Ireland, for example, Kendler et al. (29 ) reported that the risk to siblings of
schizophrenic probands is 9.2%, consistent with the rates reported by Gottesman (18 ,19 ). The risk to parents of schizophrenic
probands was 1.3%, somewhat lower than the 6.0% reported by Gottesman. In general, narrower definitions in modern studies result
in somewhat lower risk figures than those reported previously. This point was demonstrated by Tsuang et al. (30 ), who reported the
risk for schizophrenia in first-degree relatives of schizophrenics as 3.2%, compared with 0.6% for relatives of nonpsychiatric controls.
Similarly, Guze et al. (31 ) reported rates of 3.6% and 0.56%, respectively. Despite the lower prevalence figures, both these studies
reconfirmed that in comparison with the general population, relatives of people with schizophrenia are at significantly greater risk
for development of the disorder. This in itself, however, is not sufficient to demonstrate a genetic basis. Additional strategies, such
as twin and adoption paradigms, are necessary to parse out genetic and environmental determinants, and these are considered next.

Twin Studies
The two types of twins are monozygotic and dizygotic. Monozygotic twins (i.e., identical twins) share 100% of their genes, whereas
dizygotic twins (i.e., ordinary brothers and sisters) share 50% of their genes. If both members of a twin pair have schizophrenia, they
are considered concordant for the disorder; if one is schizophrenic and the other is not, they are considered discordant. If
schizophrenia were caused by genetic factors alone, then concordance rates for monozygotic and dizygotic twins would be 100% and
50%, respectively. On the other hand, if it were caused essentially by environmental variables, then concordance rates for
monozygotic and dizygotic twins reared in a common environment would be similar.

The empiric data lie between these extremes but provide clear evidence for a genetic component. For example, concordance rates
from twin studies pooled by Kendler (32 ) were about 53% for monozygotic twin pairs and 15% for dizygotic twin pairs. A similar
review by Gottesman (18 ,19 ) demonstrated median concordance rates of 46% and 14% for monozygotic and dizygotic pairs,
respectively. Interestingly, monozygotic twins reared apart have about the same concordance rates as do twins reared together (33 ).
At the same time, monozygotic concordance rates lower than 100% demonstrate an important role for environmental factors. When
they obtained quantitative estimates of the relative roles of genetic and environmental factors by translating genetic concordance
rates into “heritabilities” (the proportion of the variance between individuals that is attributable to genetic factors), Kendler and
Diehl (34 ) found that about 70% of the variance could be attributed to genetic factors in a series of twin studies. Several recent
studies in which DSM-III, DSM-III-R, or DSM-IV diagnostic criteria were used provided even higher estimates of heritability, in the
range of 80% to 86% (35 ,36 ,37 and 38 ).

Studies of twins discordant for schizophrenia have also shed light on the role of genetic and environmental factors. Gottesman and
Bertelson (39 ), for example, followed the Danish schizophrenic twin sample of Fischer (40 ). They reasoned that if genetic liability
is transmitted to the unaffected member of the twin pair, but not expressed because of environmental factors, then their offspring
should demonstrate the same genetic liability. This hypothesis was supported when 24 children of unaffected co-twins showed a
monozygotic concordance rate of 17%, a rate almost identical to that in the offspring of the twins who had schizophrenia. In
contrast, although the risk for schizophrenia in the offspring of dizygotic twins with schizophrenia was similar to the risk in
monozygotic twins, the risk in the offspring of unaffected dizygotic twins was only about 2%. What type of environmental factors
might contribute to the risk for schizophrenia? A variety of possibilities exist, but adverse events occurring early in development
(e.g., gestation, birth) have received the most attention recently, in part because the occurrence of such events during that period
may have particularly far-reaching biological consequences. McNeil et al. (41 ) showed, for example, a relationship of smaller
hippocampi and larger ventricles to complications of labor and delivery in the ill member of monozygotic twin pairs discordant for
schizophrenia. Tsujita et al. (42 ) showed evidence of postzygotic genomic discordance in discordant twins, which could influence
subsequent transcription in one or more genes. It is thus evident from twin studies with a variety of designs that both genetic and
environmental factors underlie the expression of schizophrenia. Adoption studies, considered below, further support this conclusion.

Adoption Studies
Like twin studies, adoption studies can disentangle genetic and environmental causes of disease (43 ). An important series of studies
was performed in Denmark, thanks in part to its system of national registers. Among these, Kety et al. (44 ) studied 5,500 children
from the Greater Copenhagen area who were separated from their biological families and adopted between 1923 and 1947. Although
schizophrenia or a related disorder developed in only 1.9% of the control adoptees, schizophrenia or a related illness was diagnosed
in 8.7% of the adoptees who were separated from a schizophrenic parent. When the biological relatives of schizophrenic and control
subjects were compared, 5.8% of the relatives of schizophrenic probands had (definite or probable) schizophrenia, in comparison
with 0.9% of the control relatives. The rates of schizophrenia did not differ between the adoptive relatives of the schizophrenic and
nonschizophrenic
 P.674

adoptees. Moreover, children born to nonschizophrenic parents but raised by a schizophrenic parent did not show rates of
schizophrenia above those predicted for the general population.

Limitations of adoption studies include the possibility of transmission by the mother during pregnancy or delivery of a liability for
schizophrenia via nongenetic biological, or other type of environmental, factors, even if the child is adopted away soon after birth.
However, Kety et al. (45 ) found that 13% percent of paternal half-siblings of schizophrenic adoptees had schizophrenia, in
comparison with only 2% of paternal half-siblings of nonschizophrenic adoptees. Because paternal half-siblings have different
mothers, these results cannot be attributed to in utero (environmental) effects. Many of Kety’s findings were replicated recently for
schizophrenia (45 ) and for certain schizophrenia spectrum conditions (46 ) in a sample drawn from the rest of Denmark.

Data from the Finnish adoption studies (47 ,48 ) provide additional support for both genetic and environmental influence in the
transmission of schizophrenia. Together, family, twin, and adoption studies show consistently that the biological relatives of people
with schizophrenia themselves show higher rates of schizophrenia and related disorders when compared with appropriate control
groups, regardless of whether they are raised by biological or adoptive parents.

EPIDEMIOLOGY OF SPECTRUM DISORDERS

Part of "49 - Molecular and Population Genetics of Schizophrenia "

The term related disorders is used to describe schizophrenic illness of (generally) lesser severity. In fact, genetic studies provide
evidence for a spectrum of disorders that are similar to schizophrenia and caused by the same genes. A disorder is considered to be
in the schizophrenia spectrum if it occurs more frequently among the biological relatives of schizophrenic patients than it does
among the relatives of people who do not have schizophrenia. Many of the behavioral genetic methodologies used to delineate
genetic and environmental factors in the etiology of schizophrenia (e.g., family, twin, and adoption studies) have also provided
evidence of a genetic etiology in schizophrenia spectrum conditions. Evidence for inclusion in the schizophrenic spectrum is
considered next for several candidate disorders.

Psychotic Spectrum Disorders

In about 9% of the first-degree relatives of schizophrenic patients, a psychotic disorder develops that does not meet the criteria for
either schizophrenia or a mood disorder (18 ,49 ). Two prominent examples are schizoaffective disorder and psychosis not otherwise
specified (NOS). As the name suggests, the term schizoaffective disorder describes patients with features of both schizophrenia and
affective disorders (also known as mood disorders), although subgroups may exist in which either schizophrenia or affective
symptoms predominate. Psychosis NOS is a residual diagnostic category for patients with psychotic symptoms who do not fit into a
more narrowly defined category. In many cases, the NOS designation serves as a temporary diagnosis for patients with new onset of
disease until the course of their symptoms reveals their true diagnosis.

Both schizoaffective disorder and psychosis NOS are more common among the relatives of schizophrenic patients than among the
relatives of nonschizophrenic persons. For example, in a survey of family history and family, twin and adoption studies, Prescott and
Gottesman (33 ) found that 13 of 15 studies demonstrated evidence of a familial/genetic component for schizoaffective disorder.
Consistent with this finding, monozygotic twins show higher concordance rates for schizoaffective disorder than do dizygotic twins
(36 ,50 ).

Nonpsychotic Spectrum Disorders

Personality Disorders
Milder forms of schizophrenic illness are characterized by nonpsychotic symptoms, such as poor social relationships, anxiety in social
situations, and limited emotional responses. Less frequently, mild forms of thought disorder, suspiciousness, magical thinking,
illusions, and perceptual aberrations are also present. These symptoms are observed most frequently in three personality disorders,
including schizotypal, schizoid, and paranoid personality disorders. Several studies found that (DSM) cluster A personality disorder
traits often precede the onset of psychosis in subjects in whom schizophrenia subsequently develops (51 ,52 ). Moreover, in the New
York high-risk project (53 ), offspring of schizophrenic mothers demonstrated elevated rates of these personality disorders when
they were considered together, although not separately.

Most studies of familial prevalence in the biological relatives of schizophrenic patients have related schizotypal personality disorder
to the schizophrenia spectrum more strongly than they have either schizoid or paranoid personality disorder (54 ,55 ). Evidence in
favor of including schizotypal personality disorder is consistent across family (56 ,57 and 58 ), adoption (45 ), and twin studies
(59 ,60 ). Although not all studies have detected a higher rate of schizotypal personality disorder among relatives of schizophrenic
probands (54 ), most investigations, and particularly those with large samples, show higher rates of schizotypal personality disorder
among the relatives of index cases with schizophrenia than among the relatives of control subjects (61 ). The incidence of the
disorder in schizophrenic families has been estimated at between 4.2% and 14.6% (57 ,58 ,62 ,63 ). In contrast, results for schizoid
and paranoid personality disorders have been somewhat more controversial and contradictory, with positive findings sometimes
occurring in combined paranoid–schizotypal or schizoid–schizotypal
 P.675

samples (55 ). Thus, although some symptoms may overlap between schizotypal, schizoid, and paranoid personality disorders,
schizotypal personality disorder is currently the strongest candidate among this group for a nonpsychotic, relatively mild condition
that is related genetically to schizophrenia.

Schizotaxia
Paul Meehl (64 ) first used the term schizotaxia in 1962 to describe a genetic vulnerability to schizophrenia. He suggested that a
subtle but widespread neurointegrative defect results from this vulnerability that predisposes individuals to the development of
either schizotypy or schizophrenia, depending on the protection or liability afforded by environmental circumstances. Later, Meehl
reformulated the concept to allow for the possibility that some people with schizotaxia would not progress to either schizophrenia
or schizotypal personality disorder, although most would (65 ). Eventually, the term schizotypy entered the psychiatric
nomenclature in the form of schizotypal personality disorder. Schizotaxia did not, although the term was used in a general sense by
researchers to describe the liability for schizophrenia. Now, almost 40 years after the concept was introduced, a broad literature
shows that the liability for schizophrenia can be characterized clinically by deficits or abnormalities in psychiatric,
neuropsychological, neurobiological, and psychosocial domains in nonpsychotic, first-degree relatives of people with schizophrenia.

Psychiatric features in such relatives frequently include negative symptoms (e.g., asociality and anhedonia) that are qualitatively
similar to, but quantitatively milder than, those often seen in schizophrenia (66 ). Positive symptoms, however, are usually less
evident in these relatives than they are in schizophrenia or schizotypal personality disorder. Neuropsychological impairments in
biological relatives of people with schizophrenia are also similar to those in patients with schizophrenia, but are generally of lesser
severity (67 ,68 ,69 ,70 and 71 ). The most extensively documented of these impairments involve working memory/attention, verbal
memory, and concept formation/abstraction.

We recently suggested a reformulation of Meehl’s concept of schizotaxia that focuses on these features of negative symptoms and
neuropsychological deficits (67 ). In addition to specifying the clinical consequences of schizotaxia more specifically, our view of the
concept differs from Meehl’s in a few other respects. Among these is whether schizotaxia always or even usually progresses to
schizotypal personality disorder or schizophrenia. Our empiric analyses suggest that the basic symptoms of schizotaxia occur in 20%
to 50% of adult relatives of patients with schizophrenia (68 ,69 ). This rate is considerably higher than the rates of schizophrenia or
schizotypal personality disorder likely to develop in first-degree relatives (≤ 10% for each condition), which suggests that schizotaxia
does not lead inevitably to schizophrenia or schizotypal personality disorder. Our view of schizotaxia also differs somewhat from
Meehl’s formulation in that we include both genetic and nongenetic, adverse biological alterations occurring early in development
(e.g., pregnancy and birth complications) in our conception of the syndrome.

Tsuang et al. (72 ) recently described a set of specific criteria for schizotaxia, but the clinical characterization is still evolving and
the syndrome requires independent validation and further research before it can be used clinically (73 ). These criteria underlie a
conception of schizotaxia as a neurodevelopmental condition resulting from genetic and adverse environmental (e.g., pregnancy or
delivery complications) factors. Current criteria in adult, nonpsychotic, first-degree relatives of schizophrenic patients include
moderate or greater levels of negative symptoms and neuropsychological deficits (as described above). The concept of schizotaxia
demonstrates considerable utility because it accounts for clinical deficits in a sizable proportion of relatives who may not otherwise
meet the criteria for a schizophrenia-related disorder. Moreover, because schizotaxia may be considered as a risk factor for
schizophrenia, as well as a clinically meaningful syndrome in its own right, its recognition may eventually facilitate the development
of early intervention and prevention strategies.

MODE OF INHERITANCE
Part of "49 - Molecular and Population Genetics of Schizophrenia "

Given the evidence outlined above for a substantial genetic contribution to the etiology of schizophrenia, methods such as complex
segregation analysis (74 ) can be used to identify the most likely mode of inheritance. Commonly, a mixed model (75 ) comprising
both major gene and polygenic effects is compared with the submodels of a single major locus and polygenic inheritance. However,
large sample sizes are required to distinguish between models, especially the polygenic and mixed models, so that the practical
usefulness of this approach has been limited to date.

Based on complex segregation analysis and related approaches, the pattern of risks in family and twin studies has been found to be
incompatible with a single locus accounting for all the genetic liability to schizophrenia (76 ,77 and 78 ). However, it has not been
possible to distinguish between a polygenic and a mixed model (77 ). The pattern of risks in family studies, in which the risk
decreases rapidly as the degree of genetic relatedness decreases, is also compatible with a model of multiple loci with epistasis
(interaction between genes) (79 ). However, the number of susceptibility loci, the disease risk conferred by each locus, and the
degree of interaction between loci all remain unknown. The contribution of individual genes to the familiality of a disorder can be
expressed in terms of (s (i.e., the relative risk to siblings resulting from possession of the disease allele (79 ). Risch (79 ) has
calculated that the data for recurrence risks
 P.676

in the relatives of probands with schizophrenia are incompatible with the existence of a single locus having a value of (s greater
than 3. Unless extreme epistasis (interaction between loci) exists, models with two or three loci having values of (s of 2 or less are
more plausible. It should be emphasized that these calculations are based on the assumption that the effects of genes are
distributed equally across the whole population. It is quite possible that genes of larger effect are operating in a subset of patients—
for example, those from families with a high density of illness.

These quantitative genetic investigations provide strong evidence that genetic factors increase the risk for schizophrenia. However,
although it is possible to state that, as a group, siblings of individuals with schizophrenia, for example, have a roughly 10-fold
increased risk in comparison with the general population, it is not currently possible to translate this figure to the level of risk for a
particular sibling in a particular family. Similarly, heritability estimates refer to variations in liability to schizophrenia in the general
population and have no simple meaning for an individual. Another important point is that risk to related individuals does not directly
equate with genetic risk because some relatives carry one or more susceptibility alleles for schizophrenia but remain unaffected
throughout their lives. In other words, the accumulation of susceptibility alleles, environmental risk factors, and complex
interactions between risk factors probably all play a role in determining who becomes ill. Therefore, to quantify genetic risk, it is
necessary to identify the susceptibility loci themselves at the molecular level.

MOLECULAR GENETICS: LINKAGE STUDIES

Part of "49 - Molecular and Population Genetics of Schizophrenia "

The first generation of systematic molecular genetic studies of schizophrenia effectively ignored the evidence for genetic
complexity and targeted large, multiply affected pedigrees for analysis. This was done in the hope that such families, or at least a
proportion of them, were segregating genes of sufficiently large effect that they could be detected unequivocally in this way. This
approach has been successful in other complex disorders—Alzheimer disease, for example, in which mutations in three genes, APP,
PS1, and PS2, are now known to cause rare forms of the disorder. In such cases, the disease is of unusually early onset and is
transmitted through multiplex pedigrees in an autosomal dominant fashion (80 ,81 and 82 ).

Unfortunately, it has not proved possible to identify a phenotypic trait analogous to age at onset in Alzheimer disease by which to
classify multiplex families segregating schizophrenia. Studies of such large families also initially produced positive findings in
schizophrenia (83 ), but unfortunately these could not be replicated. The reasons for this have become clear as data from
systematic genome scans have accumulated; highly penetrant mutations causing schizophrenia are at best extremely rare and quite
possibly nonexistent (84 ,85 ). The false-positives were largely the consequence of a combination of multiple testing and the use of
statistical methodology and significance levels derived from work on single-gene disorders.

Despite the failure to identify regions of unambiguous linkage in multiply affected families, modest evidence for several regions has
been reported in more than one data set. Areas implicated for which supportive data have also been obtained from international
collaborative studies include chromosomes 6p24-22, 8p22-21, and 22q11-12 (86 ,87 ; see refs. 84 and 88 for review). A number of
other promising areas of putative linkage are also currently under investigation by international consortia. These include 13q14.1-32
(89 ,90 and 91 ), 5q21-31 (92 ,93 ), 18p22-21 (94 ), 10p15-11 (95 ,96 and 97 ), 6q (98 ,99 ), and 1q21-22 (100 ). However, in each
case, both negative and positive findings have been obtained, and in only two cases, those of chromosomes 13q14.1-32 and1q21-22,
did any single study achieve genome-wide significance at values of p of .05 (90 ,100 ).

These positive findings contrast with those from a large systematic search for linkage in which a sample of 196 affected sibling pairs,
drawn typically from small nuclear families rather than extended pedigrees, was used (101 ). The results of simulation studies
suggest that the power of this study is greater than 0.95 to detect a susceptibility locus of (s = 3 with a genome-wide significance of
0.05, but only 0.70 to detect a locus of (s = 2 with the conservative assumption that a locus lies midway between two adjacent
markers. This study yielded evidence at the level of the definition of Lander and Kruglyak (102 ) of “suggestive” linkage to
chromosomes 4p, 18p, and Xcen. However, none of the findings approached a genome-wide significance of 0.05, corresponding to
Lander and Kruglyak’s definition of “significant” linkage.

The findings from linkage studies of schizophrenia to date demonstrates several features that are to be expected in the search for
genes for complex traits (103 ,104 ,105 and 106 ). First, no finding is replicated in all data sets. Second, levels of statistical
significance are unconvincing and estimated effect sizes are usually modest. Third, chromosomal regions of interest are typically
broad [often > 20 to 30 centimorgan (cM)].

At the present time, therefore, the linkage literature supports the predictions made by Risch (79 ); it is highly unlikely that a
commonly occurring locus of effect size [(s] greater than 3 exists, but suggestive evidence implicates a number of regions,
consistent with the existence of some susceptibility alleles of moderate effect [(s = 1.5 to 3]. Moreover, encouraging results in
several chromosomal regions suggest that rarer alleles of larger effect may be segregating in some large, multiply affected families.

Linkage methods in sample sizes that are realistically achievable can detect smaller genetic effects than those in the studies to date.
For example, it is possible to detect alleles with values of (s of 1.5 to 3 in a sample of 600 to
 P.677

800 affected sibling pairs (107 ,108 ). We therefore suggest that priority should now be given to collecting such samples with a
robust clinical methodology that is comparable across all interested research groups. However, if liability to schizophrenia is entirely
a consequence of the operation of many genes of small effect, then even these large-scale studies will be unsuccessful.

CANDIDATE GENE ASSOCIATION STUDIES

Part of "49 - Molecular and Population Genetics of Schizophrenia "

Once genes of smaller effect than (s =1.5 are sought, the number of affected family members required becomes prohibitively large
(107 ,108 and 109 ). For this reason, many researchers have tried to take advantage of the potential of candidate gene association
studies to identify such loci (109 ,110 ). Although a potentially powerful means of identifying genes of small effect, association
studies are not without their problems. First, for a complex and poorly understood disorder such as schizophrenia, the choice of
candidate genes is limited largely by the imagination and resources of the researcher. This places a stringent burden of statistical
proof on positive results because of low prior probability and multiple testing (111 ). Second, case–control association studies have
the potential to generate false-positives because of population stratification. This problem can be addressed by using family-based
association methods (112 ), but because of stigma, adult age at onset, and the disruptive effects of mental illness on family
relationships, family-based samples may be unrepresentative in addition to limited in size. Consequently, family-based studies may
introduce more spurious results than do case–control studies (113 ). It would seem unwise, therefore, to discard the case–control
study design, which has served epidemiology so well through the years. A third problem common to all molecular genetic studies in
complex diseases is that they are prone to type 2 errors simply because they are often underpowered, and therefore to draw
satisfactory conclusions from negative studies, larger sample sizes are required than have typically been used to date in psychiatric
genetics (111 ). Fourth, even with larger samples, it is by no means certain that a given replication study will be sufficiently
powered to replicate a particular effect. This is because variations may be noted in the contribution of a given susceptibility allele
in different patient populations as a result of different allele frequencies at the locus of interest or at interacting loci. Further
potential for heterogeneity occurs if the association with the marker is a result of tight linkage with the true susceptibility allele, or
if different subtypes of the disease exist. Given that all the above factors may influence power, and that none of the above is known
in advance, it is difficult to obtain an accurate measure of the power of a replication study. Because we cannot specify accurately
the prior probability of a candidate gene, nor know the true power for replication, it is difficult to draw definitive conclusions from
conflicting findings. However, the purpose of experiment is to reject a null hypothesis, and in the face of uncertainty, the burden of
proof remains with the proponents of a particular candidate gene.

Most candidate gene studies have been based on neuropharmacologic studies, which suggests that abnormalities in monoamine
neurotransmission, in particular dopaminergic and serotoninergic systems, play a role in the etiology of schizophrenia. Overall, the
results in this extensive literature are disappointing, but it should be noted that the sample sizes in many of the older studies would
now generally be regarded as inadequate, particularly in view of the fact that the polymorphic markers in question did not in
themselves represent functional variants and that few genes have been systematically screened even for common functional variants.
However, more promising reports of candidate gene associations have recently appeared, three of which are considered here.

Serotonin 5-HT -Receptor Gene

2A

Many novel antipsychotic drugs affect the serotoninergic system. The first genetic evidence that serotoninergic receptors may play a
role in schizophrenia came from a Japanese group reporting an association between a T-to-C polymorphism at nucleotide 102 in the
5-HT2A-receptor gene in a small sample (114 ). A large European consortium comprising seven centers and involving 571 patients and
639 controls then replicated this finding (115 ), which was further replicated with use of a family-based design (116 ). Although
many other studies followed with mixed results, a recent metaanalysis of all available data from more than 3,000 subjects supports
the original finding (p = .0009), and this does not appear to be a consequence of publication bias (117 ).

Since this metaanalysis was undertaken, a few further negative reports have followed, but none has approached the sample sizes
required. If we assume homogeneity and if the association is true, the putative odds ratio (OR) for the C allele can be expected to
be around 1.2 in any replication sample. Sample sizes of 1,000 subjects are then be required for 80% power to detect an effect of
this size, even at a relaxed criterion of p = .05. Thus, the negative studies are effectively meaningless, but it is also true that the
evidence for association, even in the metaanalysis (p = .0009), is not definitive if genome-wide significance levels are required
(109 ). At present, all we can conclude is that the evidence favors association between the T102C 5-HT2A polymorphism and
schizophrenia, but the most stringent burden of proof has not yet been met.

If the association is real, it is unlikely that the T102C polymorphism is the susceptibility variant because this nucleotide change does
not alter the predicted amino acid sequence of the receptor protein, nor is it in a region of obvious significance for regulating gene
expression. T102C is
 P.678

in complete linkage disequilibrium with a polymorphism in the promoter region of this gene, but no evidence has as yet been found
that this has a functional effect either (116 ). Recent evidence of polymorphic monoallelic expression of the 5-HT2A gene points to
the possible existence of sequence variation elsewhere that influences gene expression (118 ), and this may be the true
susceptibility variant.

D3 Dopamine-Receptor Gene
Association has been reported between schizophrenia and homozygosity for a Ser9Gly polymorphism in exon 1 of the D3 dopamine-
receptor gene (DRD3) (119 ). As with the 5-HT2A association, the results have now been confirmed in several independent samples,
including one family-based study (120 ), but several negative studies have also been reported. Metaanalysis of data from more than
5,000 individuals has revealed a small (OR = 1.23) but significant (p = .0002) association between homozygosity at Ser9Gly and
schizophrenia (120 ). Again, this cannot easily be ascribed to selective publication (120 ). Because it is a fairly uncommon genotype,
we cannot be certain that homozygosity for the 9Gly allele alone is not associated with an increased risk for schizophrenia, and
therefore it is not certain that the findings at D3 are an example of heterosis. However, a plausible biological explanation for D3
heterosis has been put forward in that possession of two different molecular forms of the receptor may allow the dopaminergic
neuron to respond more flexibly (119 ).

At present, then, the status of the D3 dopamine-receptor gene is similar to that of the 5-HT2A-receptor gene—that is, the balance of
evidence at present favors association, but the null hypothesis still cannot be confidently rejected. Those wishing to replicate or
reject these findings should bear in mind that to obtain power greater than 0.80 to detect an effect of this size at a criterion of p
= .05, a sample of 1,500 cases and 1,500 controls is required. So far, no other polymorphisms have been found that might explain
the putative D3 association, but several new polymorphisms have been identified in previously unknown exons 5′ to the exon
referred to above as exon 1 (121 ). These are currently being tested to establish whether variants in this region in linkage
disequilibrium with the Ser9Gly polymorphism provide a more functionally plausible explanation of the association with
schizophrenia.

ANTICIPATION AND TRINUCLEOTIDE REPEATS

Part of "49 - Molecular and Population Genetics of Schizophrenia "

The term anticipation, the phenomenon by which the age at onset of disease becomes earlier from one generation to the next, was
first described in connection with severe mental disorder (122 ). A series of recent studies applying modern diagnostic criteria have
now confirmed that the inheritance of schizophrenia is at least consistent with the presence of anticipation, although ascertainment
biases offer an alternative explanation (123 ). Because pathogenic expanded trinucleotide repeats are the only known genetic
mechanisms for anticipation, these findings have been taken as suggesting that such mutations may account for at least some of the
complexity in the pattern of inheritance in this disorder (124 ). This hypothesis was supported by two groups who observed that the
maximum length of the most common known pathogenic trinucleotide repeat, CAG/CTG, was greater in patients with schizophrenia
than in unaffected controls (125 ,126 ). These findings were later replicated in a European multicenter study (127 ). Unfortunately,
the early repeat expansion detection (RED) studies were followed by a series of unsuccessful attempts to identify the relevant
repeat-containing loci by a variety of methods, and by several failures to replicate the RED findings (128 ,129 and 130 ), thus
casting doubt over the CAG/CTG repeat hypothesis.

The trinucleotide repeat hypothesis was rejuvenated, however, with the report of an association between schizophrenia and alleles
of a member of the family of calcium-activated potassium channel genes, KCa3 (hKCa3/KCCN3) (131 ). For several reasons, KCa3
seemed a remarkable candidate gene for schizophrenia. First, the gene contained two CAG repeats, one of which is highly
polymorphic. Second, the family of genes to which it belongs is thought to play an important role in regulating neuronal activity,
and it was therefore considered a functional candidate gene. Third, the gene was also thought to be a positional candidate, as it
was believed to map to chromosome 22q11. Ironically, KCa3 maps not to 22q11 but to 1q21 (132 ), which is also a region implicated
by linkage studies as possibly containing a susceptibility locus for schizophrenia (100 ). Two further case–control studies have
subsequently supported the findings of Chandy and colleagues (132 ,133 ).

However, although evidence from three case–control studies lends support to the hypothesis of KCa3 as a susceptibility gene for
schizophrenia, in other respects the case for this gene as a candidate is less certain. First, the RED data cited above lend no support
to KCa3 as a candidate because the polymorphic trinucleotide repeat in this gene is far too short to account for the RED associations
(133 ). Second, a series of case–control and family studies have failed to replicate the findings (134 ,135 ,136 ,137 ,138 ,139 and
140 ). Thus, we are back to our familiar position in candidate gene analysis; although the data are insufficient to draw firm
conclusions, we believe at present that the case for KCa3 remains firmly with the null hypothesis.

What then is the status of the original associations between large CAG/CTG repeats and schizophrenia? It has been reported that
large CAG/CTG RED products (repeat size > 40) are explained by repeat size at two autosomal loci, one at 18q21.1 and the other at
17q21.3 (141 ,142 ). If the explanation is correct, then it follows that one or both of these loci should be associated with
schizophrenia.
 P.679

Unfortunately, data from Vincent and colleagues (128 ) and unpublished data from Cardiff unequivocally show that expansions at
these loci are not responsible for the RED associations. However, in both samples, only around 50% of large CAG/CTG repeats
detected by RED could be explained by polymorphisms at these two loci, which suggests that at least one further locus is responsible
for the RED data, a possibility supported by two recent studies on protein extracts from schizophrenic tissues (143 ).

HIGH RATES OF SCHIZOPHRENIA IN ADULTS WITH VELOCARDIOFACIAL

SYNDROME
Part of "49 - Molecular and Population Genetics of Schizophrenia "

Velocardiofacial syndrome (VCFS), also known as DiGeorge or Shprintzen syndrome, is associated with small interstitial deletions of
chromosome 22q11 in 80% to 85% of cases (144 ). First described by Shprintzen and colleagues (145 ), VCFS has an estimated
prevalence of 1/4,000 births (146 ). Distinctive dysmorphology, congenital heart disease, and learning disabilities characterize the
syndrome, although considerable phenotypic variability occurs. As the first recognized cohort of children with VCFS was followed
into adolescence and early adulthood, evidence began to accumulate for a high prevalence of major mental illness. Early reports
suggested that psychiatric disorders had developed in more than 10% of the cohort, which mostly resembled chronic schizophrenia
with paranoid delusions, although operational criteria were not used (147 ). In a follow-up study of teenagers (age 17 years) in which
DSM-III-R criteria were used, Pulver et al. (148 ) reported that 11 (79%) of their sample of 14 patients had been given a psychiatric
diagnosis: 29% had schizophrenia (22%) or schizoaffective disorder (7%), 29% had simple or social phobia, 21% had depression, and
14% had obsessive-compulsive disorder. More recently, Papolos et al. (146 ) reported that of their sample of 15 children and 10
adults, four (16%) had psychotic symptoms and 16 (64%) met DSM-III-R criteria for a spectrum of bipolar affective disorders. Although
none had schizophrenia, the two oldest members of their patient cohort (ages 29 and 34 years) both had schizoaffective disorder.

To try to gain a more precise determination of the prevalence and nature of psychopathology in adults with VCFS, rather than rely
on clinical diagnosis, Murphy and colleagues (149 ) recently evaluated 50 cases with a structured clinical interview to establish a
DSM-IV diagnosis. Fifteen patients with VCFS (30%) had a psychotic disorder, with 24% (n = 12) fulfilling DSM-IV criteria for
schizophrenia. In addition, six (12%) had major depression without psychotic features. They were unable to replicate the findings of
Papolos and colleagues (146 ) of a high prevalence of bipolar spectrum disorders in VCFS. However, these workers studied a small
sample that included few adults, and in view of the fact that their oldest cases satisfied criteria for schizoaffective disorder, it is
possible that the psychotic phenotype in VCFS varies with age. Prospective studies are now required to test this hypothesis.

The current balance of evidence favors the view that the high prevalence of psychosis results from hemizygosity for a gene or genes
at chromosome 22q11 rather than ascertainment bias or a nonspecific association with a low intelligence quotient (IQ) (149 ). In
particular, the prevalence of psychosis and schizotypy in VCFS appears to be much greater than that seen in most other congenital
abnormalities affecting neural development, and appears not to be correlated with the degree of intellectual impairment (149 ).
Many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder (150 ). In VCFS, defective development and
migration of mesencephalic and cardiac neural crest cells are believed to play a significant role in the pathogenesis of midfacial and
cardiac abnormalities (151 ). Consequently, it has been postulated that a gene or genes causing disruption of neural cell migration
may be a common neurodevelopmental mechanism for both VCFS and schizophrenia (152 ).

What then is the importance of 22q11 and VCFS in the etiology of schizophrenia as a whole? Karayiorgou et al. (153 ) reported that
among 100 randomly ascertained patients with schizophrenia, two were found to have a 22q11 deletion. In contrast, when subjects
with schizophrenia were selected for the presence of clinical features consistent with VCFS, 22q11 deletions were identified in 20%
to 59% of cases (154 ,155 ). These findings suggest that a small proportion of cases of schizophrenia may result from deletions of
22q11, and that clinicians should be vigilant, especially when psychosis occurs in the presence of dysmorphology, mild learning
disability, or a history of cleft palate or congenital heart disease (156 ). The question of whether genetic variation in 22q11 confers
susceptibility to schizophrenia in cases without a deletion is more difficult to answer. As we have seen, the results of some linkage
studies suggest the presence of a schizophrenia susceptibility locus on 22q. However, the linkage findings tend to point telomeric to
the VCFS region (86 ,157 ). Nevertheless, modest evidence for linkage to the VCFS region has also been claimed (90 ,158 ,159 ), and
as we have noted above, linkage mapping in complex diseases is somewhat imprecise. It remains possible that the relationship
between VCFS and “typical” schizophrenia is less direct, with little common ground between the genetic and neurodevelopmental
mechanisms involved but with convergence on identical or at least similar psychopathologic syndromes.

Another important question concerns the factors that determine whether schizophrenia will develop in a person with VCFS. When
adults with VCFS were tested with a quantitative measure of schizotypy, the patients with psychosis had the highest scores (149 ).
However, perhaps of greater interest, those without psychosis had intermediate
 P.680

scores in comparison with controls (149 ). If schizotypy is a trait marker for increased liability to psychosis, this suggests that the
majority if not all of those with 22q11 deletions are at increased risk for psychosis, but that other genetic or environmental factors
are required for this risk to be expressed. The genetic loci involved may reside elsewhere in the genome and include those involved
more widely in psychosis, or lie within 22q11. The occurrence of psychosis does not appear to be related to the size of the deletion
(153 ). However, it is possible that susceptibility to psychosis reflects allelic variation of a hemizygous gene or genes within the
deletion. The gene encoding catechol-O-methyltransferase (COMT), an enzyme involved in the catabolism of catecholamine
neurotransmitters, maps to the VCFS region and is therefore an obvious candidate for influencing the expression of psychosis in VCFS
probands. This gene exists in two allelic forms encoding high- and low-activity isoforms of the enzyme, and it has been suggested
that possession of the allele for low-activity COMT may be associated with the occurrence of schizophrenia in VCFS (160 ). However,
Murphy and colleagues (149 ) found no evidence for an association between the allele for low-activity COMT and either
schizophrenia or schizotypy in patients with VCFS.

FUTURE DIRECTIONS
Part of "49 - Molecular and Population Genetics of Schizophrenia "

Refining the Phenotype for Molecular Genetic Studies

The effectiveness of molecular genetic studies depends on the genetic validity of the phenotypes studied. Perhaps if we were better
at defining phenotypes, we would be better at finding genes. It is worth reiterating at this point that the commonly used diagnostic
criteria define phenotypes with high heritability. In principle, therefore, it should be possible to identify the genes predisposing to
schizophrenia, as defined by current diagnostic criteria, if sufficiently large samples are studied. However, perhaps genetic validity
could be improved by focusing on aspects of clinical variation, such as age at onset or symptom profiles, or by identifying biological
markers that predict degree of genetic risk or define more homogeneous subgroups. Unfortunately, despite much work, it has not
been possible to identify genetically distinct subtypes of schizophrenia. Instead, clinical variation is likely to reflect at least in part
a combination of quantitative variation in genetic risk for the disorder and the effect of modifying genes that influence illness
expression rather than the risk for illness per se. Examples of this phenomenon are probably age at onset and symptom pattern in
schizophrenia (161 ,162 ).

The search for trait markers aims to move genetic studies beyond the clinical syndrome by identifying indices of genetic risk that
can be measured in asymptomatic persons or by identifying markers of pathophysiologic processes that are closer to the primary
effects of susceptibility genes than are clinical symptoms—so-called intermediate phenotypes or endophenotypes. Work in this area
is developing fast; for example, candidate trait markers for schizophrenia include schizotypal personality traits, measures of
cognitive processing, brain evoked potentials, and abnormalities in eye movements (163 ). It is also hoped that advances in brain
imaging will lead to the identification of genetically valid trait markers. However, it seems unlikely that these phenotypes will
provide a rapid solution to the problem. First, we will need to ensure that the measures used are stable and determine the extent to
which they are affected by state. Second, to be of use in gene mapping, such measures will have to be practicably applied to a
sufficient number of families or unrelated patients. Third, we will need to ensure that the traits identified are highly heritable,
which will itself require a return to classic genetic epidemiology and model fitting. Finally, we cannot assume that the genetic
architecture of such intermediate phenotypes will be simple.

Efforts to improve the selection of phenotypes are also concerned with enhancing the traditional categoric approach to defining
psychiatric disorders by identifying genetically valid phenotypes that can be measured quantitatively. These can be used in
quantitative trait locus approaches to gene mapping. Work in this area has begun but still faces problems, particularly those relating
to the confounding influence of state-related effects. Perhaps the best hope of taking account of the complexity and heterogeneity
of the schizophrenia phenotype comes from new methods of analysis in which aspects of the phenotype can be entered as covariates
in linkage analyses (164 ).

Genome-wide Association Studies

In recent years, interest has increased in the possibility of systematic, genome-wide association studies (109 ,165 ). These have the
potential of allowing systematic searches for genes of small effect in polygenic disorders. Optimism has been fueled by the fact that
the most abundant form of genetic variation, the single-nucleotide polymorphism, is usually bi-allelic and potentially amenable to
binary, high-throughput genotyping assays such as microarrays (so-called DNA chips). Moreover, as sequence data accumulate, it has
become possible to contemplate the construction and application of very dense maps of hundreds of thousands of single-nucleotide
polymorphisms (165 ).

Essentially two types of genome-wide association study have been proposed: direct and indirect. In the former, association is sought
between a disease and a comprehensive catalogue of every variant that can alter the structure, function, or expression of every
single gene. In contrast, indirect studies seek associations between markers and disease that are caused by linkage disequilibrium
between the markers and susceptibility variants. The hope is that if sufficiently dense marker maps can be applied, it will be
possible to screen the whole genome systematically for evidence of linkage
 P.681

disequilibrium without actually having to screen every functional single-nucleotide polymorphism in the genome. However, a
number of uncertainties and difficulties remain. These include, in particular, the difficulty of identifying functional single-
nucleotide polymorphisms in regulatory rather than coding regions of the genome, uncertainty about the distances over which
linkage disequilibrium is maintained, and the lack at the present time of a rapid, accurate, and cheap method for single-nucleotide
genotyping (165 ).

Given these considerations, it seems clear that the era of genome-wide association studies, direct or indirect, is not yet at hand.
Instead, studies in the next few years should probably focus mainly on the direct approach utilizing single-nucleotide polymorphisms
from the coding sequence that actually alter protein structure in a wide range of functional and positional candidate genes.
Preferably, complete functional systems should be dissected by the application of sensitive methods for mutation detection,
followed by association studies in appropriately sized samples. We should also use our knowledge of functional pathways to make
predictions about likely epistasis. However, given our ignorance of pathophysiology, the expectation should be that most reported
associations will be false and resolved only by replication in large, well-characterized samples. At present, although the indirect
approach is not widely applicable at a genome-wide level, smaller-scale studies focusing on specific regions indicated by the results
of linkage studies may allow us to map loci. Additionally, such studies will generate the sort of data concerning patterns of linkage
disequilibrium in typical “association samples” that will be required to determine whether genome-wide studies are likely to be
feasible and what density of map will be required.

If the results are encouraging—that is, if linkage disequilibrium exists across useful distances in the genome—then until genotyping
technology has sufficient capacity to permit mass genotyping at low cost, linkage disequilibrium analyses at the genome-wide level
are most likely to be based on DNA pooling technologies (166 ,167 ,168 and 169 ).

Identifying Modifying Genes

It is sometimes assumed that variation in the clinical features of a disease or treatment response simply reflects pleiotropic effects
of etiologic risk factors. However, it is becoming increasingly apparent that specific genes probably exist that influence clinical
features and treatment response independent of those affecting liability. Thus, another potentially fruitful line of inquiry may be to
design studies aimed at seeking modifying rather than causative genes, as these genes may in themselves allow novel drug targets to
be identified. Evidence has already been found that variations in age at onset and symptom pattern in schizophrenia are probably
caused at least in part by modifying genes (161 ,162 ). Furthermore, interest is increasing in pharmacogenetics in psychiatry (170 ),
although with some trait markers, we need to exercise caution in the absence of genetic epidemiologic evidence that variations in
drug response are under genetic control. We should also not forget that there is no a priori reason why responses to behavioral and
psychological treatments should be less influenced by genetic factors than by pharmacologic treatments.

Animal Models
Another important challenge will be the development of suitable animal models to allow functional studies of putative disease loci
(165 ). Disorders that predominantly involve higher cognitive function, such as schizophrenia, are likely to prove difficult to model in
animals. However, certain features of the human phenotype, such as subtle abnormalities of cell migration, enlarged cerebral
ventricles, and abnormalities of information processing, including defects in prepulse inhibition, can be detected in animals (171 ).
In fact, a possible approach to producing a mouse model for at least some of the schizophrenia phenotype is suggested by the
finding of an increased prevalence of schizophrenia in VCFS. Currently, attempts are under way to produce transgenic mice in which
the syntenic region of mouse chromosome 16 is deleted (172 ). These animals should be investigated closely for neuroanatomic and
behavioral phenotypes of possible relevance to schizophrenia, and such studies are already yielding encouraging findings (173 ).

Functional Studies
The most important and most obvious implication of identifying genetic risk factors for schizophrenia is that it will inspire a new
wave of neurobiological studies from which, it is hoped, new and more effective therapies will emerge. However, although the
unequivocal identification of associated genetic variants will represent a great advance, many years of work will be required before
this is likely to translate to routine clinical practice. An early problem will be to determine exactly which genetic variant among
several in linkage disequilibrium within a given gene is actually responsible for the functional variation. Even when a specific variant
within a gene can be identified as the one of functional importance, functional analysis, in terms of effect at the level of the
organism, is likely to be particularly difficult for behavioral phenotypes in the absence of animal models. An extra level of
complexity is that we will need to be able to produce model systems, both in vivo and in vitro, that allow gene–gene and gene–
environment interaction to be studied.

Genetic Nosology
Although the development of new therapies will take time, it is likely that the identification of susceptibility genes will
 P.682

have an earlier effect on psychiatric nosology. If genetic risk factors are correlated with clinical symptoms and syndromes, it should
be possible to study heterogeneity and comorbidity to improve the diagnosis and classification of psychosis. The prospects will also
be enhanced for identifying clinically useful biological markers as an aid to diagnosis, so that we can move beyond the current
situation of making diagnoses based entirely on clinical signs and symptoms. Improvements in diagnostic validity will clearly
facilitate all avenues of research into these disorders. However, in the present context, we should point out that improvements in
diagnosis and classification should enhance our ability to detect further genetic and environmental risk factors, so that a positive
feedback between nosology, epidemiology, and molecular genetics can be envisaged.

Molecular Epidemiology
The identification of genetic risk factors can be expected to provide a new impetus to epidemiologic studies of schizophrenia by
allowing researchers to investigate the ways in which genes and environment interact. Studies of this kind will require large,
epidemiologically based samples together with the collection of relevant environmental data. This work could start now with DNA
being banked for future use, although in schizophrenia, the identification of plausible environmental measures might require clues
from the nature of the genetic risk factors yet to be identified. A major theme in relation to this work will be the bringing together
of methodologies from genetics and epidemiology, which have traditionally adopted somewhat differing analytic approaches (174 ).
Treating susceptibility alleles as risk factors in an epidemiologic context will allow estimates of effect sizes within a population to
be made. Accounting for specific genetic effects will also facilitate the search for independent environmental factors and the
investigation of potential gene–environment interactions. Scientific validity is likely to be enhanced by ensuring as far as possible
that control samples are drawn from the same base population as patients. In addition, the use of incident cases should guard
against the risk of identifying loci related to confounds, such as chronicity of illness, rather than susceptibility. Phenotypic
assessment is likely to benefit from a prospective element to studies, which counteracts the tendency of patients to forget historical
details and the difficulty of making observed ratings retrospectively from case records. However, the price of improved scientific
rigor is likely to be considerably more expensive studies because of the longer period and larger number of investigators that will be
required to ascertain detailed data from thousands of subjects.

Genetic Testing
A further implication concerns genetic testing. This is a complex area that raises a number of ethical issues, which have been
discussed elsewhere (175 ). However, the potential for predictive testing has probably been overstated, given that susceptibility to
schizophrenia almost certainly depends on the combined effects of predisposing and protective alleles at a number of loci and their
interaction with the environment. Consequently, until we have a comprehensive molecular understanding of the etiology of
schizophrenia, the predictive value of genetic testing is likely to be low. This applies, for example, to apolipoprotein E testing for
late-onset Alzheimer disease, and has led to a recommendation that such testing not be performed in asymptomatic persons (175 ).
Indeed, even when all the susceptibility genes for schizophrenia have been identified, it will still not be possible to predict the
development of disease with certainty until the relevant genetic and environmental risk and modifying factors have also been
identified and the nature of the various interactions understood. Such interactions may be complex and unpredictable (165 ).
However, other possible roles of genetic testing are likely to be of greater value to patients and clinicians. For example, it might be
possible to optimize treatment choices by testing genes found to influence treatment responses in psychiatric disorders and so
provide more individualized treatment.

CONCLUSIONS
Part of "49 - Molecular and Population Genetics of Schizophrenia "

Attempts to identify the genes that predispose to schizophrenia face formidable challenges arising from both genetic and phenotypic
complexity. Research to date has largely excluded the possibility that genes of major effect exist even in a subset of families.
Evidence has been obtained of the location of some genes of moderate effect, but none of these findings can be regarded as
conclusive, and proof in each case will probably have to await identification of the susceptibility locus itself. The clearest molecular
genetic risk factor for schizophrenia that has been identified to date is deletion of a gene or several genes on chromosome 22, which
can markedly increase the risk for schizophrenia. However, fairly strong data suggest that allelic variation in genes encoding the 5-
HT2A and D3 dopamine receptors confer a small degree of susceptibility.

As in other common diseases, it is hoped that advances will come through the use of a new generation of genetic markers and new
methods of genotyping and statistical analysis (165 ). However, successful application of these methods requires access to large,
well-characterized patient samples, and the collection of such data is a priority at the present time. We need to focus research on
the development and refinement of phenotypic measures and biological markers. Success will also depend on the traditional medical
disciplines of clinical description and epidemiology, and on our ability to integrate these with genetic approaches.

ACKNOWLEDGMENTS
Part of "49 - Molecular and Population Genetics of Schizophrenia "
 P.683

Preparation of this chapter was supported in part by National Institute of Mental Health grants 1 R01MH41879-01, 5 UO1 MH46318-02, and 1
R37MH43518-01 to Dr. Ming T. Tsuang; the Veterans Administration Medical Research, Health Services Research, and Development and Cooperative
Studies Programs; and a NARSAD Distinguished Investigator Award to Dr. Tsuang. Dr. Owen is supported by the United Kingdom Medical Research
Council. Dr. Tsuang wishes to acknowledge the assistance of Drs. Stephen Faraone and William Stone, and Dr. Owen wishes to acknowledge the
assistance of Drs. Michael O’Donovan and Alastair Cardno in the preparation of this manuscript.

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50
Animal Models Relevant to Schizophrenia Disorders

Mark A. Geyer

Bita Moghaddam

Mark A. Geyer: Department of Psychiatry, School of Medicine, University of California at San Diego, La Jolla, California.

Bita Moghaddam: Departments of Psychiatry and Neurobiology, Yale University School of Medicine, New Haven, Connecticut.

Animal models used to study schizophrenia include both models of the full syndrome and models of specific signs or symptoms. As
reviewed elsewhere (1 ), models are commonly explored initially because of indications of so-called face validity, but they are
evaluated scientifically in terms of their construct and etiologic and predictive validity with respect to both clinical phenomena and
responsiveness to antipsychotic drugs. Here, models are organized by the manipulations used to mimic the clinical phenomena. Thus,
in some of these models, only specific dependent measures are utilized, whereas others are evaluated by using a range of
dependent measures.

A model is defined as any experimental preparation developed to study a particular condition or phenomenon in the same or
different species. Typically, models are animal preparations that attempt to mimic a human condition, in our case the human
psychopathology associated with the group of schizophrenia disorders. In developing and assessing an animal model, it is important
to specify the purpose intended for the model because the intended purpose determines the criteria that the model must satisfy to
establish its validity. At one extreme, one can attempt to develop an animal model that mimics the schizophrenia syndrome in its
entirety. In the early years of psychopharmacology, the term animal model often denoted such an attempt to reproduce a
psychiatric disorder in a laboratory animal. Unfortunately, the group of schizophrenia disorders is characterized by considerable
heterogeneity and a complex clinical course that reflects many factors that cannot be reproduced readily in animals. Thus, the
frequent attempts to model the syndromes of schizophrenia in animals usually met with failure and so prompted skepticism
regarding this entire approach.

At the other extreme, a more limited use of an animal model related to schizophrenia is to study systematically the effects of
antipsychotic treatments. Here, the behavior of the model is intended to reflect only the efficacy of known therapeutic agents and
so lead to the discovery of related pharmacotherapies. Because the explicit purpose of the model is to predict treatment efficacy,
the principle guiding this approach has been termed pharmacologic isomorphism (2 ). The fact that such models are developed and
validated by reference to the effects of known therapeutic drugs frequently limits their ability to identify new drugs with novel
mechanisms of action. Similarly, an important limitation inherent in this approach is that it is not designed to identify new
antipsychotic agents that might better treat the symptoms of schizophrenia refractory to current treatments.

Because of the complexity of schizophrenia, another approach to the development of relevant animal models relies on focusing on
specific signs or symptoms associated with schizophrenia, rather than mimicking the entire syndrome. In such cases, specific
observables that have been identified in schizophrenic patients provide a focus for study in experimental animals. The particular
behavior being studied may or may not be pathognomonic for or even symptomatic of schizophrenia, but it must be defined
objectively and observed reliably. It is important to emphasize that the reliance of such a model on specific observables minimizes a
fundamental problem plaguing animal models of the syndrome of schizophrenia. Specifically, the difficulties inherent in conducting
experimental studies of schizophrenic patients have limited the number of definitive clinical findings with which one can validate an
animal model of schizophrenia. The validation of any animal model can only be as sound as the information available in the relevant
clinical literature (3 ). By focusing on specific signs or symptoms rather than syndromes, one can increase the confidence in the
cross-species validity of the model. The narrow focus of this approach generally leads to pragmatic advantages in the conduct of
mechanistic studies addressing the neurobiological substrates of the behavior in question. By contrast, in models intended to
reproduce the entire syndrome of schizophrenia, the need for multiple simultaneous endpoints
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makes it relatively difficult to apply the invasive experimental manipulations required to establish underlying mechanisms.

Another approach to the development of animal models is based more theoretically on psychological constructs believed to be
affected in schizophrenia. Such identification of underlying psychological processes or behavioral dimensions (2 ,3 ) involves the
definition of a hypothetical construct and the subsequent establishment of operational definitions suitable for experimental testing
of the validity of the construct. Constructs such as selective attention, perseveration, sensorimotor gating, and working memory
have been used in this manner in schizophrenia research. This approach is most fruitful when conceptually or procedurally related
experiments are undertaken in both the relevant patient population and the putative animal model. In other words, studies of
appropriate patients are needed to establish the operational definitions of the hypothetical construct and the relevance of the
construct to schizophrenia. In concert, parallel studies of the potentially homologous construct, process, or dimension are required
to determine the similarity of the animal model to the human phenomena. An important and advantageous aspect of this approach
is that the validation of the hypothetical construct and its cross-species homology can be established by studies of normal humans
and animals in addition to studies of schizophrenic patients and experimentally manipulated animals. Thus, this approach benefits
from the existing literature relevant to the hypothetical construct on which the model is based. In a sense, this approach explicitly
recognizes that the experimental study of schizophrenia in humans involves as much of a modeling process as does the study of the
disorder in animals.

 PHENOTYPIC CHARACTERIZATION OF ANIMAL MODELS

 PHARMACOLOGIC MODELS
 DEVELOPMENTAL MODELS
 GENETIC MODELS
 CONCLUSIONS
 ACKNOWLEDGMENTS

PHENOTYPIC CHARACTERIZATION OF ANIMAL MODELS

Part of "50 - Animal Models Relevant to Schizophrenia Disorders "

Behavioral measures have been used extensively for establishing the validity of animal models of schizophrenia. Some of these
measures, such as horizontal locomotion, do not correspond to schizophrenic symptomatology and have been primarily useful for
providing a functional measure of the antidopaminergic activity of neuroleptics. Other behavioral measures, such as disruption of
prepulse inhibition or impaired attentional set shifting, resemble characteristics of schizophrenia. These measures are useful for
establishing the construct and predictive validity of putative animal models.

In addition to behavioral assessments, cellular and molecular markers that are based on described changes in human postmortem
and imaging studies are potentially useful measures for establishing the validity of animal models. Furthermore, because of the
inherent limitations of modeling in laboratory animals some of the most prominent behavioral abnormalities of schizophrenia, such
as delusions and hallucinations, cellular and molecular characterizations can complement behavioral investigations in evaluating
developmental and genetic models of schizophrenia.

Behavioral Phenotypes

Locomotor and Stereotypy

Changes in locomotor activity in rodents have often been used to assess both models of schizophrenia and the effects of
antipsychotic treatments. The original impetus for the use of locomotor activity measures was derived from the psychostimulant
models based on the dopamine hypothesis of schizophrenia, as reviewed elsewhere (3 ,4 and 5 ). These models arose because of the
apparent similarity (i.e., “face validity”) between the symptoms of schizophrenia and the effects of high doses of amphetamine in
presumably normal humans (6 ). Cross-species studies in animals treated with psychostimulants revealed both locomotor
hyperactivity and, at higher doses, striking stereotyped or perseverative behaviors, which were seen as having face validity for the
stereotyped behavior induced by amphetamine in humans (4 ,6 ,7 ). Measures of locomotor hyperactivity have been used extensively
to characterize the effects of both dopaminergic psychostimulants and N-methyl-D-aspartate (NMDA) antagonists, such as
phencyclidine (PCP), although PCP-induced hyperactivity differs markedly in qualitative features from that produced by
dopaminergic psychostimulants. Although patients with schizophrenia are not typically hyperactive, they often exhibit perseverative
or stereotyped behaviors. Hence, many studies in rodents have focused on the forms of stereotypy produced by psychostimulants.

Gating Measures
Clinical observations in schizophrenic patients have identified deficiencies in the processing of information, including an inability
automatically to filter or “gate” irrelevant thoughts and sensory stimuli to prevent them from intruding on conscious awareness.
Hence, theories of schizophrenic disorders often conceptualize the common aspect of these disorders as involving one or more
deficits in the multiple mechanisms that enable normal persons to filter or gate most of the sensory stimuli they receive (8 ,9 and
10 ). In the most classic measure of filtering deficits, numerous studies have observed deficits in the habituation of startle responses
in schizophrenic patients (e.g., 12,31,102), which may reflect failures of sensory filtering leading to disorders of cognition. A more
specific class of such mechanisms is referred to as sensory or sensorimotor gating. Theoretically, impairments in either filtering or
gating lead to sensory overload and cognitive fragmentation. It is also possible that the mechanisms that subserve experimental
examples of filtering or gating are also responsible for the gating of cognitive information. The hypothetical construct of
sensorimotor
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gating has been operationalized and explored in both human and animal studies. The validity of this gating construct has been
assessed most thoroughly by means of an operational measure based on cross-species homologies in the startle reflex—namely, the
prepulse inhibition (PPI) of startle paradigm. In a conceptually related approach, analogous measures of event-related potentials are
used across species to study sensory gating in the P50 event-related potential condition–test paradigm.

Habituation
Habituation refers to the decrement in responding when the same unimportant stimuli or cognitions occur repeatedly in the absence
of any contingencies. Habituation is considered to be the simplest form of learning and is essential for the development of selective
attention. Although habituation can be assessed with a variety of behavioral measures, the most common approach has been to
study the gradual decrease in the startle response elicited by a series of tactile or acoustic stimuli in humans, rats, or mice. In
patients with schizophrenia or schizotypy, deficits in startle habituation have been reported with the use of either modality of
startling stimuli (9 ,11 ,12 and 13 ). A striking advantage of the startle habituation measure is the fact that extremely similar
behavioral tests can be conducted in both humans and experimental animals.

Prepulse Inhibition
The PPI paradigm is based on the fact that a weak prestimulus presented 30 to 500 milliseconds before a startling stimulus reduces,
or gates, the amplitude of the startle response. The generality and reliability of this robust phenomenon is clear; PPI is observed in
many species, PPI is evident both within and between multiple sensory modalities when a variety of stimulus parameters are used,
and PPI does not require learning or comprehension of instructions. Virtually all the evidence available supports the belief that PPI is
homologous from rodents to humans, unlike most other cross-species comparisons based on often dubious arguments of similarity or,
at best, analogy. As reviewed elsewhere (14 ,15 ), several laboratories have reported significant deficits in PPI in schizophrenic,
schizotypal, and presumably psychosis-prone subjects with the use of a variety of testing procedures and stimulus parameters.
Nevertheless, PPI deficits are not unique to patients in whom schizophrenia has been diagnosed; they are also observed in other
psychiatric disorders involving abnormalities of gating in the sensory, motor, or cognitive domains.

P50 Gating
In the P50 sensory gating paradigm, two acoustic clicks are presented in rapid succession, usually 500 milliseconds apart. In normal
persons, the P50 event-related potential to the second click is reduced or gated relative to the event-related potential to the first
click. Schizophrenic patients and their first-degree relatives exhibit less sensory gating (16 ). An analogous form of sensory gating is
studied in rodents based on the N40 event-related potential generated from the hippocampus (17 ).

Latent Inhibition
Latent inhibition is a relatively complex paradigm that is conceptually related to the gating theories of schizophrenic disorders.
Latent inhibition refers to the observation that repeated exposures to a sensory stimulus (i.e., habituation) retard the rate at which
a subject subsequently acquires a stimulus–response association based on this stimulus (18 ). Deficits in latent inhibition have been
reported in schizophrenic patients (19 ), although it appears that such deficits may be limited to acute episodes of schizophrenia
(19 ,20 ). This limitation has diminished interest in latent inhibition as a model for schizophrenia.

Social Behavior
Social withdrawal is included among the negative symptoms of schizophrenia and is often one of the earliest symptoms to occur.
Models of social isolation have been studied in both monkeys (21 ) and rats (22 ). Naturally, given the importance of language in
human social interactions, the species-specific differences in social behavior limit the direct comparisons that can be made across
species.

Cognitive Measures
Cognitive deficiencies played a prominent role in the original description of schizophrenia by Kraepelin and distinguish the diagnosis
of schizophrenia from manic-depressive and other forms of psychosis. Cognitive deficits are reported across all subtypes of
schizophrenia and include impairments of attention, working memory, verbal memory, set shifting, and abstraction. Severe
cognitive deficits appear to be a major factor contributing to impaired social and vocational functioning and treatment outcome
(23 ). Current modes of therapy for schizophrenia (i.e., dopamine D2 or dopamine D2/serotonin 5-HT2 antagonists, which effectively
reduce the positive symptoms of schizophrenia in the majority of patients) have minimal beneficial effects on cognitive functioning.
Typical antipsychotic drugs (e.g., haloperidol, chlorpromazine) may in fact lead to a deterioration in cognitive functions in
schizophrenia patients (24 ) by producing so-called secondary deficit symptoms. Although some reports suggest that the atypical
antipsychotic drug clozapine and the new generation of antipsychotics (e.g., olanzapine and risperidone, which target several
subtypes of dopamine and serotonin receptors) may improve cognitive function, this effect is relatively small and has not been
reproducible across laboratories (25 ,26 and 27 ). Thus, an important future direction of preclinical research relating to
schizophrenia
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is the design of animal models and novel treatments that target cognitive dysfunctions associated with this disorder. However,
establishing the validity of animal models of cognitive deficits of schizophrenia and designing new pharmacologic approaches to the
treatment of symptoms depends on appropriate behavioral paradigms for laboratory animals. These must provide as good an analogy
as possible to the empiric measures on which schizophrenic patients are impaired. Unfortunately, the ratings of symptoms commonly
assessed in clinical studies are of little value in this context.

The limited cognitive capacity of laboratory animals hinders the design of cognitive tasks that can be considered entirely
“analogous” to relevant experimental paradigms, such as the Wisconsin Card Sorting Test and Continuous Performance Test.
Therefore, most of the research involving cognitive tasks that are relevant to schizophrenia has been conducted in monkeys.
Nevertheless, it is possible to design behavioral paradigms in rodents that can evaluate cognitive constructs “comparable” with
those measured in many human experimental paradigms (40 ,41 and 42 ).

Among the most common of animal cognitive tasks are those with a working memory component (i.e., the ability to guide behavior
by forming internal representations of stimuli that are no longer present in the environment). Human psychological tests of working
memory, such as the “n-back” task (28 ), on which patients with schizophrenia exhibit an impairment (29 ), provide a measure of
delay-dependent retention of mental representation. Several rodent tasks of working memory, such as delayed matching or
nonmatching to sample (30 ) and discrete trial delayed alternation (31 ), contain important elements of human experimental
paradigms and are used routinely to understand the cellular basis of working memory.

Schizophrenic patients, like patients with overt frontal lobe damage, display profound deficits in tasks that require behavioral
flexibility, strategy shifting, and response to environmental feedback. These tasks include the Wisconsin Card Sorting Test (32 ), the
Category Test (33 ), and the Tower of Hanoi Task (34 ), all of which involve an ability to utilize knowledge or feedback to change or
shape behavior. Several interesting tasks have been characterized for evaluating behavioral flexibility and strategy-shifting ability in
the rat. Of note is a maze-based strategy-shift task described by Ragozzino et al. (35 ). This task requires rats first to learn either a
response strategy (unidirectional turn) or a visually cued place-discrimination strategy (black vs. white maze arm) to obtain a food
reward. Following acquisition of the initial strategy, the rats are required to shift to the alternative strategy (e.g., response strategy
to visual cue strategy) or to reverse the reward contingency within a strategy (e.g., right turn response to left turn response) to
receive the food reward. This task is particularly useful in that it allows the experimenter to assess task acquisition, behavioral
flexibility within and between strategies, and perseverative behavior, all of which appear to be relevant to the cognitive deficits
associated with schizophrenia.

Attentional deficits are among the hallmarks of the clinical phenomenology of schizophrenia (36 ,37 ). One of the best characterized
rodent attentional tasks is the Five-Choice Serial Reaction Time task, which was designed by Robbins and co-workers (38 ) based on
the human Continuous Performance Test of Attention (39 ). The basic form of this task requires the rat to detect brief flashes of
light occurring in one of five holes and provides a steady-state procedure in which the effect of manipulations can be determined
against a baseline of stable performance. One advantage of this task is that it allows for a number of manipulations that test several
variables on which patients with schizophrenia show deficits during the Continuous Performance Test, such as perseverative
responding and omission errors.

The behavioral tests outlined here, albeit not without limitations, provide important tools for establishing the construct validity of
animal models of schizophrenia. Although relatively few studies (e.g., 19,75,87) have utilized these measures to characterize animal
models, their use may be important for defining novel pharmacologic approaches for the treatment of cognitive deficits associated
with schizophrenia.

Cellular and Molecular Phenotypes

Cellular and molecular markers, identified by morphologic findings from postmortem studies, are being used increasingly to establish
the validity of animal models of schizophrenia. Although a review of the schizophrenia postmortem literature is beyond the scope of
this chapter, some of the findings relevant to animal models include abnormalities in the neuronal organization of the prefrontal
cortex, such as a reduction in cortical volume (e.g., 7,43), altered laminar distribution of neurons that contain the enzyme
nicotinamide adenine dinucleotide phosphate diaphorase (44 ), and reduced neuropil and elevated neuronal density (45 ). In
addition to cytoarchitectural findings, postmortem evidence of abnormalities in cortical neurotransmitter function has also been
demonstrated. These abnormalities include alterations in γ-aminobutyric acid (GABA) neurons or other indices related to GABA-
mediated neurotransmission (46 ,47 and 48 ), decreased density of tyrosine hydroxylase immunoreactive axons (49 ), and changes in
gene expression of the NR2B and NR1 subunits of the NMDA receptor (50 ,51 ). These markers have been used primarily in validating
developmental animal models. Examples include alterations in cortical cell migration and neurogenesis in the gestational
malnutrition model (52 ) and reduced thickness of neocortex and hippocampus after prenatal exposure to the influenza virus (53 ) or
the antimitotic agent methyazoxymethanol (54 ).

Functional findings from imaging studies performed on patients with schizophrenia also have the potential of helping
 P.693

to establish the predictive and construct validity of animal models. Of note are studies demonstrating an exaggerated dopamine
release in response to amphetamine in patients with schizophrenia (55 ,56 ). These findings have been suggested to support the
validity of the amphetamine sensitization model of schizophrenia (57 ).

PHARMACOLOGIC MODELS
Part of "50 - Animal Models Relevant to Schizophrenia Disorders "

The most common approach for developing animal models has been to exploit pharmacologic treatments or “drug-induced states”
that produce schizophrenia-like symptoms in nonschizophrenic humans. These models generally have some predictive or construct
validity and have been instrumental in establishing three of the most prominent theories of schizophrenia: the dopamine hypothesis,
serotonin (or serotonin–dopamine) hypothesis, and glutamate hypothesis.

Dopamine-Agonist Models
The most widely studied class of drug-induced models of schizophrenia is based on the behavioral effects of psychostimulant drugs
such as amphetamine. Although the models that evolved from this approach have demonstrated considerable predictive validity in
terms of pharmacologic isomorphism, current thinking now indicates that the original appearance of face validity was actually
somewhat misleading. In recent years, the dopamine hypothesis of schizophrenia has evolved into the narrower hypothesis that the
mesolimbic dopamine system, distinct from the nigrostriatal dopamine system, is most relevant to schizophrenia. The nigrostriatal
dopamine system is now seen as most relevant to the dyskinetic side effects of antipsychotic treatments. The mesolimbic system
appears to mediate the locomotor-activating effects of lower doses of amphetamine, whereas the nigrostriatal system mediates the
stereotypies that predominate at higher doses (4 ). Thus, the stereotypies originally proposed to have the most face validity for the
human condition now appear to be more closely linked neurobiologically to phenomena that are considered side effects of the
clinical treatments. Because schizophrenic patients are not generally considered to be motorically hyperactive, the amphetamine-
induced hyperactivity that is mediated by what is believed to be the most relevant neurobiological substrate has seldom been
considered to mimic the human disorder. Note that the failure of the model to have face validity has in no way weakened its utility
in neurobiological research, which is based on the etiologic and predictive validity of the model. In fact, virtually any of the
behavioral effects of amphetamine in rodents, including either locomotor hyperactivity or stereotypy, have a high degree of
pharmacologic isomorphism as models for the efficacy of dopamine-antagonist treatments for schizophrenia (4 ,5 ); thus, the
predictive validity of these models appears limited to dopaminergic treatments.

Both dopamine agonists and antagonists have been studied in rat paradigms used to assess latent inhibition. Most of these paradigms
utilize three distinct stages: preexposure, conditioning, and expression. Drugs are typically given during the preexposure or
conditioning stages, or both. Hence, different drugs can produce different profiles depending on the stage at which they exert their
effect. Such complexity does not arise in the testing of latent inhibition in patients with schizophrenia, in whom the condition being
tested is necessarily present at all stages of the test paradigm. In both rats and healthy humans, amphetamine disrupts latent
inhibition in a haloperidol-sensitive manner (18 ). In contrast, direct dopamine agonists, such as apomorphine, do not alter latent
inhibition. One of the most interesting aspects of the latent inhibition paradigm is that both typical and atypical antipsychotics can
actually improve latent inhibition in rats when testing parameters are adjusted to yield low levels of latent inhibition in control
animals. Hence, the latent inhibition paradigm differs from most other models in rats in that it is possible to assess the effects of a
putative antipsychotic drug in a latent inhibition paradigm without first having to disrupt performance by the administration of a
drug or some other manipulation.

As reviewed in detail elsewhere (14 ,15 ), the sensorimotor gating deficits assessed by measures of PPI of startle in schizophrenia-
spectrum patients are mimicked in rats by the activation of dopamine systems. Thus, drugs such as the direct dopamine agonist
apomorphine and the indirect dopamine agonists D-amphetamine and cocaine impair PPI in rodents. As in patients with
schizophrenia (58 ), the apomorphine-induced disruption of PPI in rats is not modality-specific, being seen when acoustic prepulses
are used to inhibit either acoustic or tactile startle (59 ). The D2-receptor family appears to mediate the apomorphine disruption of
PPI in rats because it is blocked by D2 antagonists, largely insensitive to D1 antagonists, and reproduced by the D2 agonist quinpirole,
but not by the D1 agonist SKF 38393. Within the D2 family of receptors, the D2 subtype appears to be the most relevant to these
effects. In comparisons of knockout mice lacking either D2, D3, or D4 dopamine receptors, the effects of amphetamine on PPI were
absent only in the D2-subtype knockouts (60 ). In addition to being blocked by typical antipsychotics, the apomorphine-induced
disruption of PPI is reversed by the atypical antipsychotics clozapine, olanzapine, and quetiapine (15 ,61 ), which lack neuroleptic
properties in some behavioral assays. For example, clozapine fails to reverse amphetamine- and apomorphine-induced stereotypy in
rats, or apomorphine-induced emesis in dogs (4 ). The ability of antipsychotics, including atypical antipsychotics, to restore PPI in
apomorphine-treated rats strongly correlates with their clinical potency (r = .99) (61 ). In addition to its sensitivity, the specificity
of the PPI model for compounds with antipsychotic efficacy is supported by
 P.694

the fact that it predicts no such efficacy for a wide variety of other psychiatric drugs. Thus, the PPI paradigm appears to be
sensitive to both typical and atypical antipsychotics, but—when used with the dopamine agonist apomorphine—this paradigm clearly
fails to make the important distinction between these two classes of antipsychotic agents. Thus, converging evidence indicates the
important involvement of dopaminergic systems, acting via D2-family receptors, in the control of PPI. These findings in rats parallel
the deficits in PPI observed in schizophrenic patients (58 ), which are also reported to be corrected by both typical and atypical
antipsychotics (62 ,63 ).

It has been suggested that the increased stereotypy and submissive behavior produced by amphetamine in monkeys may mimic the
stereotypy and paranoid ideation in schizophrenic patients. Accordingly, both these behavioral effects can be prevented by
pretreatment with the antipsychotics haloperidol and chlorpromazine (21 ). In contrast, the social isolation induced by amphetamine
in monkeys is generally regarded as related to the social withdrawal seen in patients with schizophrenia. As in schizophrenic
patients, the animals actively avoid other animals, and these effects cannot be reversed by typical antipsychotic drugs such as
haloperidol and chlorpromazine (21 ). Although few novel antipsychotics have been tested in this model, both clozapine and
quetiapine have been shown to reverse amphetamineinduced social isolation in monkeys.

One aspect of the psychostimulant model that has generated considerable interest involves the dosage regimens required for
amphetamine or related drugs to produce psychotic-like behavior in psychiatrically healthy humans. Because of the widespread
belief that amphetamine-induced psychosis is produced only by repeated exposure to the drug (6 ,64 ), many preclinical researchers
have directed their attention to the behavioral effects of amphetamine that are augmented or sensitized by repeated administration
of the drug. A review of the available clinical literature, however, reveals that chronic exposure is not required and that psychotic
episodes can be produced by acute administration of amphetamine or related drugs (5 ). The complex and limited nature of the
clinical data seems to have led to mistaken interpretations that have inordinately influenced a large proportion of the basic research
in this area. Although it is clear that tolerance to the psychosis-inducing effects of amphetamine does not occur in humans, it is not
clear that sensitization is required for these effects. Hence, although an animal model based on the effects of chronic amphetamine
could be invalidated if tolerance were observed, the development of sensitization does not provide evidence supporting the
relevance of the model to schizophrenia. Indeed, it appears that the animal models having the greatest amount of predictive
validity are those based on the effects of the psychostimulant that are evident after acute administration (4 ,5 ).

Serotonin-Agonist Models
Soon after the initial reports of the behavioral effects of lysergic acid diethylamide (LSD), researchers began to explore the idea
that the class of drugs represented by LSD might appropriately be called psychotomimetics, or even psychotogens. This hypothesis
was engendered by the similarities between the effects of LSD on perception and affective lability and the symptoms of the early
stages of psychoses such as schizophrenia (65 ). Recent studies systematically comparing hallucinogen-induced psychotic states with
the early stages of psychotic disorders have confirmed substantial overlap in the two syndromes (66 ). Despite many clear
similarities, two major differences prompted the dubious albeit widely accepted conclusion that this class of drugs does not provide
a useful model of schizophrenia (67 ,68 ). First, tolerance was found to develop rapidly to the subjective effects of LSD-like drugs,
whereas the symptoms of schizophrenia persist for a lifetime. Second, the hallucinations produced by LSD and related drugs are
typically visual rather than auditory, as is characteristic of schizophrenia. These two observations weaken the predictive validity of
the hallucinogen model of the syndrome of schizophrenia.

Initial interest in hallucinogens was spurred by the possibility that abnormalities of biochemistry might lead to the endogenous
production of such compounds and hence be responsible for some psychotic symptomatology. For example, the transmethylation
hypothesis posited that serotonin could provide a substrate for the endogenous production of hallucinogens similar to N,N-
dimethyltryptamine (DMT) (68 ). Initially, this etiologically based model was dismissed because of the rapid tolerance associated
with traditional hallucinogens such as LSD and mescaline. Nevertheless, recent studies indicate that no tolerance occurs to the
subjective effects of DMT in humans (69 ), which suggests that DMT may differ from other hallucinogens and that this model may
still be viable. Indeed, different mechanisms may be involved in the various actions of the different hallucinogenic drugs, as
suggested by the lack of cross-tolerance to DMT in human subjects made tolerant to LSD (70 ). Hence, further studies are warranted
to provide the objective evidence needed to evaluate adequately the model of psychosis based on the hypothesis of an endogenous
psychotogen.

Furthermore, it remains possible that these drugs may be psychotomimetic and therefore have relevance as models of some aspects
of psychotic episodes in humans. Recent suggestions of serotoninergic abnormalities in schizophrenia (71 ) and of 5-HT2A-receptor
contributions to the clinical efficacy of atypical antipsychotics (72 ) have revitalized interest in this possibility. Hallucinogens are
now believed to produce their characteristic subjective effects by acting as 5-HT2A agonists (73 ). Many of the newer atypical
antipsychotic drugs are clearly potent 5-HT2A antagonists (72 ). With regard to specific abnormalities exhibited by patients
 P.695

with schizophrenia, evidence indicates that the study of hallucinogen action may provide useful animal models. For example, both
schizophrenic and schizotypal patients exhibit deficits in startle habituation (9 ,11 ,12 and 13 ). Hallucinogenic 5-HT 2A agonists such
as LSD and mescaline produce similar deficits in startle habituation in rats (59 ,74 ). Conversely, opposite behavioral effects are
produced by 5-HT2A antagonists (74 ), including some antipsychotics (72 ). Similarly, the PPI-disruptive effects of hallucinogenic 5-
HT2-receptor agonists are blocked by the selective 5-HT2A antagonist M100907, but not by the dopamine blocker haloperidol (74 ).
Furthermore, in keeping with the similarities between acute psychotic states and the syndrome induced by hallucinogens, latent
inhibition is also disrupted by LSD and other serotoninergic hallucinogens (75 ), as it is in acutely ill schizophrenic patients. These
effects can be blocked by the putative antipsychotic M100907 (75 ). Thus, the effects of hallucinogens on habituation, PPI, and
latent inhibition in animals have some predictive validity with regard to both specific abnormalities exhibited by patients in the
early stages of schizophrenia and the effects of antipsychotics (74 ). The construct validity of this model is based on compelling
evidence that both the symptoms of schizophrenia and the effects of hallucinogens reflect exaggerated responses to sensory and
cognitive stimuli, theoretically resulting from failures in normal filtering or gating processes such as habituation, PPI, or latent
inhibition (1 ,3 ,9 ). Accordingly, 5-HT2A antagonism by itself might be effective in the treatment of certain forms of schizophrenia.
Indeed, a rather selective 5-HT2A antagonist, M100907, appears to have efficacy as an antipsychotic in some patients with
schizophrenia, despite having negligible affinity for dopamine receptors (76 ). This finding suggests the possibility of a
nondopaminergic mechanism for a treatment of subtypes of schizophrenia and provides important support for the predictive validity
of the hallucinogen model of psychosis.

Glutamatergic Models
Dysfunctional glutamate neurotransmission has been implicated in schizophrenia, primarily because noncompetitive antagonists of
the NMDA subtype of glutamate receptors, including PCP and ketamine, produce a behavioral syndrome in healthy humans that
closely resembles symptoms of schizophrenia and is frequently misdiagnosed as acute schizophrenia (77 ,78 ). The syndrome includes
positive symptoms, such as paranoia, agitation, and auditory hallucinations; negative symptoms, such as apathy, poverty of thought,
and social withdrawal; and cognitive deficits, such as impaired attention and working memory. The remarkable similarity of PCP-
precipitated behaviors with the diverse array of symptoms associated with schizophrenia has prompted the use of PCP (and its
analogue ketamine) in pharmacologic models of schizophrenia in both basic and clinical studies. Notably, whereas psychotic
episodes are generally associated with prolonged abuse of amphetamine, a single exposure to PCP or ketamine can produce the
cognitive deficits and several symptoms listed above in healthy humans. Thus, acute exposure to these compounds is considered a
useful pharmacologic tool for producing some aspects of schizophrenic symptomatology in the laboratory animal.

Several interesting aspects of this model distinguish it from monoamine-based models. For example, the behavioral effects of PCP
and related compounds are not, for the most part, mediated by increased dopamine transmission and therefore are not blocked by
typical antipsychotics (see below). Similarly, in normal human volunteers, the psychotomimetic effects of ketamine are not blocked
by typical antipsychotics, but they are reduced significantly by the prototypal atypical antipsychotic clozapine (79 ). Therefore, this
model may be especially useful for testing the effectiveness of atypical and perhaps even novel antischizophrenia drugs. In fact, the
first non-monoaminergic ligands (including a glycine-site agonist and a metabotropic glutamate-receptor agonist), which have
recently entered clinical trials, have been based on preclinical PCP models (41 ,80 ). Another attractive aspect of the NMDA
antagonist model is that, unlike the dopamine-based models, it has strong construct validity for studying the cognitive and
attentional deficits in schizophrenia. In laboratory animals, NMDA antagonists impair working memory, set shifting, and other
cognitive functions that are related to schizophrenia (31 ). More importantly, in clinical studies, direct comparison of schizophrenic
patients with healthy volunteers receiving subanesthetic doses of ketamine have indicated no significant difference in scores for
thought disorder between the two groups (81 ).

In rats and monkeys, noncompetitive NMDA antagonists, including PCP and ketamine, produce a range of behavioral abnormalities
that have important relationships to schizophrenic symptomatology. These drugs produce both locomotor hyperactivity and
stereotyped behaviors. Although they also increase dopamine neurotransmission in limbic regions (82 ), their motor-activating
effects appear to be dopamine-independent (83 ). At rather low doses, PCP retards habituation of the startle response without
affecting startle reactivity (84 ), a pattern similar to that seen in parallel studies in schizophrenic patients (9 ). Also as in
schizophrenia, PCP-treated rats exhibit marked deficits in social behavior. Although typical antipsychotics have no reliable effect on
the PCP-induced disturbance in social behavior in rats, the atypical antipsychotics clozapine, sertindole, and olanzapine appear to
reverse the effects partially (22 ). In terms of sensorimotor gating measures, PPI is reduced or eliminated in rats by psychotomimetic
noncompetitive NMDA antagonists, including PCP, dizocilpine (MK-801), and ketamine (14 ,15 ). As with apomorphine and as in
schizophrenia, both intramodal and cross-modal PPI is sensitive to noncompetitive NMDA antagonists (59 ). In contrast to
 P.696

the effects of dopamine agonists on PPI, but in keeping with the results of studies of the subjective effects of ketamine in humans,
the PPI-disruptive effects of NMDA antagonists are not reversed by typical antipsychotics such as haloperidol or selective D1 or D2
antagonists. Importantly, these effects are reversed by the atypical antipsychotics clozapine, olanzapine, quetiapine, and
remoxipride (14 ,15 ). These findings raise the possibility that the PCP-induced disruption of PPI may be a useful model for
identifying compounds with atypical antipsychotic potential.

In addition to acute dosing with PCP and ketamine, withdrawal from repeated administration of PCP has been proposed to be a
useful model for some aspects of schizophrenia (85 ). Although this model lacks some of the important characteristics of acute
models, such as lack of an effect on PPI, it produces an enduring cognitive impairment that is highly relevant to schizophrenic
symptomatology.

DEVELOPMENTAL MODELS
Part of "50 - Animal Models Relevant to Schizophrenia Disorders "

The best-characterized animal model in this class is that proposed by Lipska and Weinberger (86 ,87 ), which involves neonatal
excitotoxic lesions of the ventral hippocampus. These lesions produce postpubertal behavioral disturbances, such as increased
spontaneous, amphetamine-induced, and NMDA antagonist-induced locomotion. They also produce potentiated apomorphine-
induced stereotypies, disruption of PPI, reduced cataleptic response to haloperidol, impaired working memory, and hypersensitivity
to stressful stimuli. Furthermore, this manipulation results in alterations in some cellular and molecular markers that may have
relevance to schizophrenia (88 ), such as reduced expression of the glutamate transporter excitatory amino acid transporter 1 (EAAT1)
and glutamic acid decarboxylase (GAD67). To the limited extent that they have been tested, dopamine antagonists, including classic
and atypical antipsychotic drugs, ameliorate the behavioral abnormalities produced by neonatal ventral hippocampal lesions. It will
be important in the future to examine the predictive power of this model for the identification of antipsychotic drugs more
thoroughly with measures that are not sensitive to the effects of antipsychotic drugs in sham-lesioned rats.

Other strategies that have been used to disrupt early cortical development include systemic exposure to L-nitroarginine, a nitric
oxide synthase inhibitor that disrupts neuronal maturation (89 ), or the antimitotic agent methyazoxymethanol (54 ,90 ). These
models produce morphologic changes relevant to schizophrenia, such as altered neurogenesis and reduced cortical volume. They
also produce some of the behavioral characteristics associated with schizophrenia, such as stereotypy, cognitive impairments, and
deficits in PPI. As yet, the predictive validity of this model in terms of sensitivity to antipsychotic treatments remains to be
determined.

Isolation rearing of rats has also been used as a manipulation to generate models related to schizophrenia and models of depression
and attention-deficit/hyperactivity disorder (ADHD). In the context of schizophrenia, the focus has been on the disruptions of PPI
rather than the locomotor hyperactivity observed in isolation-reared rats. Indeed, comparisons among different strains of rats
indicate that both effects are strain-dependent but appear in different strains (91 ,92 and 93 ). Thus, as with a variety of
pharmacologic manipulations, locomotor hyperactivity and deficient PPI are readily dissociable behavioral phenomena, even though
both have been used in animal models related to schizophrenia. In several laboratories, isolation-reared rats have been shown to
exhibit a neuroleptic-reversible deficiency in PPI in comparison with group-reared controls (91 ,94 ). This effect of isolation rearing
appears to be specific to development; similar isolation of adult rats fails to produce the deficit in PPI observed in isolation-reared
rats (95 ). Furthermore, as in the most common form of schizophrenia, the PPI deficits are not evident before puberty but emerge at
about that time (96 ). Converging evidence for an influence of isolation rearing on gating mechanisms in adulthood stem from the
observation that the rat analogue of the P50 sensory gating deficit of schizophrenia is also seen in isolation-reared rats (97 ).
Because these deficits in PPI and P50 gating are not associated with concomitant deficits in latent inhibition (95 ), which occurs only
in acutely ill schizophrenic patients (19 ), it would appear that the isolation-rearing model is more relevant to chronic than to acute
schizophrenia. In addition to reversals by typical antipsychotics (haloperidol, raclopride), reversals of the isolation-induced deficits
in PPI by clozapine, risperidone, quetiapine, olanzapine, and the putative antipsychotic M100907 have been observed (94 ,98 ). Thus,
PPI deficits in isolation-reared rats may be a valuable paradigm that—like the apomorphine-induced disruption of PPI—is sensitive,
but not specific, in its ability to identify compounds with atypical antipsychotic properties. The potential advantage of the isolation-
rearing model, as of other models involving developmental perturbations, is that it does not rely on the administration of a drug or
the introduction of an artificial lesion to produce the behavior of interest. When the behavior studied in the model is induced by a
drug, such as a dopamine or serotonin agonist, the model is typically effective in identifying the corresponding antagonist. Indeed,
most of the animal models of schizophrenia have relied on dopaminergic psychostimulants and have proved to be largely limited to
the detection of dopamine antagonists. The major message of the fact that clozapine is effective, even at doses that achieve low
levels of dopamine receptor occupancy, is that new treatments can be identified for patients with schizophrenia, and that these
novel treatments may not involve dopamine antagonism. The isolation-rearing manipulation presumably produces a deficit in PPI by
virtue of a substantial reorganization of neural circuits through the course of development.
 P.697

Hence, such a model has the potential to identify completely novel antipsychotic treatments simply because it does not require the
administration of a drug.

GENETIC MODELS
Part of "50 - Animal Models Relevant to Schizophrenia Disorders "

Genetic contributions to schizophrenia have been clearly established in family studies. Although the focus of considerable research,
the application of linkage analyses to schizophrenia has not generally proved successful, perhaps because schizophrenia does not
represent a single phenotype. Nevertheless, it remains possible that genetic approaches will lead to etiologically based models

Strain Differences
Genetic factors appear to be critical determinants of both sensory and sensorimotor gating in rats. For example, some inbred strains
of mice are deficient in gating of the N40 event-related potential (17 ), which is the rodent analogue of the P50 gating deficit seen
in schizophrenia. Indeed, a linkage between the P50 gating deficit in patients with schizophrenia and a specific chromosomal marker
associated with the gene for the α7 subunit of the nicotinic acetylcholine receptor has been demonstrated in a series of elegant
studies (16 ). The potential power of cross-species studies of specific behavioral abnormalities in psychiatric disorders is exemplified
by the parallel between these human linkage studies and the observation that the strain of mice that is most deficient in gating of
the N40 event-related potential is also the most deficient in α7-nicotinic receptors (17 ). Such parallel investigations in patients and
animals provide an exemplar for the application of modern molecular biological techniques to the generation and validation of
animal models of psychiatric disorders. However, this genetically related deficit in sensory gating does not extend to studies of
sensorimotor gating as measured by PPI of the startle response. Thus, mice in which the α7-nicotinic receptors have been deleted by
genetic engineering exhibit normal levels of PPI (99 ). Nevertheless, other evidence indicates that PPI is regulated by genetic factors.
For example, strain-related differences in the dopaminergic modulation of PPI have been reported (100 ). More relevant to the
recent indications that PPI deficits are evident in family members of schizophrenia patients (101 ), Ellenbroek et al. (102 ) utilized
pharmacogenetic selective breeding to produce strains of rats that were either sensitive (APO-SUS) or insensitive (APO-UNSUS) to
the effects of apomorphine on gnawing behavior. Within either a single generation or after many generations of selective breeding,
APO-SUS rats and their offspring exhibited significantly less PPI and latent inhibition than did APO-UNSUS rats. Apparently, the
physiologic substrates that regulate behavioral sensitivity to apomorphine (presumably some feature related to dopamine-receptor
transduction) are associated with substrates that regulate both PPI and latent inhibition, which are transmitted genetically. In
another approach, comparisons of several inbred strains of rats identified some strains that exhibit deficits in PPI (103 ). Because
these strains did not exhibit hearing impairments, the genetically determined deficit in PPI likely represents a deficit in
sensorimotor gating processes.

Genetically Modified Animals

Other examples of nonpharmacologically based models relevant to schizophrenia are emerging from the field of molecular biology,
in which genetic engineering is being used to generate transgenic and knockout animals. In the absence of established candidate
genes, the use of mutant animals in models of schizophrenia has focused on the identification of phenotypic differences in behaviors
considered relevant to the clinical disorder (i.e., validity has been sought primarily in the dependent measure rather than in the
independent variable). For example, schizophrenia-like deficits in PPI of startle have been observed in specific strains of mice (104 )
and in “knockout” mice in which specific genes have been deleted (105 ). The focus of genetic engineering in the mouse is beginning
to prompt extensions of pharmacologic studies from the rat to the mouse. Although much more such work is needed, it is already
abundantly clear that species differences in pharmacologic effects between mice and rats will complicate the application of some
schizophrenia-related rat models to mice. For example, in rats, antipsychotic drugs by themselves have minimal effects on PPI, in
contrast to their marked suppression of locomotor activity and ability to improve latent inhibition. Hence, rat PPI models can
identify antipsychotic effects only if a drug reverses the effects of a disruption in PPI produced by another drug, a lesion, or a
developmental manipulation such as isolation rearing. In mice, however, it appears that antipsychotics improve PPI in mice that
have not been manipulated (106 ). This important difference means that it may be easier to detect antipsychotic effects in mice,
but also that it will be much more difficult to demonstrate a reversal of a PPI deficit produced by an experimental manipulation.

In an approach that is distinctly different from the candidate gene approach, genetically modified mice have been used to test
specific hypotheses of relevance to animal models of schizophrenia. For example, although most pharmacologic evidence in rat had
implicated the D2 subtype of the family of dopamine receptors in the PPI-disruptive effects of dopamine agonists, gene knockout
mice proved useful in testing this conclusion more definitively. Ralph et al. (60 ) tested the effects of amphetamine on PPI in D2-,
D3-, and D4-receptor knockout mice and corresponding wild-type mice. Only the mice lacking the D2 subtype of receptor failed to
show the normal effect of amphetamine on PPI. Although knockout manipulations are confounded
 P.698

by developmental adaptations, such a study takes advantage of the specificity that represents the fundamental strength of the
knockout technology, as no dose of a drug can ever inactivate all of a given receptor without interacting with other receptors at the
same time.
Another model with relevance to the etiology and pathophysiology of schizophrenia involves the NMDAR1 (NR1) “knock-down” line of
mutant mice (107 ). These animals display exaggerated spontaneous locomotion and stereotypy in addition to deficits in social and
sexual interactions. Interestingly, preliminary studies indicate that some of these behavioral abnormalities may be ameliorated with
a single dose of haloperidol or clozapine. This is an intriguing model that, with further characterization, may advance our
understanding of the long-term effects of congenital NMDA-receptor hypofunction. Nevertheless, its relevance to schizophrenia may
be questioned by the fact that no evidence has been found in schizophrenia for abnormalities in genes that express subunits of the
NMDA receptor (108 ). Furthermore, these animals, as would be expected, show no behavioral reaction to the NMDA antagonists PCP
and dizocilpine. Schizophrenic patients, on the other hand, exhibit a profound exacerbation of preexisting symptoms after exposure
to a single dose of PCP (109 ) or ketamine (110 ).

CONCLUSIONS
Part of "50 - Animal Models Relevant to Schizophrenia Disorders "
Establishing the construct, etiologic, and predictive validity of animal models relevant to schizophrenia-related disorders is limited
by the paucity of rigorous experimental data derived from clinical studies. The major source of validation remains the ability of
established antipsychotic drugs to demonstrate efficacy, measured by broadly defined clinical scales in heterogeneous groups of
patients. Specific measures of clinical subtypes, clinical course, and symptom-specific treatment effects that can be translated into
relevant animal models are needed to overcome the limitations inherent in relying on global assessments of treatment efficacy for
validity assessment. The objective study of such measures in translational research is critical for the eventual identification of new
antipsychotic treatments. Such treatments not only could help patients who are resistant to treatment with typical antipsychotics
but also could help in the treatment of the negative and cognitive symptoms that do not appear to be treated adequately even by
the newest generation of atypical antipsychotics.

ACKNOWLEDGMENTS
Part of "50 - Animal Models Relevant to Schizophrenia Disorders "
This work was supported by National Institutes of Health (NIH) research grants DA02925 (MG), MH42228 (MG), MH52885 (MG),
MH48404 (BM), and MH 01616 (BM); the VISN 22 Mental Illness Research, Education, and Clinical Center (MG); and the Veterans
Administration National Center for Schizophrenia (BM).

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51
Endophenotypes in Studies of the Genetics of Schizophrenia

David L. Braff

Robert Freedman

David L. Braff: Department of Psychiatry, University of California at San Diego, La Jolla, California.

Robert Freedman: Department of Psychiatry and Pharmacology, University of Colorado, Denver, Colorado.

The power and appeal of the molecular biology mantra, “DNA to RNA to protein,” to explicate cell biology comes from its universal
appearance and application in all species, from microorganisms to human beings. Based on this mantra, the genomes of viruses,
bacteria, fruit flies, and now humans are being mapped and sequenced, so that all the genes and, ultimately, their corresponding
biological activity can be identified. As these genes are identified, it is reasonable to ask how this information can be related to the
inheritance of risk for psychiatric illness. For a bacterial enzyme, genetic coding of the amino acid sequence of proteins can be
closely associated with a functional change in enzymatic activity. For a complex psychiatric illness, as defined by DSM-IV criteria,
the relationship is obviously not as straightforward. Psychiatric illnesses such as schizophrenia are generally conceptualized as
multifactorial and most likely reflect the combined influence and interactions of both genetic and nongenetic factors. Furthermore,
there is no reason to presuppose that only one gene is responsible for a complex psychiatric disorder such as schizophrenia, as there
is in some simple mendelian illnesses. Persons who are ill may differ in more than one gene from the rest of the population, and
different sets of genes may be associated with illness in different populations. Thus, how best to use the power of molecular
genetics to understand the inheritance and pathophysiology of complex genetic psychiatric illnesses remains an enigma that is only
now beginning to be solved.

In the simplest and most commonly used strategy of molecular genetics that is applied to complex psychiatric disorders, it is
assumed that the distribution of illness in a family represents the effect of a single gene, and techniques of genetic analysis are used
to identify that gene. This approach does not necessarily overlook the complexity of psychiatric illness, but it assumes that the
effect (i.e., signal) of a single gene will be discerned in a complex, “noisy” genetic background if samples sizes are large enough or
if the population is sufficiently homogeneous (e.g., 1). An attractive feature of this approach is that the search for genes is not
constrained by preexisting hypotheses about the biology of the illness, which, in the case of schizophrenia, is still unclear. A second
commonly applied strategy is, in fact, the opposite approach; an assumption is made about the biology of the illness and then
candidate genes associated with that biology are examined to determine if they are mutated. Both approaches have been successful
to a limited extent for explicating the genetics of schizophrenia. Replicable linkages for schizophrenia have been obtained at several
locations (e.g., chromosomes 1, 6, 8, 13, 15, and 22), but genetic mutations have not as yet been identified at these sites (2 ). On
the other hand, DNA mutations have been found in candidate genes such as NURR1, the gene for the receptor for retinoic acid, a
pathway critical in neuronal development, but these mutations seem to be found in only a small proportion of schizophrenic patients
(3 ).

This chapter describes a third approach, which attempts to make use of the power of the molecular biology mantra by identifying
brain dysfunctions that may be caused by a single genetic abnormality. The rationale comes from the mantra itself. If discrete
genetic abnormalities are associated with schizophrenia, then each of them should cause a specific protein change that is reflected
in a corresponding discrete functional abnormality. Even if several genes are abnormal, along with additional environmental factors,
the functional abnormality resulting from each gene should generally be identifiable. Theoretically, the relationship between these
functional abnormalities and genes, discovered either by genetic linkage or by candidate gene analysis, should be stronger than the
association to the illness itself because the illness itself results from a mixture of genetic and nongenetic abnormalities that may
vary between different individuals and families. As is true for the other approaches described above, this approach has not yet led
to the identification
 P.704
of the genes that are associated with and that may even cause schizophrenia in most cases. Nevertheless, the strategy has been useful for gene
discovery in other complex illnesses, such as colon cancer and hemochromatosis. In colon cancer, the formation of multiple polyps, rather than
cancer itself, has been found to be the genetically heritable trait (4 ), and in hemochromatosis, a high serum level of iron, rather than the clinically
recognized illness, has been found to be the more penetrant heritable trait (5 ).
Endophenotype is often used as the descriptive term for these discrete, genetically determined phenotypes that may be part of a complex illness.
The search for endophenotypes is not straightforward because no a priori criterion can be used to decide if a particular element of schizophrenia or
any other psychiatric illness reflects the effect of a single gene. Putative endophenotypes have ranged from clinical characterizations, such as the
presence of schizotypy in relatives of schizophrenic patients (6 ), to the neurophysiologic and neuropsychological measures described in this chapter,
to structural measures of specific, functionally important regions of the brain and ventricular size. Because none of these phenotypes has yet led to
the identification of a specific molecular deficit, it has not been proved that any one of them is actually linked to a specific genetic abnormality. In
this context, even if endophenotypes turn out to be multiple, rather than single, gene phenomena, their genetic architecture, even as complex
endophenotypes, may turn out to be simpler than schizophrenia in certain families. The sections below outline the stage of investigation for a
number of putative phenotypes, from presence in schizophrenia probands and their relatives to statistically significant genetic linkage to a
chromosomal locus.
Several points must be considered in the assessment of endophenotypes. First, because these are putative genetic traits, their biology begins at
conception, so that by the time they are measured in adulthood, their expression may have been modified by such factors as development, aging,
brain injury, and medication and substance abuse and. Second, most genes expressed in the brain are expressed in many different brain areas, so
that their ultimate functional expression may involve much more than the simple phenotype being measured. Third, many genes expressed in the
brain are also involved in the development of neurons, so that their most important functional effects may have occurred prenatally. Fourth,
according to Mendel’s second law, every genetic trait segregates independently in a family, so that if schizophrenia is a multifactorial trait, some
siblings should express specific phenotypes independently of other phenotypes. These siblings may be better subjects for characterizing the
phenotype than the patients themselves, whose multiple deficits may obscure the unique phenotype. Finally, because the aim of genetics generally
is to identify affected individuals who have or do not have a particular genetic abnormality, the measurement of the putative phenotype must
clearly separate most affected and unaffected individuals, regardless of whether a quantitative or discrete variable is used. The range of effect sizes
for several putative endophenotypes is shown in Table 51.1 , which reflects another point. The measurement of endophenotypes is in itself a
complex endeavor in which modest-appearing paradigmatic manipulations lead to significant shifts in the signal of the dependent measure being
assessed.

TABLE 51.1. ENDOPHENOTYPES AND THE GENETICS

OF SCHIZOPHRENIA: EFFECT SIZE DIFFERENCES
BETWEEN SCHIZOPHRENIA SPECTRUM GROUPS AND
NORMAL COMPARISON SUBJECTS

The search for endophenotypes takes advantage of genetic strategies to evaluate the current state of understanding the pathophysiology of
schizophrenia. The initial endophenotype was schizotypy, which was proposed to be a pure expression of schizotaxia, the genetic predisposition for
schizophrenia. Schizotypy itself does not generally show mendelian segregation, so that the likelihood that it reflects a single genetic trait is now
considered small. However, the presence of schizotypy in family members has been related to linkage of schizophrenia at a specific chromosomal
locus in a subset of families, so that a reexamination of schizotypy as an endophenotype in some families may once again be productive. Inhibitory
interneurons have increasingly become a focus of interest in the biology of schizophrenia. Many of the endophenotypes described below are attempts
to demonstrate inhibitory neuronal function by means of psychophysiologic and neurophysiologic techniques. Structural phenotypes have been
limited to the measurement of brain volume. As functional brain-imaging techniques become more advanced, so that specific neuronal functions can
be demonstrated, it is likely that these techniques will also be used. Magnetic resonance spectroscopy of the amino acids associated with neuronal
function, such as N-acetylasparate, is an example (7 ).

 CANDIDATE ENDOPHENOTYPES FOR GENETIC STUDIES

 SUMMARY
 ACKNOWLEDGMENTS

CANDIDATE ENDOPHENOTYPES FOR GENETIC STUDIES

Part of "51 - Endophenotypes in Studies of the Genetics of Schizophrenia "
Given the considerations discussed above, it is important to stress that although the DSM-IV diagnostic criteria for schizophrenia may be clinically and
administratively useful, they are not likely to be optimally useful as phenotypes in genetic studies (8 ,9 ,10 and 11 ). The search for and use of new,
nondiagnostic, non–DSM-IV-based candidate endophenotypes parallels our search for the corresponding candidate genes in complex human genetic
disorders such as schizophrenia. Also, we understand that in accounting for the genetic diathesis or vulnerability to schizophrenia, we are
“accounting” for, at most, 50% to 70% of the variance of the disorder; the remaining variability resides in nongenetic “second hits,” such as neonatal
or in utero neural damage to the developing hippocampus (12 ,13 ,14 and 15 ) or other factors. A plethora of studies indicate that in addition to
mutant genes, a second level of environmental or other generalized or specific stressor probably must act as a second hit in the central nervous
system. An example of the result of this need for a second hit is illustrated by the fact that clinically “unaffected” relatives of patients with
schizophrenia have endophenotypic markers of abnormalities in some or all of the measures listed below but do not have the disorder of
schizophrenia. Therefore, it appears clear that some nongenetic contributions (not necessarily reflected by these endophenotypes) are crucially
important in the expression of some forms of this elusively heterogeneous and complex disorder of schizophrenia. In searching for non–diagnosis-
based “candidate endophenotypes,” we are not alone in schizophrenia research because many disorders, from diabetes to hypertension to bipolar
disorder, also present the same conundra and difficult conceptual issues.
In schizophrenia research, it seems reasonable to classify candidate endophenotypes into structural and functional abnormalities. Because of limits
of chapter length, we do not discuss structural endophenotypes (e.g., widely dispersed, decreased, generalized gray matter; decreased superior
temporal gyrus volume; deficits of hippocampal or temporal lobe volume (16 ); gray matter volume or organizational abnormalities in various
subsections of the prefrontal cortex, most significantly the dorsolateral prefrontal cortex) that may be useful in genetic studies (17 ,18 ). It is
important to note that such structural abnormalities may be correlated with some of the functional abnormalities discussed below. In Table 51.1 ,
some functional endophenotypes are listed, along with estimates of the effect sizes of deficits of each in schizophrenic patients, clinically
unaffected relatives of schizophrenic patients, and schizotypal patients in comparison with normal subjects. In addition, reasonable and well-
understood neural substrates for these measures are known, as discussed. Table 51.1 summarizes what we have selected as important and
representative (but not all-inclusive) candidate endophenotypes for genetic studies in schizophrenia, with an emphasis on the information-processing
abnormalities that have assumed a central role in the search for candidate endophenotypes (10 ,11 ). Identifying these endophenotypes for genetic
studies is only a first step; after they have been identified, complex strategies must be employed, as discussed above and below, to conduct linkage,
association, and other genetic studies on the candidate endophenotypes so that we can identify the candidate genes that are likely contributing to
the endophenotypes (and their neural substrates) present in the complex disorder of schizophrenia. In the next section, we describe possible
functional endophenotypes, with an emphasis on gating and oculomotor abnormalities.
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Abnormalities of Sensorimotor Gating

The concept of deficits of sensory gating in schizophrenia derives from the clinical observation that patients report failures of
information processing characterized by poor sensory gating—an inability to screen out trivial stimuli and focus on salient aspects of
the environment so as to process information smoothly and successfully navigate through life (10 ,19 ,20 ). Gating functions are
commonly assessed with P50 suppression and prepulse inhibition (PPI) of the startle response.

P50 Suppression
Initial studies at the University of Colorado have identified P50 suppression as an important candidate endophenotype in studies of
schizophrenia (21 ,22 ,23 and 24 ). In a typical paradigm, P50 suppression occurs when two clicks are presented with a 500-
millisecond interval between them. The small P50 event potential wave elicited by the click stimulus can be identified when many
trials (e.g., 30 to 100 or more) are performed; this number of trials plus various filtering strategies provides investigators with a
robust signal-to-noise ratio for identifying and quantifying the P50 wave. A P50 wave is generated to the first click and another to
the second click. Across multiple studies, it has been found that the second P50 wave is normally suppressed; suppression can
probably be attributed to the activation of inhibitory processing and circuitry by the first P50 stimulus. In normal subjects, the
second P50 wave typically is diminished by 80% in comparison with the first wave (Fig. 51.1 ).

FIGURE 51.1. Pairs of auditory clicks are presented to subjects and EEG is averaged across trials. The P50 component of the
auditory event-related potential is measured in response to the first and second clicks.

Initial studies (21 ,22 ,23 and 24 ) demonstrated an expected failure of suppression in schizophrenic patients, consistent with
theories of failed inhibitory function or impaired sensorimotor gating in schizophrenia (25 ). These studies of deficits in P50
suppression in schizophrenic patients have been widely replicated (26 ,27 ,28 ,29 ,30 and 31 ). The failure of P50 suppression in
schizophrenic patients is not necessarily specific to this one disorder. For example, Franks et al. (32 ) reported that P50 suppression
is also deficient in patients with acute mania but “normalizes” with time, whereas the deficits of P50 suppression are more
persistent in schizophrenic patients (32 ). This finding is consistent with the idea that genetic “diatheses” may be shared between
schizophrenia and mania. P50 suppression deficits have also been shown (and replicated) in “clinically unaffected” family members
of schizophrenic patients (24 ,31 ,33 ,34 and 35 ). The P50 suppression abnormalities of these family members normalize following
administration of the cholinergic nicotinic receptor stimulant nicotine (36 ), as do those of schizophrenic patients (37 ). This finding
has raised interest in the critical importance of the cholinergic system in P50 suppression, and some of the cholinergic
neurobiological substrates of P50 suppression deficits have been elucidated.

As discussed in the section on PPI , suppression is probably the function of a more wide-ranging neural circuitry prominently
involving hippocampal structures (38 ). The use of P50 suppression as a candidate endophenotype in genetic studies is probably the
most advanced of any of the endophenotypes we discuss here; a specific linkage of P50 suppression with a genetic marker at the
locus of the α7 subunit of the nicotinic receptor gene (11 ) has been identified in the first study linking a candidate endophenotype
of information processing in schizophrenia to a specific
 P.707

chromosomal region. It is important to stress that these types of studies do not identify a “schizophrenia endophenotype,” but
rather the linkage of deficits in P50 suppression (characteristic of schizophrenia) to a specific chromosome region. Future studies
will have to identify the specific genetic deficit(s) (e.g., specific single-nucleotide polymorphisms) associated with abnormalities of
P50 suppression.

In terms of our assessment of candidate endophenotypes and genetic studies, it is important to note that medications have an
influence on P50 suppression abnormalities in schizophrenia. It appears that atypical antipsychotic medications may reverse the P50
suppression deficits in schizophrenic patients (39 ,40 ,41 and 42 ). If these initial results continue to be confirmed, the search for
candidate endophenotypes will be complicated by the fact that atypical (and perhaps, in some circumstances, typical) antipsychotic
medications are increasingly being utilized as first-line agents in the treatment of schizophrenia. We may thus face the circumstance
of examining schizophrenic patients whose P50 suppression deficits have been “normalized” and then conducting family studies in
which these deficits may appear in unaffected relatives of schizophrenic patients. Should this occur, genetic statistical strategies
will have to be utilized that will allow us to “exclude” the “normalized” schizophrenic patient from analysis and utilize only
clinically unaffected family members in genetic (e.g., linkage) studies. Much more information will be generated in the next several
years, and the use of what we would term “null proband” strategies may be necessary as schizophrenic patients who are not
medicated or are neuroleptic-naïve become more difficult to ascertain and are replaced by patients treated with atypical
antipsychotic medications. In addition, the use of drug withdrawal strategies to unmask endophenotypic markers has come under
increasing criticism (43 ) and is becoming more difficult to justify ethically in comparison with other promising research strategies
(e.g., 44).

Prepulse Inhibition of the Startle Response

Since 1978 (45 ), PPI deficits of the startle response have been consistently identified in schizophrenic patients. PPI of the startle
response occurs as follows. Normally, an intense and powerful sensory stimulus elicits a whole-body startle response in almost all
mammals. This rapid, intense sensory stimulus may be sound or light, or it may be tactile (e.g., an air puff). When a weak
prestimulus precedes the startling stimulus by approximately 100 milliseconds, PPI occurs. Schizophrenic patients and their relatives
show deficits in PPI (45 ,46 ,47 ,48 ,49 ,50 ,51 ,52 ,53 and 54 ) (Fig. 51.2 ). This is the second commonly studied form of
sensorimotor gating (along with P50 suppression) (see ref. 25 for a discussion of gating abnormalities in schizophrenia). PPI is being
increasingly used in schizophrenia research. It is important to distinguish the PPI paradigm described above from a similar but quite
distinct paradigm in which attentional allocation to the prepulse (55 ) is used in an attempt to increase the degree of PPI. The PPI
paradigm typically used in schizophrenia research, described above, is a “neutral” or “uninstructed” paradigm that largely taps into
involuntary and automatic information processing (56 ); the instructed paradigm is different because subjects attend to the prepulse
and thus the paradigm identifies so-called voluntary attentional deficits. This section describes the more widely studied
uninstructed PPI.

FIGURE 51.2. Across all prepulse-to-pulse

intervals tested, the schizophrenic patients

showed a loss of gating effect of the prepulse

that preceded the startle stimulus. (From Braff

DL, Grillon C, Geyer MA. Gating and habituation

of the startle reflex in schizophrenic patients.

Arch Gen Psychiatry 1992;49:206–215, with

permission.)

Much like deficits of P50 suppression, PPI deficits are not unique to schizophrenia. PPI deficits are characteristic of a “family” of
disorders in which cognitive, sensory, and motor information undergoes a failure of gating. Patients with gating disorders include
those with schizophrenia (45 ,46 ,47 ,48 ,49 ,50 ,51 ,52 and 53 ), obsessive-compulsive disorder (with obsessive and ungated ideas)
(57 ), and Huntington syndrome (58 ) and Tourette syndrome (59 ) (with ungated motor activity).

The clinical correlates of PPI in schizophrenia comprise a rich database. PPI deficits have been correlated with distractibility (60 ),
perseverative responses on the Wisconsin Card Sorting Test (61 ), and most prominently thought disorder (62 ), especially when PPI
and thought disorder are measured at the same time (63 ). These deficits are also associated with an earlier age of onset (51 ).
Modest correlations have been found with both positive and negative symptoms, and the symptom correlates may be associated with
subcortical dopamine hyperactivity and reciprocal frontal dopamine
 P.708

hypoactivity (47 ). An initial report has described PPI deficits in clinically unaffected family members of schizophrenic patients (54 ),
and further work is needed to understand the heritability pattern of PPI deficits in family members of schizophrenic patients. Much
of what is known about the neural substrate of PPI can be attributed to the extensive work of Swerdlow, Geyer, Braff, and their
associates. It appears that PPI is modulated mostly by the ventral cortico-striato-pallido-thalamic (CSPT) circuitry originally
described by Swerdlow and Koob (64 ), based on the pioneering work of Penney, Alexander, and Young on the dorsal loci of the CSPT
circuits. The circuitry cannot be described in detail here, but lesion infusion studies and a variety of other strategies have
established the animal model of PPI deficits in schizophrenia as a robust area of study (65 ,66 and 67 ). For example, in rat pups
with ventral hippocampal lesions, PPI levels are normal until adolescence, when PPI deficits appear (68 ,69 ), a finding that supports
the neurodevelopmental model of PPI deficits as it applies to an integrated model of schizophrenia. Apomorphine used as a
dopamine D2 agonist induces PPI deficits that are reversible with typical or atypical antipsychotic medications. Phencyclidine
induces PPI deficits that are differentially reversed by atypical (but not typical) antipsychotic medications. The interested reader is
referred to Swerdlow et al. (70 ) for further discussion of these issues.

Some initial results utilizing between-subjects rather than the more compelling within-subjects designs indicate that PPI deficits in
schizophrenic patients may be reversed or “normalized” by antipsychotic medications (51 ,53 ); however, no linkage studies have
utilized PPI, although the genetic contributions to PPI have been elucidated by the fact that PPI levels differ in different rat strains
(71 ), and differential sensitivity to PPI deficits has been observed in these strains (72 ,73 and 74 ). The increasing use of isolation
rearing (75 ,76 ,77 and 78 ) and knockout mice (79 ,80 ,81 and 82 ) in PPI studies will undoubtedly yield much more information
about the neural and genetic contributions to PPI. In parallel, human linkage studies (already in progress) are being conducted.

Oculomotor Function
Oculomotor function is another important measure that has been used in schizophrenia research. As in gating, two fundamental
paradigms have been utilized: eye tracking, or smooth pursuit, and the antisaccade task. Eye-tracking dysfunction in schizophrenic
patients was first reported by Diesendorf and Dodge (83 ), and their work was later extended by numerous investigators, including
Holzman and colleagues (84 ,85 ). Across quantitative and qualitative studies, the eye-tracking deficits seen in schizophrenia have
been well documented (86 ,87 ,88 and 89 ).

Smooth-Pursuit Eye Movement

In a typical study of smooth-pursuit eye movement, a signal is presented to subjects on a computer screen and their ability to track
the target smoothly is assessed. Schizophrenic patients do not track the target smoothly; they exhibit fragmented eye movements
and resulting eye movement abnormalities, such as “catch-up” saccades. This task has been very useful in family studies; clinically
unaffected family members of schizophrenic patients also exhibit eye movement dysfunctions that typically involve an inability to
follow a target smoothly, which leads to a variety of abnormalities, including “catch up” saccades (85 ,86 ,88 ,90 ,91 ,92 ,93 and
94 ). In a series of studies, Siever et al. (95 ) and others (91 ,93 ,96 ,97 ) also reported abnormalities of smooth-pursuit eye
movement in schizotypal patients.

Antisaccade Task
The antisaccade task has also been widely employed in schizophrenia research as another oculomotor task and as a potential
endophenotype. In the antisaccade task, the subject first fixates on a centrally presented visual cue. A target stimulus is then
presented to the left or right of the fixation stimulus, and the subject is instructed to look away from the target stimulus; if the
stimulus is presented 3 degrees to the left of the fixation point, the subject is expected to look 3 degrees to the right and inhibit the
natural tendency to “follow” the target to the left. Voluntary inhibitory functions are utilized to suppress the normal tendency to
look at the target stimulus and gaze in the opposite direction. This task, like some of the other measures discussed above, uses
inhibition and is largely volitional (like smooth-pursuit eye movement) rather than automatic (like gating). Schizophrenic patients
show marked deficits in performing this task; an initial gaze directed toward rather than away from the target stimulus is
characteristic (98 ,99 ,100 and 101 ). The magnitude (i.e., effect size) of the performance deficits in schizophrenic patients and
clinically unaffected family members is large (Table 51.1 ), and the large difference in effect size between probands and normal
comparison subjects makes the antisaccade task an excellent candidate endophenotype for genetic studies (101 ,102 ). Within the
schizophrenia spectrum, it is notable that antisaccade deficits occur in family members of schizophrenic patients and in patients
with schizotypal personality disorder (96 ,101 ,103 ,104 ). In this way, the antisaccade deficit meets the second criterion for a
candidate endophenotype—that is, a candidate endophenotype should appear in clinically unaffected family members of
schizophrenic patients (and perhaps in schizotypal patients).

Neuropsychological Tasks
A plethora of candidate endophenotypes have been derived from the neuropsychological literature. It is well-known that
schizophrenic patients exhibit a wide range of neuropsychological deficits (105 ,106 ) and that these deficits extend to clinically
unaffected family members (107 ). Deficits have
 P.709

been reported in several important domains: (a) executive function, as assessed by the Wisconsin Card Sorting Test (108 ); (b)
working memory, as assessed by the Letter–Number Span (109 ), and (c) thought disorder, commonly derived from the processing of
stimuli from the Rorschach Test to yield the Thought Disorder Index (110 ) and the Ego Impairment Index (111 ). These cognitive
dysfunctions are frequently found in family and twin studies in clinically unaffected family members
(112 ,113 ,114 ,115 ,116 ,117 ,118 ,119 ,120 and 121 ), schizotypal patients (122 ,123 ,124 ,125 and 126 ), and clinically unaffected
monozygotic twins discordant for schizophrenia itself (127 ). The neural substrates of many of these abnormalities are well
understood and are being rapidly explicated because these tasks are very well suited to performance during functional brain imaging
(e.g., 128). For example, it appears that the Wisconsin Card Sorting Test relies on dorsolateral prefrontal cortex (128 ,129 ,130 and
131 ) and related distributed circuit structures. Working memory utilizes a complex neural substrate that includes the prefrontal
cortex and related structures. It important when working memory is utilized to be clear about whether the test assesses simple
delayed recall (transient online storage) or the more complex storage, manipulation, and recall (executive functioning) of
visuospatial or verbal memory; these are two distinct neuropsychological processes that probably utilize at least partially distinct
neural substrates influenced by at least partially different sets of genes (132 ). Functional imaging experiments in thought disorder
are more preliminary, and the neural substrate of thought disorder is now being explicated.

Continuous Performance Task

The Continuous Performance Task (CPT) is another measure that has been widely applied in the study of schizophrenic patients
(133 ,134 ,135 and 136 ). In the basic form of this task, the subject is presented with a string of stimuli and asked to identify target
stimuli from among background or noise stimuli. The CPT is thought to tap into the function of vigilance; schizophrenic patients
commonly have significant deficits in the CPT. The CPT also has the advantage that it can be presented in a variety of “degraded”
forms in which the signal-to-noise ratio of the stimulus to be identified is attenuated through a variety of parametric manipulations;
these make the vigilance and identification task more difficult to perform and may correspondingly increase group separation
between schizophrenic patients, their clinically unaffected relatives, and appropriate comparison subjects. The CPT is one of the
most thoroughly studied tasks in schizophrenia, in no small degree because of the pioneering work of Nuechterlein et al. (135 ) and
others (137 ,138 ) who have consistently demonstrated CPT deficits in schizophrenic patients. The fact that unaffected relatives of
patients with schizophrenia (139 ,140 ,141 ,142 ,143 ,144 and 145 ) and schizotypal disorder (146 ,147 and 148 ) have parallel
deficits supports the utility of the task as a candidate endophenotype for genetic studies of information-processing deficits in
schizophrenia. These deficits appear to measure stable markers of schizophrenia that may be associated with a genetic vulnerability
to the illness and are seen in neuroleptic-naïve, first-break, and neuroleptic-withdrawn schizophrenic patients and their siblings
(142 ).

Span of Apprehension
The utility of Span of Apprehension as a candidate endophenotype, like that of the others measures discussed in this chapter, is
supported by a vast amount of literature, only a brief summary of which can be presented here. In its most simplified form, Span of
Apprehension refers to the number of items that can be apprehended or attended to and subsequently recalled at one time from an
array of stimuli. The interested reader is referred to a particularly scholarly discussion by Asarnow et al. (149 ). As in the other
measures discussed here, the Span of Apprehension has yielded a pattern of interesting results that makes the task another
excellent candidate endophenotype in schizophrenia. Additionally, Span of Apprehension deficits have been found in clinically
unaffected family members of schizophrenic patients (150 ,151 and 152 ) and in patients with schizotypal disorder (153 ,154 ).
Recently, in a study of normal twins, Bartfai et al. (155 ) reported a significant genetic component in the Span of Apprehension task,
which further strengthens its utility in genetic studies of schizophrenia.

Visual Backward Masking

In Visual Backward Masking, a simple target stimulus presented with a tachistoscope (156 ,157 and 158 ) or, more recently, a
computer (159 ,160 ) is followed by a complex, usually powerful masking stimulus of interlocking Xs that overlap the area of target
presentation. A subject reports the target stimulus when it is presented alone. As the masking stimulus appears closer in time to the
target (e.g., 100 milliseconds after the target, like the interstimulus interval in PPI experiments), the subject is no longer able
identify the target stimulus. Schizophrenic patients are subjected to the effects of the mask on target identification at an
interstimulus interval at which normal subjects have little trouble distinguishing the target stimulus (161 ). Explanations for the
masking effect extend from integration of the mask with the target stimulus to interruption of target stimulus processing by the
mask (161 ,162 ,163 and 164 ). Whatever the mechanism, the phenomenon is readily identified as a marker in schizophrenic
patients (158 ,165 ,166 ,167 and 168 ), family members of schizophrenic patients (159 ,160 ,169 ), and schizotypal patients
(170 ,171 ,172 and 173 ). The specificity of the deficit is unclear, although in one study, manic patients at the height of psychosis
showed visual masking deficits that were reversible over time with treatment. In this study, it was reported that the deficits of
schizophrenic patients with a good prognosis, who
 P.710

typically respond to treatment, may also be reversible over time (174 ). The underlying neural mechanism linked to masking deficits
involves the dorsal and ventral information-processing substrates that are supported by magnocellular and parvocellular neurons
(175 ,176 ). Because both clinically unaffected family members and schizotypal patients exhibit deficits of Visual Backward Masking,
it may well serve as an important candidate endophenotype in genetic studies.

P300 Event-Related Potential

Since the initial reports of Callaway and colleagues (177 ,178 ,179 and 180 ) of P300 deficits in schizophrenia, a large number of
investigators have identified their topography, lateralization, and neural basis. The results of these studies make the P300 event-
related potential an excellent candidate endophenotype for understanding the genetic basis of the deficits.

Schizophrenic patients have long been known to have deficits in the P300 component of the event-related potential (177 ,178 ,181 ).
The P300 wave occurs about 300 milliseconds after stimulus presentation and is commonly thought to reflect the apportionment of
attention to a stimulus that is relatively novel or rich in information. Many paradigms utilize a series of rather neutral stimuli and
then use an “attention-grabbing” stimulus to elicit a large P300 wave. Although the variability of the latency properties of the P300
event-related potential wave may account for part of the diminution in schizophrenia, repeated studies report that schizophrenic
patients show a decreased P300 wave amplitude over time (182 ,183 ,184 and 185 ). The fact that these deficits are also found in
unaffected family members of schizophrenic patients (186 ,187 ,188 ,189 and 190 ) and in schizotypal patients (191 ,192 and 193 )
supports the utilization of the P300 wave as a candidate endophenotype. The original work of Callaway et al. (178 ) and more recent
studies by McCarley and associates (182 ,194 ,195 and 196 ) have contributed to an understanding of the P300 neural circuitry that
supports the generation of this wave. It appears that the P300 wave is generated from the temporal lobes, perhaps the superior
temporal gyrus of the brain. Along with a diminution of the P300 wave in schizophrenic patients, the volume of the superior
temporal gyrus gray matter is also diminished. Lateralization findings indicate that it is probably the left P300 wave that is
differentially diminished in schizophrenic patients, matching the volume depletion of the left superior temporal gyrus.

Older studies of reaction time and exciting new and evolving studies of mismatch negativity (197 ) offer a wide range of potentially
useful endophenotypes that may prove to be especially interesting in schizophrenia research.

SUMMARY
Part of "51 - Endophenotypes in Studies of the Genetics of Schizophrenia "

A multitude of interlocking studies, only some of which have been reviewed here, point to information-processing deficits and
closely related inhibitory abnormalities as excellent candidate endophenotypes for genetic studies of schizophrenia. The heritability
of several of these candidate endophenotypes has already been assessed in genetic studies. In addition, the neuronal mechanisms of
many of the endophenotypes are currently being investigated through neurophysiologic studies in both humans (e.g., functional
imaging) and related animal models. The ultimate utility of physiologic endophenotypes may be to correlate the wealth of emerging
but nonfunctional genetic information with the critically important and functionally significant underlying neurobiology of these
endophenotypes. The task of identifying genes that convey a risk for schizophrenia is now under way, generally with the use of
either the clinical phenotype of schizophrenia or risk-related endophenotypes. Findings that many of the linkage sites are positive
for both schizophrenia and bipolar disorder (e.g., 198) will undoubtedly stimulate a reexamination of what aspect of psychotic
psychopathology is being transmitted at each genetic locus, and which nongenetic factors, such as neonatal ventral hippocampal
lesions, interact with these genes to produce schizophrenia (e.g., 68) versus bipolar disorder. Additionally, within cohorts of
schizophrenic patients, some of these information-processing endophenotypes overlap with each other, both behaviorally and in
terms of their underlying neural substrates. This allows for the exciting possibility of constructing “composite” phenotypes
consisting of neurologically coherent combinations of more than one of these identified markers (102 ).

As the molecular mantra states, the fundamental unit of genetic transmission is an abnormality in the structure or expression of a
protein. Presumably, most of those protein abnormalities affect neuronal functions that can be measured as changes in physiologic
functions, such as the endophenotypes we have described above. The elucidation of how different genetic abnormalities, singly or in
combination, contribute to the neuronal pathophysiology of psychosis may help to redefine the nosology of psychotic illnesses and
point the way to new treatment approaches. The power and value of endophenotypes is that they illuminate genetically mediated
risk/vulnerability factors that often interact with nongenetic factors to produce the syndrome of schizophrenia. Thus, in the pool of
genetic strategies and techniques that can be used to understand complex genetic psychiatric disorders (199 ,200 ,201 and 202 ),
endophenotype-based strategies play an important and informative role. In colon cancer, the inherited genetic factor is familial
polyposis rather than cancer itself (4 ). In parallel, it is quite likely that failures of information processing/inhibition are the
genetically transmitted risk factors that interact with nongenetic factors to produce the clinical disorder of schizophrenia. Thus, the
identification and genetic analysis of these endophenotypes should prove particularly valuable in understanding the genetic basis of
schizophrenia. These studies will also facilitate a fuller understanding of how genetic
 P.711
and nongenetic factors interact to produce this devastating illness and, it is hoped, point the way to more effective treatments.

ACKNOWLEDGMENTS
Part of "51 - Endophenotypes in Studies of the Genetics of Schizophrenia "
This work was supported in part by grants from the National Institute of Mental Health (MH42228) and the Department of Veteran Affairs (VISN 22
MIRECC; Mental Illness Research, Education, and Clinical Center).

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200. Barondes SH. Report of the National Institute of Mental Health’s genetics workshop: introduction. Biol Psychiatry
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201. Freedman R, Adler LE, Leonard S. Alternative phenotypes for the complex genetics of schizophrenia. Biol Psychiatry
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202. Hyman SE. Introduction to the complex genetics of mental disorders. Biol Psychiatry 1999;45:518–521.

203. Cadenhead KS, Light GA, Geyer MA, et al. Sensory gating deficits assessed by the P50 event-related potential in subjects
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204. Holzman PS, Coleman M, Lenzenweger MF, et al. Working memory deficits, antisaccades, and thought disorder in relation
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52
Neurochemistry of Schizophrenia: Glutamatergic Abnormalities

James H. Meador-Woodruff

Joel E. Kleinman

James H. Meador-Woodruff: Department of Psychiatry, University of Michigan, Ann Arbor, Michigan.

Joel E. Kleinman: Clinical Brain Disorders Branch, National Institutes of Mental Health Neuroscience Center, Washington, DC.

Multiple neurotransmitters have been implicated in schizophrenia. Dopamine is the neurotransmitter most often hypothesized to be
associated with the pathophysiology of schizophrenia for two reasons. First, dopaminergic agonists can cause or exacerbate
psychotic symptoms. Second, the correlation between antipsychotic efficacy and D2 dopamine-receptor blockade is excellent. For
these reasons, a number of postmortem studies have focused on the dopaminergic system in schizophrenic brain. Although the
results of these studies have generally been negative, the few positive findings have rarely been replicated, with the notable
exception of increased striatal D2-receptor expression, which may be secondary to prior neuroleptic treatment. These studies of
dopaminergic abnormalities in postmortem brain in schizophrenia have been recently reviewed (1 ,2 ).

Given the lack of findings associated with the dopamine system in the brain in schizophrenia, the elucidation of other potential
neurotransmitter substrates of this illness has been an area of recent investigation. Glutamatergic dysfunction has been
hypothesized to occur in schizophrenia, and this has been one of the most active areas of neurotransmitter research in this illness
during the past few years. In this chapter, the glutamate hypothesis of schizophrenia is reviewed, the complexity of the molecules
associated with the glutamate synapse is outlined, and postmortem neurochemical data suggesting glutamatergic abnormalities in
schizophrenia are presented.

 GLUTAMATE AND SCHIZOPHRENIA

 GLUTAMATE-RECEPTOR SUBTYPES
 ABNORMALITIES OF GLUTAMATE RECEPTORS IN SCHIZOPHRENIA
 GLUTAMATE TRANSPORTERS
 OTHER NEUROMODULATORS AND MARKERS
 CONCLUSIONS

GLUTAMATE AND SCHIZOPHRENIA

Part of "52 - Neurochemistry of Schizophrenia: Glutamatergic Abnormalities "

Several lines of evidence have implicated glutamatergic dysfunction in schizophrenia. Dissociative anesthetics, especially
phencyclidine (PCP) and ketamine, can cause psychotic symptoms in normal humans (3 ,4 ), and worsen these symptoms in persons
with schizophrenia (5 ,6 and 7 ). Unlike catecholamine agonists, PCP can produce both the positive and negative (deficit) symptoms
associated with this illness. PCP and related compounds are uncompetitive inhibitors of the N-methyl-D-aspartate (NMDA) subtype of
glutamate receptor. Hence, this pharmacologic literature has been interpreted as suggesting that schizophrenia may be associated
with decreased NMDA-receptor activity (5 ,8 ).

Several other reasons make a glutamate-receptor hypothesis of schizophrenia attractive. Schizophrenia is believed to have a
neurodevelopmental component, and the NMDA receptor is critical in guiding axons to their targets in development (9 ). Further,
NMDA receptors may be important in processes that lead to synaptic pruning seen in adolescence, which has been hypothesized to
be abnormal in schizophrenia (10 ). Cognitive functioning depends on the plasticity mediated in part by NMDA receptors, and
schizophrenics often have cognitive deficits (11 ). Finally, the reduction of gray matter in several brain regions seen in schizophrenia
has been suggested to be the result of neurotoxicity mediated by NMDA receptors (12 ). A constellation of symptoms, findings, and
hypotheses of schizophrenia can be parsimoniously explained by NMDA-receptor dysfunction.

The NMDA receptor is one of multiple subtypes of the glutamate receptor, however, and all these subtypes have functional
interrelationships. Thus, although NMDA-receptor abnormalities have been hypothesized in schizophrenia, apparent NMDA-receptor
dysregulation could be associated with abnormalities of another receptor subtype that interacts with the NMDA receptor, which in
turn results in a breakdown of normal glutamatergic transmission in schizophrenia.

GLUTAMATE-RECEPTOR SUBTYPES
Part of "52 - Neurochemistry of Schizophrenia: Glutamatergic Abnormalities "

The four classes of glutamate receptors are functionally and pharmacologically distinct (Fig. 52.1 and Fig. 52.2 ). The ionotropic
P.718
 P.719

glutamate receptors, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), kainate, and NMDA are each composed of four or five subunits that form ligand-gated ion
channels. The metabotropic glutamate receptors (mGluRs) are all seven transmembrane-domain, G protein-coupled receptors (13 ,14 ).

FIGURE 52.1. Diagram of a typical glutamatergic synapse. Recent data suggest that
glutamatergic transmission requires three cells: a presynaptic glutamate-releasing cell, a
presynaptic glial cell that releases the endogenous agonist for the glycine co-agonist site
(recently reported to be D-serine), and a postsynaptic neuron. The various glutamate
receptors and transporters are differentially expressed by these three distinct cell
populations. The glutamate uptake transporter EAAT3 (excitatory amino acid transporter
3), which is not shown on this figure, appears to be expressed primarily on the cell body
and dendrites.

FIGURE 52.2. Subtypes of glutamate receptors. Three families of ionotropic

glutamate receptors (N-methyl-D-aspartate, AMPA, and kainate) are known,
each of which is composed of distinct subunits and has identifiable binding
sites. The metabotropic receptors cluster into three groups, members of
which share pharmacologic and structural features.

The AMPA-receptor subunits are derived from a family of four genes that have been named GluR1 through GluR4. The transcripts from each of these genes are expressed in one of two
isoforms, termed flip and flop, that result from alternative splicing. In addition, the final subunit protein of the AMPA receptor subunits has amino acids at specific locations in the ion
channel that can vary according to RNA editing (13 ,14 ). Thus, a potential exists for considerable heterogeneity in the final assembled AMPA receptors, based on subunit composition
and post-translational modification. The assembled AMPA receptors contain several binding sites: one for glutamate, another at which competitive antagonists such as CNQX (6-cyano-
7-nitro-quinoxalindione) act, and yet another where desensitization modulators exert their influence. Subunit composition appears to confer unique pharmacologic properties to the
final receptors (15 ,16 ,17 ,18 and 19 ). For example, decreased calcium influx in AMPA receptors that contain the GluR2 subunit drastically diminishes the electrophysiologic activity
of these receptors.

Kainate receptors are also ligand-gated ion channels composed of subunits derived from genes for the low-affinity GluR5 through GluR7 and high-affinity KA1 through KA2 subunits
(13 ,14 ). The transcripts associated with these five subunits also undergo alternative splicing and editing. Final assembled kainate receptors may be composed of five identical
subunits, or they may be heteromers composed of low- and high-affinity subunits, with pharmacologic properties that differ from those of low-affinity or high-affinity homomers.

The NMDA receptor subunits are encoded by five genes termed NR1 and NR2A through NR2D (13 ,14 ). An NR3 gene has also been identified, although this subunit appears to be
expressed primarily during early development (20 ,21 and 22 ). NR1 is expressed as one of eight isoforms because of alternative splicing of exons 5, 21, and 22 (13 ,14 ,23 ,24 ). As in
the case of the AMPA and kainate receptors, transcription of the NR1 subunit presents an important level for the regulation of the expression of functional NMDA receptors. This
regulation can influence certain properties of the final functional NMDA receptors, including the pharmacology of their binding sites.

The pharmacologic regulation of the NMDA receptor depends on the unique combination of binding sites (13 ,14 ). A primary agonist site exists for the binding of glutamate. A separate
glycine co-agonist site must also be occupied before glutamate can activate the ion channel; recent reports suggest that D-serine produced by astrocytes is the endogenous ligand for
this site (25 ,26 ,27 and 28 ). Modulatory binding sites for polyamines, protons, neuropeptides including dynorphin, and zinc have also been identified. Additionally, magnesium ions
block the ion channel of the NMDA receptor complex at physiologic concentrations. This blockade is voltage-dependent; partial depolarization of the cell membrane extrudes the
magnesium ion. Therefore, presynaptic glutamate release and postsynaptic pre-depolarization are both required for NMDA receptor activity. Finally, a site within the ion channel itself
is associated with the binding of uncompetitive antagonists of the NMDA receptor, such as PCP, ketamine, and MK-801. These antagonists are use-dependent (i.e., the ion channel must
be opened for these compounds to bind to the receptor), so cooperativity between multiple sites is necessary for occupancy by uncompetitive antagonists.

These binding sites are associated with different subunits, and their affinities can vary depending on subunit composition. NR1 homomers have been shown to form glycine binding sites,
but an NR2 subunit appears to be required to form both glutamate and MK-801 binding sites (29 ,30 ,31 and 32 ). Further, receptors containing NR2A subunits have a higher affinity for
compounds that bind to the glutamate agonist site, whereas receptors with NR2A or NR2B subunits have higher affinities for MK-801 binding than do receptors with NR2C or NR2D subunits
(31 ). In addition, NMDA receptors containing particular NR1 splice variants have a higher affinity for MK-801 than do receptors with others, irrespective of NR2 co-assembly (33 ).
Receptors with NR2B subunits are associated with a higher affinity for polyamine modulators (31 ,34 ). Therefore, differential subunit combinations confer unique binding properties to
the NMDA receptors and probably are associated with subtle electrophysiologic differences within a population of NMDA receptors.

Eight mGluRs have been cloned and are grouped (group I, group II, and group III) based on pharmacology, sequence homology, and linkage to signal transduction pathways
(35 ,36 ,37 ,38 ,39 and 40 ). These mGluRs belong to a unique subset of G protein-coupled receptors with seven transmembrane domains and large, extracellular amine termini. When
expressed in heterologous systems, group I mGluRs have been shown to stimulate phospholipase C, phosphoinositide hydrolysis, and the formation of cyclic adenosine monophosphate
(cAMP) (41 ,42 ,43 and 44 ). In heterologous systems, groups II and III mGluRs inhibit forskolin-stimulated cAMP formation and adenylyl cyclase, possibly via a Gi protein
(39 ,40 ,45 ,46 ). The metabotropic receptors have been the target of considerable recent interest because a functional relationship appears to exist between the group II
metabotropic and NMDA receptors (47 ).

Each glutamate receptor subtype appears to have a unique role in glutamatergic neurotransmission. Glutamate receptors interact at multiple levels, as AMPA, kainate, and
metabotropic receptors all affect NMDA-receptor activity. Accordingly, although the NMDA receptor is typically hypothesized to be dysregulated in schizophrenia, disturbances of any
of the glutamate receptors could result in a condition
 P.720

that produces the appearance of an abnormally functioning NMDA receptor.

ABNORMALITIES OF GLUTAMATE RECEPTORS IN SCHIZOPHRENIA

Part of "52 - Neurochemistry of Schizophrenia: Glutamatergic Abnormalities "

Given the possibility of glutamate-receptor dysfunction in schizophrenia, the expression of all four families of the glutamate
receptor have been studied in schizophrenic brain. As would be expected, these investigations have primarily targeted limbic
regions that have been implicated in schizophrenia, particularly limbic cortex, striatal areas, medial temporal lobe structures, and,
more recently, the thalamus. These investigations have also targeted multiple levels of gene expression, including subunit
messenger RNA (mRNA) and protein levels, and final binding sites have been studied. In the following sections, the studies that have
been published for each receptor subtype in postmortem brain in schizophrenia are reviewed.

AMPA Receptors
Of all of the glutamate receptors in schizophrenia, the AMPA receptor has been studied the most, as summarized in Table 52.1 .
When the AMPA-associated subunits were first cloned, Harrison et al. (48 ) examined the expression of the mRNA encoding the GluR1
subunit in medial temporal lobe structures in schizophrenia. A consistent decrease in the expression of this subunit transcript was
found in hippocampal regions, an abnormality that was statistically significant only in the CA3 region. These investigators
subsequently extended their finding and demonstrated that GluR1-subunit mRNA is decreased in multiple hippocampal subfields
(dentate gyrus, CA3, and CA4) and also in the subiculum (49 ). They also reported that GluR2-subunit mRNA is decreased in the
medial temporal lobe in schizophrenia, particularly in the parahippocampal gyrus (49 ), and continued their examination of AMPA-
receptor expression in the medial temporal lobe by determining the patterns of expression of the flip and flop isoforms of the GluR1
and GluR2 subunits. Decreased expression of GluR2-subunit
 P.721
mRNA was again found in hippocampal structures, and both the flip and flop variants were reduced, the flop to a greater extent (50 ).

TABLE 52.1. AMPA RECEPTOR

BINDING AND SUBUNIT
EXPRESSION IN
SCHIZOPHRENIA

Several studies have examined the expression of the AMPA-subunit proteins in the medial temporal lobe in schizophrenia. Using quantitative
immunocytochemical analyses, Eastwood et al. (51 ) reported decreased expression of the AMPA subunits in medial temporal lobe structures. In
particular, GluR1 immunoreactivity was noted to be significantly reduced in the parahippocampal gyrus, and combined GluR2/3 immunoreactivity was
decreased in the CA4 subfield of the hippocampus. On the other hand, Breese and co-workers (52 ) found no differences in GluR1, GluR2, or GluR3
immunoreactivity in schizophrenia when they used Western analysis in hippocampal samples.
AMPA-receptor binding has also been studied in medial temporal lobe structures. Using [3H]CNQX to label the AMPA receptor, Kerwin et al. (53 )
noted decreased binding to the AMPA receptor in the schizophrenic hippocampus, particularly in the CA3 and CA4 subfields. More recently, Gao and
colleagues (54 ) found decreased [3H]AMPA binding in CA2, but not in other hippocampal fields or associated structures. The convergence of these
data is that AMPA-receptor expression is decreased in the medial temporal lobe in schizophrenia, a decrease that involves alterations of subunit gene
expression in addition to the final binding site.
Although the medial temporal lobe data are the most robust, AMPA-receptor expression has also been examined in other brain regions in
schizophrenia. In two studies, none of the AMPA-associated subunit transcripts were changed in striatal subregions (caudate, putamen, and nucleus
accumbens) in schizophrenia (55 ,56 ). To date, subunit protein levels have not been reported in striatal regions. Binding to the AMPA receptor has
been determined in striatal regions, but results have not been consistent. Although Noga and colleagues (57 ) reported an increase in AMPA binding,
determined with [3H]CNQX, in caudate, putamen, and accumbens in schizophrenia, no differences in [3H]AMPA binding were found in striatal regions
in schizophrenia in three other reports (55 ,58 ,56 ).
The cortex has also been studied for alterations of AMPA-receptor expression in schizophrenia. In one study, no differences in the expression of any
of the AMPA-associated subunit mRNAs were found in prefrontal or occipital cortex in schizophrenia (55 ), although Sokolov (59 ), using reverse
transcriptase polymerase chain reaction (RT-PCR), reported decreased GluR1 mRNA in superior frontal gyrus. Breese et al. (52 ) found no differences
in GluR2 or GluR3 protein in cingulate cortex as determined by Western analysis. Several groups have studied [3H]AMPA binding in cortical areas in
schizophrenia (55 ,60 ), with generally negative results.
Recently, the neurochemical anatomy of the thalamus has become a subject of interest in schizophrenia research. The AMPA receptor is expressed in
multiple nuclei of the human thalamus. In a recent report (61 ), although [3H]AMPA binding was not different in limbic thalamic nuclei in
schizophrenia, the transcripts encoding the GluR1 and GluR3 subunits were both found to be reduced in the face of normal levels of GluR2 and GluR4
mRNA. These results suggest that alterations in the stoichiometry of subunit composition may be associated with the AMPA receptor in the
schizophrenic thalamus.

Kainate Receptors
The kainate receptor has been the subject of study in the brain in schizophrenia, as summarized in Table 52.2 . Although the medial temporal lobe
has been the best-studied region in the schizophrenic brain for AMPA-receptor expression, fewer studies have systematically focused on the kainate
receptor in these structures. Porter and colleagues (62 ) found decreased expression of GluR6 and KA2 mRNA in several hippocampal regions, results
paralleling similar data for the AMPA subunits in the medial temporal lobe. In this same study, GluR6 mRNA was not found to be changed in the
schizophrenic cerebellum. Only one study to date has examined any of the kainate subunit proteins; GluR5 was studied by Western analysis and was
not changed in schizophrenic hippocampus (52 ), although the antisera used in this study cross-reacts with GluR6 and GluR7.

TABLE 52.2. KAINATE

RECEPTOR BINDING AND
SUBUNIT EXPRESSION IN
SCHIZOPHRENIA

Kainate-receptor expression has been examined in multiple cortical regions. Sokolov (59 ) has published data suggesting that GluR7- and KA1-subunit
transcripts are decreased in the superior frontal gyrus in schizophrenia, similar to the decreases this investigator noted for some of the subunits
associated with the AMPA and NMDA receptors. In a recent study examining transcripts of kainate-receptor subunits in the prefrontal cortex (63 ), a
shift in subunit stoichiometry was found in multiple cytoarchitectural regions of the prefrontal cortex, with increased expression of GluR7 mRNA and
decreased expression of KA2 mRNA in the face of normal expression of the other kainate subunits. In this same study, no changes in transcripts of
kainate-receptor subunits were noted in Brodmann area 17.
Several studies have examined the expression of transcripts of the kainate-receptor subunit in subcortical structures. Two reports (56 ,63 ) noted no
alterations of these subunits in multiple striatal regions in schizophrenia. On the other hand, a recent study noted decreased levels of KA2 mRNA but
normal levels of other transcripts of kainate-receptor subunits in limbic thalamic nuclei in schizophrenia.
Kainate-receptor binding has been studied in multiple brain regions in schizophrenia by several independent groups. All these studies have used
[3H]kainate to label this receptor. In general, kainate-receptor binding has been reported to be altered in multiple cortical areas in schizophrenia
(63 ,64 and 65 ). Data on the expression of kainate binding sites in medial temporal lobe structures are inconsistent;
 P.722

one study reported decreased [3H]kainate binding in the hippocampus and parahippocampal gyrus (53 ), but another found no
differences in binding in medial temporal lobe structures (54 ). Although kainate-receptor binding has been reported to be abnormal
in cortical structures, it has not been found to differ in subcortical regions in schizophrenia; [3H]kainate is unchanged in both striatal
subregions (56 ,57 ,63 ,65 ) and limbic thalamic nuclei (61 ) in this illness.

NMDA Receptors
Although most hypotheses of glutamatergic dysfunction in schizophrenia invoke the NMDA receptor, relatively few studies of this
receptor subtype have been carried out to date (Table 52.3 ). Only several studies have been published that examine the expression
of the NMDA subunits in schizophrenic brain, and all these have focused on mRNA levels. In a comprehensive examination of all the
NMDA subunits in prefrontal cortex, Akbarian et al. (66 ) found no absolute differences between controls and schizophrenic patients
for any of the NMDA subunits, but the contribution of NR2D to the total pool of NR2 transcripts was elevated in the schizophrenic
patients. Recently, Gao et al. (54 ) found an altered stoichiometry of NMDA subunits in hippocampus, with decreased NR1 and
increased NR2B mRNA expression but normal NR2A expression, in schizophrenia. Several other studies have been published in which
only the NR1 transcript was measured; in one study, this molecule was reported to be decreased in superior temporal cortex (67 ),
and in another, it was decreased in superior frontal cortex (59 ).

TABLE 52.3. NMDA RECEPTOR BINDING AND SUBUNIT EXPRESSION IN

SCHIZOPHRENIA
Several recent studies have examined the expression of the NMDA receptor subunits in subcortical structures in schizophrenia. In one
study (56 ), NR1, NR2A, NR2B, NR2C, and NR2D mRNAs were measured in the caudate, putamen, and nucleus accumbens in schizophrenia;
no significant differences were found in comparison with control striata. On the other hand, significant reductions of NR1, NR2B, and
NR2C transcripts (but not of NR2A and NR2B transcripts) were found in dorsomedial and anterior thalamic nuclei in this disorder (61 ).

Because of the myriad binding domains of the NMDA complex, studies of receptor binding are difficult to interpret and are subject
to the selection of radioligand. Further, it has become apparent that certain subunit compositions are associated with specific
binding sites, so it is possible that some but not all binding sites on the NMDA receptor are altered in schizophrenia. The best-
studied of the
 P.723

NMDA-associated sites is the ion channel/PCP site. In general, studies in which [3H]MK-801 was used have been relatively
unimpressive. In an early study (68 ), increased [3H]MK-801 binding was reported in the schizophrenic putamen, but no differences
were noted in frontal cortex or multiple medial temporal lobe regions, including the hippocampus, amygdala, and entorhinal cortex.
A more recent study (57 ) found no differences in caudate, putamen, or nucleus accumbens. The ion channel site has also been
studied with the ligand [3H]TCP, and again minimal changes were noted. In one study (69 ), no changes were found in multiple
cortical areas, putamen, or cerebellum. A subsequent report (70 ) observed no differences between controls and schizophrenic
patients in hippocampus, amygdala, or polar frontal cortex (Brodmann area 10), but increased [3H]TCP binding was noted in
orbitofrontal cortex (Brodmann area 11) in the schizophrenic patients.

The other NMDA-associated binding sites have been studied more recently. The primary agonist site for glutamate has been studied
with [3H]glutamate in the hippocampus, and no differences have been found in schizophrenia (53 ,54 ). The glycine co-agonist site
has also been studied. Using [3H]glycine, Ishimaru and colleagues (71 ) reported increased binding in multiple cortical areas in
schizophrenia. Recently, the glycine site was studied in striatum with [3H]L-689,560, and increased binding was noted in putamen,
but not caudate or accumbens, in schizophrenia (72 ).

Several comprehensive studies examining multiple binding sites associated with the NMDA receptor complex in subcortical structures
have recently been reported. In one of them (56 ), binding to the glutamate (measured with [3H]CGP39653) and glycine (measured
with [3H]MDL105,519) agonist sites, the intrachannel/PCP site ([3H]MK-801), and the polyamine modulatory site ([3H]ifenprodil) were
determined in caudate, putamen, and nucleus accumbens in schizophrenia. In this study, no differences were noted between
controls and schizophrenic subjects. On the other hand, a study in thalamus from this same group (61 ), in which the same ligands
were used to label the four sites, found decreased expression of binding associated with the glycine and polyamine sites, but not the
intrachannel/PCP site or glutamate binding domain in limbic nuclei, in schizophrenia. These changes in some but not all binding
sites in the thalamus were also associated
 P.724

with changes in the stoichiometry of the various NMDA-associated subunit transcripts in these nuclei.

Metabotropic Receptors
Very little has been published about this family of receptors in schizophrenic brain (Table 52.4 ). In one study, the mRNAs encoding
the metabotropic receptors mGluR3 and mGluR5 were measured in prefrontal cortex (73 ). Although mGluR3 mRNA was not changed in
schizophrenia in multiple areas of the prefrontal cortex, mGluR5 was increased in the orbitofrontal cortex (Brodmann area 11), but
not in Brodmann areas 9 or 10. Cell-level analysis revealed that this increase was secondary to increased expression of mGluR5 mRNA
in pyramidal cells in lamina III of this area of prefrontal cortex. More recently, the expression of the transcripts encoding seven of
the eight cloned metabotropic receptors was reported in schizophrenic and control thalamus (74 ). No differences were found in the
expression of the mGluRs in six different thalamic nuclei in schizophrenia in this study.

TABLE 52.4. METABOTROPIC GLUTAMATE RECEPTOR RNA EXPRESSION IN

SCHIZOPHRENIA

GLUTAMATE TRANSPORTERS
Part of "52 - Neurochemistry of Schizophrenia: Glutamatergic Abnormalities "

In addition to the glutamate receptors, other molecules at the glutamate synapse are critical for normal glutamatergic
neurotransmission (Fig. 52.1 ). At least five glutamate uptake transporters, excitatory amino transporter 1 (EAAT1) through EAAT5,
are expressed in the glutamate synapse (75 ). EAAT1 is predominantly expressed in astrocytes of the cerebellum, although expression
is also significant in the forebrain. EAAT2 is expressed in both astrocytes and neurons but has a more widespread distribution in the
brain (76 ). Severe neuropathology and epilepsy develop in knockout mice for the EAAT2 gene, which confirms its importance in
normal glutamatergic function. EAAT3 is a neuronal transporter expressed in multiple limbic regions. EAAT4 expression is restricted to
Purkinje cells of the cerebellum, and EAAT5 is confined to the retina.

Although glutamate transporters affect the function of all four glutamate receptor subtypes, the glycine transporter family may
specifically affect NMDA receptor-mediated activity. Glycine is an NMDA receptor co-agonist, and glycine transporter inhibitors
affect normal NMDA-receptor function and reverse PCP-induced behaviors (77 ,78 ,79 ,80 and 81 ). The two families of glycine
transporters are GLYT1 and GLYT2; three isoforms of GLYT1 have overlapping expression in astrocytes throughout the human brain,
whereas GLYT2 is restricted to the hindbrain and spinal cord (82 ,83 ). By altering the availability of glutamate for its receptors,
changes in the expression of the transporters may induce profound changes at the level of receptor function. Further, given that the
NMDA receptor may depend on glycine as a co-agonist, abnormal synaptic levels of this amino acid may be associated with disturbed
function of the NMDA receptor.

Initially, the quantification of glutamate uptake sites in schizophrenia preceded the identification of the EAAT subtypes, and
conflicting data have been obtained in schizophrenic prefrontal cortex and basal ganglia with use of the nonselective transporter
ligand [3H]D-aspartate (Table 52.5 ). Early studies found decreases in striatal uptake sites (84 ,85 ); however, later studies did not
replicate these findings (57 ,86 ). Similarly, increases in frontal cortical uptake sites (64 ) were not confirmed in follow-up studies
(84 ,87 ). The discrepancies in this literature may be in part a consequence of the nonselectivity of [3H]D-aspartate for the multiple
 P.725

transporter subtypes; shifts in transporter subtype expression may occur in the absence of changes in total uptake sites. Consistent
with this interpretation is the recent demonstration of decreased EAAT2 mRNA levels in prefrontal cortex of schizophrenics (73 ).
This change is in the opposite direction of that noted in previous studies examining radioligand binding to the transporters (64 ,84 ),
which suggests that a shift from EAAT2 to EAAT1/EAAT3 expression may occur in prefrontal cortex in schizophrenia.

TABLE 52.5. EXCITATORY

AMINO ACID BINDING IN
SCHIZOPHRENIA

OTHER NEUROMODULATORS AND MARKERS

Part of "52 - Neurochemistry of Schizophrenia: Glutamatergic Abnormalities "

An alternative mechanism for altering glutamate neurotransmission involves neuropeptide modulators of glutamate-mediated
neurotransmission (88 ,89 ,90 and 91 ). For instance, cholecystokinin (CCK) augments glutamate-mediated neurotransmission
(88 ,91 ). CCK is expressed in subgroups of γ-aminobutyric acid (GABA)- and glutamate-containing neurons in the entorhinal cortex
(92 ,93 and 94 ). Several postmortem studies have found abnormalities in CCK, CCK receptors, and CCK mRNA expression in
schizophrenia, both in the frontal and temporal lobes (95 ,96 ,97 and 98 ). A cell-based silver grain analysis confirmed the
involvement of layer VI, finding a reduction in the level of CCK mRNA expression per pyramidal cell (99 ). This is further supported
by other molecular studies involving the measurement of complexin I and complexin II mRNAs, which suggest preferential
involvement of excitatory pyramidal neurons in the mesial temporal lobe in schizophrenia (100 ,101 ).

A second neuropeptide neuromodulator concentrated in glutamate neurons, N-acetylaspartylglutamate (NAAG), antagonizes the
effects of glutamate at NMDA receptors (102 ). NAAG is cleaved by glutamate carboxypeptidase II (formerly referred to as N-acetyl-
α-linked acidic dipeptidase), a membrane-spanning glial enzyme, to yield glutamate and N-acetylaspartate (NAA). One study of
NAAG and glutamate carboxypeptidase II found decreased glutamate carboxypeptidase II activity in prefrontal cortex and
hippocampus and increased NAAG levels in the prefrontal cortex of schizophrenic patients relative to normal controls (103 ).
Moreover, in vivo magnetic resonance spectroscopic imaging has revealed selective reductions in NAA in the dorsolateral prefrontal
cortex and hippocampal formation of schizophrenic subjects (104 ,105 ). This suggests that NAA, a marker of neuronal integrity, may
be decreased specifically and regionally in schizophrenia secondary to decreases in glutamate carboxypeptidase II.

CONCLUSIONS
Part of "52 - Neurochemistry of Schizophrenia: Glutamatergic Abnormalities "

Converging evidence indicates that abnormalities of glutamatergic neurotransmission occur in specific brain regions in schizophrenia.
Although the hippocampus and associated structures have been the best studied, emerging data point to glutamatergic
abnormalities in other areas of the brain that are likely to be associated with the pathophysiology of schizophrenia, including limbic
cortex, striatal regions, and thalamus. Pharmacologic evidence suggests involvement of the NMDA receptor in schizophrenia, but
other studies and theoretic considerations indicate that other molecules associated with glutamatergic transmission are also
abnormal in this illness.

Studies in postmortem samples of the molecules associated with the glutamate synapse have not been conducted in a systematic
and comprehensive fashion; however, several general principles are emerging from available data. First, although abnormalities of
the glutamate synapse have been reported primarily in hippocampal regions, recent data suggest that thalamocortical circuits may
also be abnormal. Interestingly, the striatal subregions appear to be less affected than medial temporal lobe and thalamocortical
pathways. Second, all four families of glutamate receptors have been reported to be abnormal in brain in schizophrenia, although in
region- and circuit-specific patterns. Third, changes are apparent at both transcriptional and translational levels of gene expression.
Fourth, the ionotropic glutamate receptors have been studied most, and results thus far reveal changes in ionotropic receptor
binding sites in addition to subunit changes suggestive of altered stoichiometry of subunit composition. The metabotropic receptors
are just beginning to be studied, but the few available reports do suggest abnormalities of these receptors.

The literature on postmortem neurochemical studies of glutamatergic molecules in schizophrenia supports the hypothesis of
abnormal glutamatergic neurotransmission in this illness that particularly involves the ionotropic receptors. These data suggest that
novel strategies that permit the modulation of these receptors may prove to be of therapeutic utility in this illness, and may also
provide clues about the pathophysiologic substrate of schizophrenia.

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53
Neural Circuitry Approaches to Understanding the Pathophysiology of
Schizophrenia

David A. Lewis

David A. Lewis: University of Pittsburgh, Pittsburgh, Pennsylvania

 PAST AND PREVAILING PATHOPHYSIOLOGIC MODELS OF PSYCHIATRIC DISORDERS

 EXAMINING SCHIZOPHRENIA AS A DISORDER OF NEURAL CIRCUITRY
 OVERVIEW OF DPFC CIRCUITRY IN PRIMATES
 INTEGRITY OF PREFRONTAL CORTICAL CIRCUITRY IN SCHIZOPHRENIA
 OPPORTUNITIES FOR NEURAL CIRCUITRY-BASED STUDIES OF SCHIZOPHRENIA

PAST AND PREVAILING PATHOPHYSIOLOGIC MODELS OF PSYCHIATRIC

DISORDERS
Part of "53 - Neural Circuitry Approaches to Understanding the Pathophysiology of Schizophrenia "

Models of the nature of brain dysfunction in major psychiatric disorders have evolved substantially over the past several decades in
parallel with the progression in knowledge resulting from investigations of the neurobiological mechanisms that contribute to normal
cognition, emotion, and behavior. Some models of psychopathology have emphasized, in their simplest forms, the central
contribution of excesses or deficits in the functional activity of a given neurotransmitter to the disease process of interest. In
general, these models have been very useful in motivating investigations of the molecular underpinnings and biochemical functions
of the neurotransmitter systems of interest, and in spurring the development of novel psychopharmacologic agents that influence
these systems. However, in extreme cases, these models tended to view individual psychiatric disorders as the consequences solely
of the postulated disturbance in the neurotransmitter of interest, and consequently they tended to be conceptually similar to
historic views that altered levels of the four classical bodily humors produced different forms of madness. In addition to this limited
conceptual perspective, neurotransmitter-based models sometimes seemed to attribute behavioral, emotional, or cognitive
functions to neurotransmitters, instead of explicitly recognizing that neurotransmitters have defined actions on receptors, whereas
behaviors, emotions, and cognitive abilities represent emergent properties of the integrated activity of large networks of neurons.

Other models of psychopathology have emphasized the critical role of localized disturbances in individual brain regions, an approach
that, in extreme cases, has been critiqued as “neophrenology.” Although these models have been useful in stimulating studies of the
structure–function relationships of the implicated brain regions, they have been limited in a number of respects, including the
inability to account for the array of signs and symptoms that typically constitute the syndromic diagnosis of a psychiatric disorder.

These two types of models were influenced, at least in part, by extrapolations from earlier successes in the study of Parkinson’s
disease, which was then viewed as a single neurotransmitter (e.g., dopamine) disease owing to a localized neuropathology (e.g., cell
death in the substantia nigra). However, in recent years, these two general approaches have given way to neural circuitry-based
models that reflect a fuller appreciation of the fact that neurotransmitters act in an anatomically constrained fashion to produce
specific biochemical effects at the cellular level, and that the localization of function(s) is a consequence of the flow of information
processing through the neural circuits within a given brain region and those linking that region to other brain areas. These latter
types of models (1 ,2 and 3 ) incorporate the recognition that complex brain functions, such as those that are disturbed in major
psychiatric disorders, are subserved by the coordinated activity of distributed ensembles of neurons.

Consequently, the goal of this chapter is to examine the use of a neural circuitry-based approach to understanding the
pathophysiology of a psychiatric disorder, using studies of the neurobiological basis for cognitive dysfunction in schizophrenia as an
example. Specifically, this chapter: (a) considers the convergent lines of evidence that suggest that the neural circuitry involving
the dorsal prefrontal cortex is disturbed in this disorder, (b) reviews the normal organization of this circuitry as revealed through
studies in animals, (c) assesses the evidence regarding the integrity of this circuitry in schizophrenia, and (d) discusses new
opportunities
 P.730

for neural circuitry-based studies of the pathophysiology of schizophrenia.

EXAMINING SCHIZOPHRENIA AS A DISORDER OF NEURAL CIRCUITRY

Part of "53 - Neural Circuitry Approaches to Understanding the Pathophysiology of Schizophrenia "

Clues from the Clinical Syndrome

Although schizophrenia and Alzheimer’s disease (AD) both received these appellations approximately a century ago, their initial
descriptions were based on different types of data. For schizophrenia, the disorder was recognized by the presence of a
constellation of clinical features and a particular longitudinal course, whereas the identification of AD was based on
clinicopathological correlations. Indeed, the observation of neurofibrillary tangles and neuritic plaques in the cerebral cortex of the
victims of AD provided the foundation for the rich array of anatomic, biochemical, and molecular genetic studies in the past two
decades that have produced the wealth of current knowledge regarding the pathophysiology of this disease.

In contrast, studies of the pathophysiology of schizophrenia continue to depend, in large part, on clues derived from the clinical
syndrome. Although the number and diversity of the clinical features of this illness make this approach daunting, a reasonable case
can be made that the disturbances in cognition commonly seen in schizophrenia represent a core feature of the illness. For example,
at least some of the other signs and symptoms of schizophrenia may be conceptualized as secondary to the cognitive disturbances
(4 ), cognitive abnormalities can be identified during the prodromal phase of the illness and in those at increased risk for the
disorder (5 ), cognitive symptoms appear to be persistent across the course of the illness (6 ), and the severity of cognitive
impairment may be the best predictor of long-term outcome (7 ,8 ). Thus, the clinical features of schizophrenia argue for an
emphasis on the neural circuitry that normally subserves the types of cognitive functions that are disturbed in the disorder.

Clues from Developmental Features

Although the idea that schizophrenia is a late consequence of an early developmental lesion (9 ) has many merits, direct evidence
for a brain abnormality that could be explained by a mechanism operating prior to or at the onset of the clinical symptoms of the
illness, and that could not be attributed to factors associated with having the illness, has proven difficult to obtain. For example,
reports of cytoarchitectural disturbances in the entorhinal cortex of schizophrenic subjects (10 ,11 ) attracted a great deal of
attention because the reported findings were strongly suggestive of an abnormality in neuronal migration (12 ); however,
subsequent studies with larger sample sizes have both failed to confirm these reports and have provided likely methodologic
explanations for the initial findings (13 ,14 ,15 and 16 ). The report of an altered distribution of interstitial neurons in the
subcortical white matter was also conceptually very attractive because it strongly suggested an early developmental lesion (17 );
however, the majority of schizophrenic subjects appear to lack such abnormalities (18 ,19 ).

However, one of the characteristics of schizophrenia is the tendency for clinical symptoms to first appear during late adolescence or
early adulthood, and it has been argued that hypotheses of the pathophysiology of schizophrenia must accommodate this age of
onset (9 ,20 ). Although the average age of first hospitalization for patients with schizophrenia is in the early or mid-twenties for
men and women, respectively (21 ,22 ), psychotic symptoms may appear months or even years prior to hospitalization (22 ,23 and
24 ). In addition, deterioration in other areas, such as scholastic performance and sociability, precedes the onset of the overt
symptoms of schizophrenia by some time (5 ,23 ,24 ), and may represent strong predictors of the subsequent appearance of the
disorder. Thus, developmental events occurring during the second decade of life may play a critical role in the appearance of
cognitive dysfunction in schizophrenia.

Clues from Distributed Brain Abnormalities

Structural and functional neuroimaging studies in subjects with schizophrenia have implicated a number of brain regions as potential
sites of dysfunction or morphologic abnormalities. For example, certain brain regions, such as the medial temporal lobe (including
the hippocampus, amygdala, and parahippocampal gyrus), the superior temporal gyrus, the dorsal prefrontal cortex and the
thalamus, have all been shown to have reduced total volume in subjects with schizophrenia, although the magnitude of the decrease
and its consistency across studies has not been uniform (25 ). Similarly, functional imaging studies have shown alterations in the
activation of some of these brain areas under different conditions, especially when subjects are performing tasks that normally are
associated with a change in activation (26 ). Although some of the reported findings cannot be accommodated in this way, a number
of the affected regions share reciprocal connections. Thus, pathophysiologic models that account for the reported abnormalities in
two or more of these regions, and the connections that link them, may be particularly promising.

Incorporating These Types of Clues into Research Strategies for Identifying Neural
Circuitry Abnormalities
Based on the three types of clues summarized in the preceding, one can ask whether they converge or triangulate on neural circuit(s)
that may be preferentially involved in the pathophysiology of schizophrenia. Although this approach
 P.731

suggests a number of possible candidate circuits for investigation, the dorsal prefrontal cortex (dPFC) may be considered a
prototypic nodal point for circuit analysis in schizophrenia for the following reasons.
First, from the perspective of clinical clues, subjects with schizophrenia perform poorly on cognitive tasks that involve working
memory, the ability to transiently maintain information in order to guide a subsequent response (27 ). For example, individuals with
schizophrenia exhibit impairments on oculomotor delayed-response tasks (28 ), a cognitive paradigm on which nonhuman primates
with structural or reversible cooling lesions of the dPFC perform poorly (29 ). Consistent with these observations, subjects with
schizophrenia also fail to show normal activation of the dPFC when attempting to perform tasks that tap working memory (30 ).
Second, from the developmental perspective, the circuitry of the primate dPFC clearly undergoes marked refinements during
adolescence, although certainly some other brain regions that have not been as well studied are also likely to show such changes.
For example, the number of excitatory synapses in the dPFC declines by 50% during adolescence in both monkeys and humans
(31 ,32 ). In addition, substantial changes occur in markers of excitatory, inhibitory, and modulatory inputs to pyramidal neurons in
deep layer 3 of primate dPFC. The apparent laminar specificity of at least some of these changes raises the possibility that circuits
involving these pyramidal cells may be preferentially affected in schizophrenia (33 ).
Third, from the perspective of regional brain analyses, the PFC has been shown to have subtle reductions in gray matter volume in a
number of structural imaging studies of schizophrenia (25 ). The failure of other studies to detect such structural abnormalities in
the PFC has been hypothesized to be a consequence of several factors, including a reduction in total PFC volume that approximates
the level of sensitivity of MRI and the restriction of volumetric changes to certain regions or gyri of the dPFC (25 ). Consistent with
these interpretations, postmortem observations indicate that cortical thickness in the dPFC may be reduced by 3% to 12% in subjects
with schizophrenia (34 ,35 ,36 and 37 ), although these changes do not always achieve statistical significance. In addition, some
(38 ,39 and 40 ), but not all (41 ), in vivo proton spectroscopy studies indicate that N-acetyl aspartate (NAA), a putative marker of
neuronal and/or axonal integrity, is reduced in this brain region. Interestingly, the magnitude of these NAA changes in the dPFC was
correlated with the degree of impaired activation in other brain regions during working memory tasks, raising the possibility that a
neuronal abnormality in the dPFC could account for distributed functional disturbances in the working memory network (40 ).
Other lines of evidence suggest that these changes may reflect disturbances in the synaptic connectivity of the dPFC in
schizophrenia. For example, the presence of decreased phosphomonoesters and increased phosphodiesters (42 ,43 ), as measured by
P31 spectroscopy, in the PFC of schizophrenic subjects has been interpreted to reflect an increased breakdown of membrane
phospholipids, and consequently a decreased number of synapses. In addition, a recent gene expression profiling study using cDNA
microarrays found that the group of genes encoding proteins that regulate presynaptic secretory machinery were most consistently
altered (44 ). Furthermore, reduced levels of synaptophysin, an integral membrane protein of small synaptic vesicles, have also
been observed in the dPFC of subjects with schizophrenia in most (45 ,46 ,47 ,48 and 49 ), but not all (50 ), studies.
For these reasons, the next two sections of this chapter focus on a summary of the normal organization of dPFC circuitry and on a
review of the evidence suggesting that this circuitry is disturbed in schizophrenia.

OVERVIEW OF DPFC CIRCUITRY IN PRIMATES

Part of "53 - Neural Circuitry Approaches to Understanding the Pathophysiology of Schizophrenia "
Direct studies of the circuitry of the human dPFC have obvious limitations; however, the available cross-species studies indicate that
the macaque monkey brain provides an accurate and useful model system for understanding the general organization of the human
dPFC. Thus, this section reviews the constituent cell types and the patterns of intrinsic and extrinsic connectivity that characterize
the primate dPFC.

Cell Types

Pyramidal Neurons
About 70% of all cortical neurons are pyramidal cells (51 ). The majority of pyramidal cells have a characteristically shaped cell body
that gives rise to a single apical dendrite that is oriented perpendicular to the cortical surface and frequently ends in a terminal tuft
(Fig. 53.1 ). An array of shorter basilar dendrites spread in a radial fashion at the base of the cell body. Both the apical and basilar
dendrites are coated with short protrusions or spines, which represent the principal targets of most excitatory synaptic inputs to
pyramidal neurons. Because dendritic spines are actively formed or resorbed in response to changes in presynaptic inputs, dendritic
spines provide a good estimate of the number of excitatory synapses that pyramidal cells receive (52 ). Typically, pyramidal neurons
possess 6,000 to 10,000 dendritic spines (53 ). In addition to receiving one excitatory input, some dendritic spines also receive a
synapse with the features suggestive of an inhibitory input. Inhibitory terminals also synapse on the dendritic shafts, cell body, and
axon initial segment of pyramidal cells. Typically, pyramidal cells receive about 2,000 inhibitory synapses on dendritic shafts, 200 on
the cell soma, and 20 on the axon initial segment (53 ).

FIGURE 53.1. Schematic drawing illustrating

the characteristic morphologic features of
pyramidal neurons in different cortical layers.
Note that the laminar location of the cell soma
tends to be associated with the major
projection target of the principal axon. Arabic
numbers, cortical layers; WM, white matter.
Adapted from Jones EG. Laminar distribution of
cortical efferent cells. In: Peters A, Jones EG,
eds. Cerebral cortex, vol 1. New York: Plenum
Press, 1984:521–553.
 P.732

The axons of pyramidal cells typically give rise to intrinsic collaterals, which travel either horizontally or vertically within the gray
matter, and a principal axonal projection, which enters the white matter and travels to another brain region. These axons utilize
excitatory amino acids, such as glutamate, as a neurotransmitter and form synapses that have the characteristic morphology
associated with excitatory neurotransmission. These so-called Gray’s Type I synapses are characterized by the presence of small
round vesicles in the axon terminal, and a postsynaptic density that is thick and asymmetric in appearance (54 ).

Nonpyramidal Neurons
Of the other major type of cortical neurons, nonpyramidal cells, about 90% utilize the inhibitory neurotransmitter γ-aminobutyric
acid (GABA). The axons of these generally small, local circuit or interneurons, arborize within the cortical gray matter and form
Gray’s Type II synapses that are characterized by pleomorphic vesicles in the axon terminal and symmetric pre- and postsynaptic
densities. GABA neurons constitute approximately 25% of all neurons in the primate neocortex (55 ), and are comprised of about 12
distinct subtypes (56 ,57 ). Although the differences among subtypes can be described on the basis of the morphologic features of
the cell body and proximal dendrites (e.g., bipolar, multipolar, bitufted), the most discriminating and functionally meaningful
classification system is based on the organization of the axonal arbor and synaptic targets of the axon terminals. In addition, GABA
neurons are chemically heterogeneous, and separate subpopulations can be identified by the presence of specific neuropeptides or
calcium-binding proteins (58 ,59 ).

Together, these morphologic and chemical features define subpopulations of GABA neurons that appear to have different biophysical
properties and different roles in dPFC circuitry (Fig. 53.2 ). For example, GABA neurons of the chandelier class, which may also
express either the neuropeptide corticotropin-releasing factor (60 ) or the calcium-binding protein parvalbumin (61 ,62 ), are found
primarily in cortical layers 2 to 5 in monkey dPFC (56 ). The axon terminals of these neurons, which are arrayed as distinct vertical
structures (termed “cartridges”), form Gray’s Type II synapses exclusively with the axon initial segment of pyramidal
 P.733

neurons (63 ,64 ,65 ,66 ,67 and 68 ), the site of action potential generation in pyramidal cells. Each chandelier cell may contact up
to 300 pyramidal neurons within a radius of 100 to 150 μm from its cell body (69 ). Thus, chandelier cells exert critical inhibitory
control over the activity of a localized group of pyramidal neurons. In contrast, the axons of wide arbor (basket) neurons spread
horizontally for considerable distances (up to 1.0 mm) within the dPFC (56 ) and form Gray’s Type II synapses with the cell bodies,
dendritic shafts, and dendritic spines of pyramidal neurons (70 ). Wide arbor neurons may be specialized to provide inhibitory
constraints over the activity of spatially segregated populations of PFC pyramidal neurons (71 ,72 ). A third example, double
bouquet cells, which contain the calcium-binding proteins calbindin or calretinin (58 ), have radially restricted axonal arbors that
synapse with the dendritic shafts of both pyramidal and local circuit neurons (73 ).

FIGURE 53.2. Schematic drawing of the synaptic

interactions between different classes of local
circuit neurons and a layer 3 pyramidal neuron (P)
in monkey prefrontal cortex. C, parvalbumin (PV)-
labeled chandelier neuron; CRC, calretinin (CR)-
and/or CB (calbindin)-labeled Cajal-Retzius
neuron; DB, CR- or CB-labeled double-bouquet
neuron; M, CB-labeled Martinotti cell; N, CB-
labeled neurogliaform neuron; WA, PV-labeled
wide arbor (basket) neuron. Modified from Condé
F, Lund JS, Jacobowitz DM, et al. Local circuit
neurons immunoreactive for calretinin, calbindin
D-28k, or parvalbumin in monkey prefrontal
cortex: distribution and morphology. J Comp
Neurol 1994,341:95–116.

Laminar Arrangement of Neurons

The dPFC, like other cortical association regions, is composed of six layers that can be distinguished according to the size and
packing density of their constituent neurons (Fig. 53.3 ). Layer 1, which is located just below the pial surface, contains relatively
few neurons, but approximately 90% of these neurons utilize GABA. Layers 2 and 4 are thin and densely packed with small “granular”
cells. The majority of these neurons are small pyramidal cells, and GABA neurons constitute approximately 30% and 15% of all
neurons in layers 2 and 4, respectively (55 ). Layers 3 and 5, the thickest cortical layers, contain prominent pyramidal neurons with
a classic morphology. In both of these layers, the size and packing density of the pyramidal neurons is greater near their borders
with layer 4. These patterns make it possible to subdivide layers 3 and 5 into superficial (toward the pial surface) and deep zones.
Pyramidal cells in layer 6 have a modified or atypical appearance. In general, GABA cells constitute 20% to 30% of the neurons in
layer 3, and about 15% of the neurons in layers 5 and 6 (55 ).

FIGURE 53.3. Nissl-stained sections showing

laminar and regional differences in cell size in
packing density in area 46 (A) of the human
prefrontal cortex and area 17, primary visual
cortex (B). Calibration bar = 200 μm. Modified
from Lewis DA. The organization of cortical
circuitry. In: Harrison PJ, Roberts GW, eds. The
neuropathology of schizophrenia: progress and
interpretation. Oxford: Oxford University Press,
2000:235–256.

Different types of axonal projections to the dPFC terminate in certain cortical layers, and projections from the dPFC to other brain
regions generally originate from pyramidal neurons in specific lamina. Thus, the laminar specificity of abnormalities in the dPFC in
schizophrenia may reveal information about the types of connections that are affected. Nevertheless, owing to the vertical spread
of the dendrites of many neurons, alterations in afferents to one layer can influence the function of neurons whose cell body is
located in a different layer.
 P.734

Organization of Projections from the dPFC

Pyramidal neurons located in layers 2 and superficial 3 tend to project to nearby cortical regions in the same hemisphere, whereas
those in deep layer 3 frequently project to more distant ipsilateral regions or across the corpus callosum to cortical regions in the
other hemisphere (Fig. 53.1 ). Projections within the same hemisphere are termed associational projections, whereas those to the
contralateral hemisphere are termed callosal projections. Pyramidal cells in layers 5 and 6 project subcortically, with outputs from
layer 5 directed to the striatum, superior colliculus, and other subcortical structures, and those from layer 6 preferentially directed
to the thalamus. The efferent axons of pyramidal neurons tend to project to only one other brain region, although a small
percentage of these neurons do give rise to collateral projections (74 ,75 ). In contrast to these extrinsic projections, the small
pyramidal cells in layer 4 project primarily within the gray matter and relatively few send axons into the underlying white matter.

Although this laminar distribution of cortical efferents is generally accurate, many exceptions exist. For example, 25% to 30% of the
pyramidal neurons furnishing associational projections to other PFC regions are located in the infragranular layers (layers 5 and 6)
(74 ), and approximately 15% to 20% of neurons projecting to the striatum are located in layer 3 (76 ). In addition, the nature of the
cortical output to a given region may vary with the location of the cell body of origin. For example, neurons in layer 6 provide
“modulatory” inputs to cells in higher order thalamic nuclei (such as the mediodorsal thalamic nucleus, the principal source of
thalamic projections to the dPFC) as well as inputs to the thalamic reticular nucleus, which regulates thalamocortical interactions.
In contrast, thalamic projections originating in layer 5 do not innervate the reticular nucleus and appear to provide “driving”
afferent inputs to higher order nuclei (77 ).

The innervation patterns of the intrinsic axon collaterals of pyramidal cells also tend to differ across cortical layers (71 ). Pyramidal
neurons in layers 2 and 3 furnish local collaterals that arborize in the vicinity of the cell body, as well as horizontal axon projections
that spread for considerable distances through the gray matter and then give rise to discrete clusters of axon terminals in the
supragranular layers. Although pyramidal neurons in layers 5 and 6 also furnish horizontal intrinsic collaterals, these have a more
limited spread and do not terminate in spatially segregated clusters. In contrast, pyramidal cells in layer 4 furnish predominantly
vertically oriented axons, which appear to be specialized for interlaminar connections. The intrinsic axonal connections of pyramidal
neurons in layers 2 and 3 of the dPFC also appear to be specialized relative to the homologous neurons in at least some other
cortical regions. For example, although approximately 95% of the long distance intrinsic and associational axon projections of these
neurons target the dendritic spines of other pyramidal cells (78 ), the synaptic targets of the local axon collaterals of these cells are
equally divided between spines and dendritic shafts of GABA neurons (79 ).

Organization of Projections to the dPFC

The dPFC shares connections with a number of other cortical regions (Fig. 53.4 ). The inputs from these areas frequently terminate
across all cortical layers, although the different layers tend to be preferentially innervated depending upon the source of the inputs.
For example, inputs from cortical regions that have a well-developed layer 4 tend to terminate more prominently in layers 4 to 6,
whereas those that originate in regions with a poorly developed layer 4 tend to terminate in layers 1 to 3 (80 ). In some cases,
afferents from different regions (e.g., callosal inputs from the contralateral PFC and associational inputs from the posterior parietal
cortex) tend be distributed into interdigitated columns (81 ).

FIGURE 53.4. Schematic diagram of principal corticocortical connections within the frontal lobe regions, including primary
limbic connections, and between posterior sensory cortices and frontal lobe regions. Double-head arrows indicate that most
connections are reciprocal. Relatively sparse direct connections between dorsolateral prefrontal cortex and limbic structures
(hippocampal formation and amygdala nuclei) are depicted with a dashed line. OFC, orbitofrontal cortex. Modified from Kaufer
D, Lewis DA. Frontal lobe anatomy and cortical connectivity. In: Miller BL, Cummings JL, eds. The human frontal lobes:
functions and disorders. New York: Guilford Publications, 1998:27–44.

In contrast to these cortical inputs, afferents from thalamic relay nuclei, such as the medial dorsal nucleus, project dominantly to
layers deep 3 and 4, with a minor projection to layer 6 (82 ). Although afferents from the amygdala project more densely to orbital
than dorsal regions of the PFC, these tend to terminate in layers 1 and 6 (83 ).

Subcortical nuclei containing monoamines or acetylcholine also exhibit distinct laminar patterns of termination in the PFC, along
with substantial regional differences in relative
 P.735

density. Dopamine (DA)-containing axons from the ventral mesencephalon have a bilaminar distribution in the PFC (84 ), forming a
dense band in layers 1 through the most superficial portion of layer 3 and a second band of lower density in layers deep 5 and 6. In
more densely innervated regions, such as dorsomedial PFC (area 9), labeled axons are also present in high density in the middle
cortical layers, forming a third distinctive band in deep layer 3. The noradrenergic (NA) projection from the locus coeruleus exhibits
a different, and in some ways complementary, laminar innervation pattern to that of DA axons (85 ,86 ). The density of NA axons is
substantially greater in the deep cortical layers, especially layer 5, than in the more superficial cortical laminae. In particular, few
NA axons are present in layer 1, which receives a dense DA innervation. In contrast, the relatively uniform laminar distribution of
cholinergic (87 ) and serotonergic (88 ) axons contrasts with the substantial heterogeneity exhibited by both DA and NA axons.

INTEGRITY OF PREFRONTAL CORTICAL CIRCUITRY IN SCHIZOPHRENIA

Part of "53 - Neural Circuitry Approaches to Understanding the Pathophysiology of Schizophrenia "

In this section, we consider how this knowledge about the normal organization of dPFC circuitry can be used to interpret studies of
the integrity of different types of neural elements in subjects with schizophrenia. As noted, several lines of evidence support the
hypothesis that schizophrenia is associated with a decrease in the synaptic connectivity of the dPFC. However, these abnormalities
do not appear to be a consequence of a decreased complement of dPFC neurons, because several postmortem studies (34 ,37 ,89 )
have reported either a normal or increased cell packing density in the dPFC. In addition, the one study that used unbiased
approaches to determine the total number of PFC neurons did not observe a reduction in subjects with schizophrenia (90 ); however,
the approaches used in these studies probably lacked adequate sensitivity to detect reduced numbers of small subpopulations of PFC
neurons. Some studies that focused on certain neuronal subpopulations have reported decreases in density of small neurons in layer
2 (91 ) or of the parvalbumin-containing subpopulation of GABA neurons (92 ). However, the latter abnormality was not observed in
another study (35 ), and it should be noted that a reduction in neuronal density when using immunocytochemical markers might
reflect an alteration in the target protein rather than in the number of cells.

Smaller neuronal cell bodies could also contribute to the observed reduction in PFC gray matter in schizophrenia. Interestingly, two
groups have reported that the somal volume of pyramidal cells in deep layer 3 of dPFC area 9 is decreased in subjects with
schizophrenia (89 ,93 ). In addition, this reduction in somal volume may be associated with a decrease in total length of the basilar
dendrites of these neurons (94 ). In contrast, the size of GABA neurons does not appear to be reduced (35 ,95 ), although less
rigorous methods were employed in these studies.

In summary, although a reduction in neuron number cannot be completely excluded, the subtle reduction in dPFC gray matter in
schizophrenia may be attributable to a combination of smaller neurons and a decrease in dPFC neuropil, the axon terminals, distal
dendrites and dendritic spines that represent the principal components of cortical synapses. Indeed, as described in more detail
below, these two factors may be interrelated.

Candidate Sources for Synaptic Reductions

The apparent reduction in synaptic connectivity in the dPFC of subjects with schizophrenia may be attributable to one or more of
the following sources of synapses: axon terminals intrinsic to the dPFC, or afferents from other cortical regions, the thalamus, or
other subcortical locations. Although none of these sources can be excluded at present, some are more likely to be major
contributors than others. For example, one subcortical source, the DA projections from the mesencephalon, may be reduced in
number in schizophrenia as evidenced by the report of diminished densities of axons immunoreactive for tyrosine hydroxylase, the
rate limiting enzyme in catecholamine synthesis, and the DA membrane transporter in the dPFC (96 ). However, these reductions
appeared to be restricted to the deep cortical layers. Furthermore, an in vivo neuroimaging study found a reduced density of DA D1
receptors in the dPFC of subjects with schizophrenia (97 ); however, DA axons are estimated to contribute less than 1% of cortical
synapses (84 ). Consequently, even the complete loss of DA projections to the dPFC could not, in isolation, account for the observed
reductions in gray matter volume or synaptophysin protein levels. The relatively small contributions that NA-, serotonin-, and
acetylcholine-containing axons make to the total number of synapses in the dPFC also argues against disturbances in these systems
as the principal cause of reduced synaptic connectivity in this brain region.

The fact that layer 3 pyramidal cells, which give rise to a substantial number of intrinsic excitatory synapses, have reduced somal
volume in subjects with schizophrenia (89 ,93 ) may suggest that the synapses furnished by the intrinsic axon collaterals of these
neurons are reduced in number, because somal volume tends to be correlated with the size of a neuron’s axonal arbor (98 ,99 ).
Evidence for a disturbance in intrinsic connectivity is supported by recent studies using cDNA microarray profiling of the expression
of over 7,000 genes in dPFC area 9 of subjects with schizophrenia (44 ). Among 250 functional gene groups, the most marked
changes in expression were present in the group of genes that encode for proteins involved in the regulation of presynaptic
neurotransmitter release. Although these findings very likely indicate a general impairment in the efficacy of synaptic
 P.736

transmission within the dPFC in schizophrenia, whether they represent a “primary” abnormality intrinsic to the dPFC or a
“secondary” response to altered afferent drive to this brain region remains to be determined. Furthermore, because the specific
genes in this group that were most altered appeared to differ across subjects, it seems unlikely that these findings can be explained
solely by reduction in the number of intrinsic dPFC synapses. Consistent with this view, the expression of synaptophysin mRNA does
not appear to be reduced in the dPFC of subjects with schizophrenia (50 ,100 ), suggesting that the reduction in this synaptic protein
marker in the dPFC may have an extrinsic source. Consistent with this interpretation, synaptophysin mRNA levels are reduced in
cortical areas that do furnish projections to the dPFC (101 ,102 ). However, whether these transcriptional changes are present in
PFC-projecting neurons, and if so, whether they result in reduced levels of synaptophysin protein in the terminal fields of these
neurons, have not been assessed.

Decreased synaptic connectivity within the PFC might also be attributable to altered inputs from the thalamus. For example, some
structural MRI studies have revealed a reduction in thalamic volume in subjects with schizophrenia (103 ,104 ,105 and 106 ). In
addition, thalamic volume was correlated with prefrontal white matter volume in schizophrenic subjects (107 ), suggesting that a
reduction in thalamic volume was associated with fewer axonal projections to the PFC. Consistent with these observations,
postmortem studies have revealed reductions of 17% to 25% in volume and 27% to 40% in total neuron number of the medial dorsal
thalamic nucleus (MDN), the principal source of thalamic projections to the PFC (108 ,109 and 110 ). Interestingly, the available
data suggest that these abnormalities exhibit topographic specificity. For example, reduced cell numbers have been reported in the
MDN and the anterior thalamic nuclei (which project to the PFC and anterior cingulate cortex), whereas the ventral posterior medial
nucleus, a sensory relay nucleus, appears to be unaffected (109 ,110 ). In addition, within the MDN, one study indicates that neuron
number is significantly decreased in the parvocellular subdivision (which projects principally to the dPFC), but not in the
magnocellular subdivision (which projects principally to the ventral PFC) (110 ). Finally, studies in both subjects with schizophrenia
who never received antipsychotic medications (111 ) and monkeys treated for 1 year with haloperidol (112 ) suggest that these
medications do not account for the reduction in MDN neuron number. However, despite the apparent consistency of the published
studies, a deficient number of MDN neurons in schizophrenia must still be considered a preliminary finding given the relatively small
sample sizes reported to date, and the fact that potential confounds, such as comorbid conditions (e.g., alcoholism), have not been
adequately assessed.

Certainly, a reduction in cell number in the MDN could contribute both to the decrease in synaptic markers in the PFC, and, given
the dependence of working memory tasks on the integrity of thalamo–prefrontal connections (29 ), to the disturbances in working
memory observed in schizophrenia. However, in contrast to other species, the primate MDN contains both cortically projecting
neurons and local circuit neurons. Thus, it is critical to determine which subpopulation(s) of MDN neurons are affected in
schizophrenia. Interestingly, the density of neurons in the anterior thalamic nuclei that contain parvalbumin (113 ), a calcium-
binding protein present in thalamic projection neurons (114 ), is reduced in schizophrenia; however, whether this reduction
represents an actual loss of neurons, as opposed to an activity-dependent decrease in parvalbumin expression, is not known.

Within the dPFC, five other lines of evidence are also consistent with a reduction in inputs from the MDN (Fig. 53.5 ). First, a
preliminary report notes that subjects with schizophrenia, but not those with major depression, have a decreased density of
parvalbumin-labeled varicosities (putative axon terminals) in layers deep 3 and 4, the principal termination zone of thalamic
projections to the PFC (115 ). In contrast, parvalbumin-labeled varicosities were not decreased in layers 2 to superficial 3,
suggesting that the reduction in layers deep 3 and 4 might not be attributable to changes in the axon terminals of the parvalbumin-
containing subset of cortical GABA neurons present in cortical layers 2 to 5 (35 ). Thus, the laminar-specific reduction of
 P.737

parvalbumin varicosities in schizophrenia is consistent with a decreased number of MDN terminals in the dPFC, although a laminar-
specific reduction in the axon terminals of local circuit neurons cannot be conclusively excluded.

FIGURE 53.5. Schematic diagram summarizing

disturbances in the connectivity between the
mediodorsal (MD) thalamic nucleus and the dorsal
prefrontal cortex (PFC) in schizophrenia. See text
for details. Modified from Lewis DA, Lieberman JA.
Catching up on schizophrenia: natural history and
neurobiology. Neuron 2000;28:325=n334.

Second, the thalamic projections to the dPFC principally target the dendritic spines of pyramidal neurons (116 ). In experimental
animals, the elimination of presynaptic axon terminals leads to a resorption of the postsynaptic dendritic spine (117 ), suggesting
that a reduction in MDN projection neurons would be associated with decreased dendritic spine density in the dPFC. The two studies
that have examined this issue both found decreased spine density on the basilar dendrites of PFC layer 3 pyramidal neurons
(94 ,118 ), with this decrease most marked for pyramidal neurons located in deep layer 3 (94 ), those most likely to be targeted by
projections from the thalamus. Although the well-documented, remarkable plasticity of dendritic spines must be considered when
interpreting these findings, these observations are consistent with a reduction in MDN–dPFC connectivity in schizophrenia. However,
the presence of more modest reductions in spine density on pyramidal neurons in cortical layers that do not directly receive MDN
input suggest that the observed decrease in deep layer 3 may reflect the combined effect of a deficient number of thalamic and
cortical synapses (94 ).
Third, the size of layer 3 pyramidal neurons is reduced in the dPFC of schizophrenic subjects (89 ,119 ). Although the possible
relationship of these findings to a decrease in MDN inputs is less clear, studies in animals have provided evidence of denervation
atrophy of layer 3 pyramidal cells following the loss of other afferent inputs (120 ).
Fourth, in the primate visual system, monocular deprivation, which results in reduced afferent drive from the thalamus, is
associated with a decline in markers of activity in cortical GABA neurons (121 ), including decreased expression of the mRNA for
glutamic acid decarboxylase (GAD67), the synthesizing enzyme for GABA (122 ). Although the experimental manipulation of the visual
system did not involve a partial reduction in thalamic neuron number, if these findings in the visual cortex can be generalized to a
deficient number of MDN projections to the dPFC, a reduction in GAD67 in the dPFC of schizophrenic subjects might be expected.
Consistent with this prediction, both GAD67 mRNA and protein levels have been reported to be reduced in the dPFC of schizophrenic
subjects (26 ,123 ,124 ), observations supported by other evidence of reduced GABA neurotransmission in the PFC of schizophrenic
subjects. (See ref. 125 for review.)
Finally, the reduction in GAD67 mRNA expression in schizophrenia appears to be restricted to a subpopulation of PFC GABA neurons
(approximately 25% to 30%), especially those neurons located in the middle cortical layers (124 ). Consistent with this finding, other
studies suggest that the affected subpopulation of GABA neurons includes chandelier cells. The axon terminals of chandelier cells
form distinctive vertical arrays (termed cartridges) that synapse exclusively on the axon initial segment of pyramidal neurons (126 ).
Interestingly, expression of the mRNA for GABA membrane transporter (GAT-1) is also undetectable in approximately 25% to 30% of
GABA neurons, which have a laminar distribution similar to the neurons with undetectable GAD67 mRNA expression (127 ). In addition,
the density of GAT-1 immunoreactive chandelier neuron axon cartridges is decreased in the dPFC of schizophrenic subjects, with the
reduction most evident in the middle cortical layers (128 ,129 ). Thus, given the powerful inhibitory control that chandelier neurons
exert over pyramidal cell output, decreased excitatory thalamic drive to the PFC may be partially compensated for by a reduction in
chandelier cell-mediated inhibition at the axon initial segment of layer 3 pyramidal cells. This effect could occur via the local axon
collaterals of layer 3 pyramidal cells, approximately 50% of which target the dendritic shafts of GABA neurons (79 ). However, it is
important to note that other causes and consequences of the observed alterations in chandelier neurons have not been excluded.
(See ref. 124 for a discussion.)
Together, these data are all consistent with the hypothesis that schizophrenia is associated with abnormalities in the projection
from the MDN to the dPFC. As in other cortical regions, the connections between the MDN and dPFC are reciprocal, which raises the
question of whether the abnormal thalamocortical projection is paralleled by a disturbance in the corticothalamic projection.
Studies that have examined PFC neurons in layers 5 and 6, the principal location of corticothalamic projection neurons, have
generally not found evidence of a decrease in neuron size or number (34 ,89 ,91 ), although one study (95 ) did report decreased
neuronal density in layer 6 of PFC (region not specified); however, a reduced density of DA axons was observed selectively in layer 6
of dPFC area 9 in schizophrenic subjects (96 ). Interestingly, the dendritic shafts and spines of pyramidal cells are the principal
synaptic targets of DA axon terminals in layer 6, and DA appears to play a critical role in regulating the influence of other inputs on
pyramidal cell activity (130 ). Thus, a shift in DA neurotransmission in dPFC layer 6 could reflect a change in the modulation of
corticothalamic feedback in response to abnormal thalamocortical drive (96 ).
The significance of these findings depends, in part, on the extent to which they are unique to the diagnosis of schizophrenia, and
not a consequence of other factors associated with schizophrenia, such as antipsychotic drug treatment. Some of these findings have
been examined for diagnostic specificity, whereas others have not. For example, the reduction in dendritic spine density on deep
layer 3 pyramidal neurons was not found in subjects with major depressive disorder (94 ), and the reduction in density of GAT-1
immunoreactive axon cartridges was not apparent in subjects with nonschizophrenic psychiatric disorders (129 ). Similarly, to the
extent to which it has been examined, these
 P.738

findings do not appear to be attributable to the abuse of alcohol or other substances, which frequently accompanies the diagnosis of
schizophrenia, although further studies in this area certainly are needed.

Similarly, the available evidence suggests that these disturbances in thalamo-prefrontal circuitry are not attributable to treatment
with antipsychotic medications. Globally, the increase in cell packing density in the dPFC observed in subjects with schizophrenia
was not found in monkeys treated for 6 months with a variety of antipsychotic agents (131 ). Similarly, treatment of monkeys for 12
months with haloperidol and benztropine at blood levels known to be therapeutic in humans was not associated with a reduction in
the size or total neuron number of the MDN (112 ). The potential influence of antipsychotic drugs on GAT-1–labeled axon cartridges
has been examined in several ways with interesting results (129 ). The density of labeled cartridges was greater in schizophrenic
subjects who were on than off antipsychotic medications at the time of death (although both groups showed reduced levels
compared to normal controls). In addition, compared to matched control animals, the density of GAT-1-positive cartridges was
elevated in monkeys treated for 1 year with haloperidol. Thus, the convergence of these findings suggests both that the
pathophysiology of schizophrenia may actually be associated with more marked reductions in GAT-1–immunoreactive cartridge
density than those observed in postmortem studies.

OPPORTUNITIES FOR NEURAL CIRCUITRY-BASED STUDIES OF

SCHIZOPHRENIA
Part of "53 - Neural Circuitry Approaches to Understanding the Pathophysiology of Schizophrenia "

The data summarized in the preceding section suggest that neural circuitry-based approaches to the study of brain abnormalities in
schizophrenia provide: (a) a useful framework to account for the abnormalities observed in individual studies, (b) a platform for the
formulation of predictions regarding the outcome of future studies, and (c) the promise of an enhanced ability to understand the
neurobiological bases of clinical phenomena. However, a truly neural circuitry-based model of schizophrenia requires an
appreciation of the mechanistic relations among the abnormalities observed in different components of the circuitry. Specifically,
understanding the pathophysiology of schizophrenia (or any other psychiatric disorder) depends ultimately on knowing how
abnormalities in one brain region or circuitry component produce and/or result from disturbances in others, a task that involves a
consideration of cause, consequence, and compensation (132 ). Does a given abnormality represent a primary pathogenetic event
(cause), does it reflect a downstream or upstream (given the reciprocal nature of many links between brain regions), secondary,
deleterious event (consequence), or does it reveal a homeostatic response intended to restore normal brain function (compensation)?
Distinguishing among these three possibilities for each component of a neural network will be necessary for understanding the
pathophysiology of the disease as well as for developing novel therapies designed to correct causes and consequences and/or to
augment compensatory responses.

Clearly, addressing these types of questions for schizophrenia requires several additional types of investigations. First, it is essential
to further assess the normal organization of MDN–dPFC connectivity in nonhuman primates. For example, what patterns of
connectivity within the dPFC link inputs from the MDN to the neurons that provide outputs to the MDN or to other brain regions such
as the striatum? Second, MDN–dPFC circuitry needs to be further probed in subjects with schizophrenia in ways that inform an
understanding of its functional integrity. For example, what are the postsynaptic consequences in pyramidal neurons of the apparent
alterations in GABA neurotransmission in chandelier cells? Third, the direction of the pathophysiological changes in MDN–dPFC
circuitry in schizophrenia need to be assessed in experimental animal models. For example, can the observations of altered spine
density and decreased GAD67 mRNA expression in the dPFC be replicated by partial lesions of the MDN in monkeys? Do manipulations
of neurotrophin expression in dPFC layer 3 pyramidal cells result in a loss of the MDN neurons that project to the dPFC?

It is also critical to extend these types of investigations to other brain regions that may integrate MDN–dPFC circuitry with broader
neural networks. Besides the dPFC, the hippocampal formation is probably the brain region that has been most extensively studied
in schizophrenia. Multiple imaging and postmortem studies have documented a slight bilateral reduction in the volume of the
hippocampal formation (25 ,133 ), an observation supported by more recent in vivo proton spectroscopy findings of reduced
hippocampal N-acetyl aspartate in both unmedicated adult and childhood onset subjects with schizophrenia (134 ). Although initial
reports of hippocampal neuron disarray or misplaced neurons in the superficial layers of the adjacent entorhinal cortex have been
widely cited, these observations have not been replicated in other studies. (See ref. 133 for review.) Reduced hippocampal volume
also does not appear to be attributable to decreased neuronal number, but several independent studies have found reductions in
neuronal cell body size in various subregions of the hippocampus proper (135 ,136 and 137 ). In addition, there are consistent
reports of reductions in the gene products for synaptophysin and related presynaptic markers and in dendritic markers, such as
microtubule-associated protein, in certain subdivisions of the hippocampus. (See ref. 12 for review.) Thus, these observations bear
some similarity to findings in the dPFC in that disturbances in synaptic connectivity appear to be present in both regions in
schizophrenia. How these findings inform our understanding of alterations in the intrinsic connectivity of the hippocampal formation
in schizophrenia remains to be
 P.739

determined, but their possible relation to dPFC abnormalities is suggested by experimental studies in rodents that indicate that
dysfunction of the PFC appears postpubertally following perinatal lesions of the hippocampus (138 ).

As noted in the preceding section, it is also important to consider these findings within the context of the developmental time
course of schizophrenia, especially the tendency for prodromal and clinical symptoms to become evident during the second and
third decades of life. Although the adolescence-related pruning of excitatory (but not inhibitory) synapses (31 ,32 ) and their targets,
pyramidal cell dendritic spines (139 ), in the primate dPFC has been well documented, little information exists regarding the extent
to which different connections are actually affected by these changes. Interestingly, limited data suggest that the terminals of
intrinsic axon collaterals from dPFC layer 3 pyramidal cells may be more extensively pruned than associational cortical projections
to the dPFC (140 ). Knowing whether projections from the MDN are particularly vulnerable to this process might provide critical
information for hypotheses regarding the mechanisms underlying disturbances in MDN–dPFC circuitry in schizophrenia.

Another current challenge to the types of neural circuitry-based models of schizophrenia illustrated herein is to understand how the
genetic factors that confer susceptibility for the disease contribute to the observed alterations in neural circuitry. In other words,
how can molecular genetic and systems neuroscience approaches be integrated in the study of schizophrenia? Schizophrenia appears
to be a consequence of multiple interacting genes that individually may have relatively little effect; it is unlikely that all such genes
are involved in every individual who meets diagnostic criteria for the disorder. Thus, assessment of the patterns of altered gene
expression in the affected brain circuits of subjects with schizophrenia (using cDNA microarray technology or related techniques),
and comparison of the chromosomal locations of these genes with regions implicated in schizophrenia through linkage studies (141 ),
may provide convergent approaches to the identification of specific susceptibility genes. For example, as noted, a recent study of
gene expression profiling in the dPFC of subjects with schizophrenia revealed that the group of genes encoding proteins involved in
the regulation of presynaptic function were most consistently altered (44 ). In addition, although the subjects with schizophrenia
appeared to share a common abnormality in the control of synaptic transmission, they differed in terms of the specific combination
of genes that showed reduced expression, a finding that may be consistent with a polygenic model for this disorder. Interestingly, a
number of the chromosomal loci that have been implicated in schizophrenia contain genes encoding proteins related to presynaptic
function (44 ). However, this strategy, and the subsequent integration of potential susceptibility genes with neural circuitry models
of the illness, rests on the prediction that genes regulating presynaptic function are not homogeneously expressed across classes of
neurons and brain regions.

Finally, neural circuitry-based models must also be considered in relation to the clinical heterogeneity of schizophrenia. Is the
magnitude of the abnormalities within MDN–dPFC circuitry related to the age of onset or severity of cognitive impairment? Can other
clinical features be understood within the context of abnormalities in broader circuits that include connections with MDN and dPFC?
Given the limitations involved in making rigorous clinicopathologic correlations in schizophrenia, the answers to these questions may
await the development and application of in vivo imaging techniques with greater sensitivity, such as those that will permit
functional assessments at the levels of individual cortical layers or thalamic nuclei.

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54
Functional Neuroimaging in Schizophrenia

Karen Faith Berman

Karen Faith Berman: National Institute of Mental Health, Intramural Research Program, Bethesda, Maryland

 THEORETICAL PERSPECTIVE
 HISTORIC PERSPECTIVE
 TECHNICAL PERSPECTIVE
 THE FINDINGS
 QUESTIONS FOR THE FUTURE

THEORETICAL PERSPECTIVE
Part of "54 - Functional Neuroimaging in Schizophrenia "

The search for a biological basis of schizophrenia includes a long chapter in which alterations in the characteristics and localization
of neural activity are the focus. It is in this arena that functional neuroimaging has had the broadest application and greatest impact
in psychiatry. This now extensive body of work has left no doubt that schizophrenia is associated with measurable, objective signs of
altered brain function, and clinical and pathophysiologic correlations have begun to emerge. Certain cerebral concomitants of this
illness have been consistently demonstrated, although no single pathognomonic neurofunctional abnormality has been delineated
yet. Increasingly, it appears that dysfunction of a system of functionally and/or structurally interconnected cortical and limbic brain
regions is present to lesser or greater degrees, producing more or less psychopathology in individual patients, and that certain brain
regions, such as frontal cortex, may play a special role in this larger picture. Abnormal neurotransmission within these neural
circuits appears to be related to core features of schizophrenia, particularly cognitive impairment. Although it is likely that at least
some of the functional abnormalities are generative of these features and not simply a response to them, clarification of this
“chicken versus egg” issue must be a crucial component of any research program in this area, and the elucidation of the underlying
mechanisms is the most important quest of this research at present. Current functional neuroimaging has much to offer in guiding
this quest, particularly when combined with new information now available from other fields such as genetics and cognitive science.
In this light, the following sections review existing findings, delineate their relationship to other neurobiological and clinical
properties of the illness, discuss conceptual issues and controversies, examine methodologic considerations (including technical
constraints), summarize new techniques and the new approaches to research design that they allow, and finally describe the most
important areas for future research.

HISTORIC PERSPECTIVE
Part of "54 - Functional Neuroimaging in Schizophrenia "

Functional neuroimaging studies utilize the fact that neuronal activation results in regionally increased blood flow and metabolism.
This can be measured either by radiotracer methods (e.g., [positron emission tomography] PET regional cerebral blood flow [rCBF]
or cerebral glucose metabolic rate) or by a regional effect on the ratio of deoxyhemoglobin to oxyhemoglobin imaged by magnetic
resonance techniques (the blood oxygenation level dependent [BOLD] effect). This work began in earnest some 50 years ago with the
pioneering studies of Seymour Kety and colleagues who developed the first reproducible, quantitative technique for measuring
cerebral blood flow (CBF) as an indicator of neuronal activity, in humans by using nitrous oxide, an inert but soluble gas. When this
method was applied to schizophrenia (1 ), these investigators found no alteration in the overall average CBF level in patients, a
result that has largely been confirmed by more recent studies; however, this finding did not rule out the existence of
neurophysiologically meaningful changes in specific brain structures.

The next advance came in the 1970s with the establishment of rigorous methods that could differentiate the functional level of
specific cortical regions, albeit with only 2-cm anatomic accuracy at best (2 ). This method, administration of tracer amounts of
radioactive xenon, was rapidly applied to the study of schizophrenia. The resulting findings of functional abnormality in the frontal
lobe spurred a shift in focus throughout many research domains in the field that remains a prevailing force today. In the 1980s, the
advent of tomographic methods, such as single photon emission computed tomography (SPECT) and PET, which both use radioactive
compounds as tracers, brought improved interregional spatial resolution on the order of 5 to 6 mm and allowed measurement of
subcortical regional function.
 P.746

In the last decade, functional magnetic resonance imaging (fMRI) has emerged as the premier technique for neuropsychiatric
functional neuroimaging. By taking advantage of the differential paramagnetic properties of oxyhemoglobin versus deoxyhemoglobin
and the altered ratio between them that occurs when blood volume and blood flow change in response to neural activation, BOLD
fMRI uses intrinsic properties of the blood itself rather than an extrinsic contrast or tracer agent, to generate maps of brain function.
It is, thus, entirely noninvasive, and measurements can be repeated over time, conferring significant advantage in experiments
designed to address important clinical questions in schizophrenia, such as response to medication, correlation with clinical course,
differentiation of state versus trait phenomena, determination of the dynamic range of neurophysiological response, and learning.
This new methodologic advance brought further improvements in spatial resolution as well as enhanced temporal resolution, which,
although still slow (several seconds) compared to neuronal signaling (on the order of 200 ms), improved to the degree that event-
related neural activity could be recorded with anatomic precision heretofore unavailable with electrophysiologic methods.

TECHNICAL PERSPECTIVE
Part of "54 - Functional Neuroimaging in Schizophrenia "

As can be seen in the preceding brief history, over the years the sophistication of the questions that could be asked and the
hypotheses about schizophrenia that could be tested have paralleled the development of new brain imaging technologies and
analytic methods. This parallel development is evident in the evolution of the science from the search for regionally specific
pathologic function to that in neural systems, and from measures sensitive only to static pathophysiology to explorations of the
dynamic interplay among regions in those neural systems. Therefore, a brief discussion follows of the newest methodologic
approaches and latest vistas for research in schizophrenia that they offer.

New Vistas in Data Acquisition

Event-related fMRI is a relatively recent class of experimental design within functional neuroimaging that exploits the superior
temporal resolution of fMRI. Unlike previous fMRI and PET approaches that blocked together relatively long (e.g., 20 to 60 sec)
periods of similar behavioral trials or conditions and examined average neural response over that time period, event-related fMRI
documents the neural response during each individual trial or short behavioral period. This approach has much in common with
traditional evoked-potential electrophysiology and offers advantages in experimental design. Because different trial types can be
randomly intermixed and then separated for analysis, order effects and habituation can not only be controlled for, but also
explicitly investigated. A particular advantage for research in schizophrenia is that neural activity during correct and incorrect trials
can be measured separately and compared, allowing more incisive study of the mechanism of cognitive failure and better
experimental control of potential confounds based in performance discrepancies that often occur between patient and control
groups. Eventrelated fMRI has very recently come into wide use in neuroimaging of cognitive systems in healthy subjects, but as of
this writing has had only limited application to the study of schizophrenia.

Multimodal neuroimaging, which seeks to determine the relationship of the neurofunctional abnormalities in patients to other
neurobiological features, will have an increasingly important role in delineating the mechanism of schizophrenia. In this approach,
blood flow and other measures such as MR spectroscopy, neuroreceptor measurements, and electrophysiology (with MEG or EEG) are
determined in the same patients. One example of the richness of the data that can be gleaned is the use of PET or fMRI to measure
blood flow in conjunction with EEG or MEG. PET and fMRI allow localization of the brain regions that work together during cognition
(spatial resolution 3 to 6 mm), but provide relatively little temporal information (temporal resolution in seconds). On the other hand,
EEG and MEG have relatively poorer spatial resolution, but provide fine time resolution (i.e., milliseconds). Combining these
methods, together with the application of the advanced computational cross-registration and source localization techniques that
now exist, provides exponentially more information than any of these techniques alone. For example, this allows the determination
of the sequence in which various finely localized regions are activated during cognition and the testing of the hypothesis that this
sequence of events is altered in schizophrenia. Although this specific multimodal approach has not been applied in schizophrenia,
other examples are described in the following.

New Vistas in Data Analysis

The analytic approaches for the two data collection modalities discussed, event-related fMRI and fusion of spatial and temporal
neurofunctional data (i.e., PET or fMRI with EEG or MEG), are in their infancy and beyond the scope of this chapter. Another recent
set of analytic methods addresses the growing appreciation that the transduction between neural and mental events is not
anatomically segregated (i.e., one brain region per one mental or cognitive activity), but rather that even the simplest of cognitive
activities requires coordinated actions across widely distributed components of neural systems (interregional integration of neural
function). Questions about functional integration and coordinated interregional activity are likely to have particular relevance for
schizophrenia, as discussed in later sections. Although the segregational view can be
 P.747

tested with univariate statistics (multiple t-tests or ANOVAs), hypotheses about interregional integration require multivariate
approaches. The influence that one brain region exerts or experiences from another is most simply examined via the correlations
between brain activity measures for the two anatomic structures; as an operational definition two brain regions can be considered
to be functionally coupled if their activities are correlated (3 ); however, this approach cannot elucidate how other nodes in the
network mediate these relationships. To model this mediation requires analysis of the covariance matrix of regions studied in its
entirety. Several new methods have been developed. These include structural equation modeling (SEM) and eigenimage analysis.
SEM, used in conjunction with acknowledged anatomical models, can characterize and quantify the “functional connectivity” among
the multiple components of neural systems. A model of known or hypothesized anatomic pathways is defined first; then a functional
model of interest is tested against this model by iterative fitting of the interregional correlational weights. It should be borne in
mind that the regional components examined are preselected based on putative pathways and the results from this approach are
only as good as the model. In contrast, eigenimage analysis is a data-driven approach that examines patterns of correlation across
the entire brain and how they vary in a time-series, revealing distributed brain systems and their temporal dynamics. Single value
decomposition or principal component analysis is used to present the percentage of variance accounted for by different patterns of
activity or spatial modes, and canonical variates analysis, conceptually similar to factor analysis, can be used to extract connectivity
patterns across the entire brain that are most different between the studied groups. Thus, these recently developed methods permit
characterization of normal and altered neural connectivity using neuroimaging.

THE FINDINGS
Part of "54 - Functional Neuroimaging in Schizophrenia "

The explosion of functional neuroimaging studies of schizophrenia has resulted in many “findings” and many discrepancies.
Nonetheless, several trends spanning the various experimental and methodologic techniques were apparent several years ago (4 ).
First, when brain activity or metabolism is averaged across the entire brain (i.e., “global” function), patients have relatively normal
values, unlike in primary degenerative disorders. Second, when scanned during so-called resting conditions (i.e., with no cognitive
or motor activities required and no specific sensory input), abnormalities are inconsistent when seen. Although patients may have
relatively normal regional patterns of resting brain activity, there appear to be associations between specific resting regional CBF
patterns and symptom profiles (5 ). Changes in lateralization of brain function also have been described (6 ,7 ). Third, when scanned
during cognitive activation, patients tend to be different from normal controls. In particular, they show abnormal prefrontal activity
(8 ) during tests involving working memory (i.e., the system used to hold information in temporary storage to complete a task). They
show deficits in cingulate cortex as well as alterations in frontal–temporal and other intracortical functional relationships (12 ,13 )
during other cognitive tasks, such as cued verbal recall (9 ) and the Stroop test (10 ), and in some studies at rest (11 ). In general,
most of these findings have been reproduced in acute, untreated patients, thus excluding a primary role for medication artifacts.

More recent functional brain imaging studies in schizophrenia have focused on: (a) further characterization of the locales and
cognitive and behavioral context of neurophysiologic deficits in schizophrenia; (b) delineation of the relationship of the deficits to
clinical symptoms and other neurobiological features of the illness; and, most important; (c) attempts to elucidate the
pathophysiologic mechanism(s) of the deficits. The following section reviews these areas of observation and others. The most
enlightening of these results have emerged from experimental paradigms designed to actively engage neural systems in cognitive or
other activities; the most consistent findings concern the function of the prefrontal cortex.

Frontal Lobe Circuits

The frontal lobes have played a prominent role in formulations of schizophrenia since the conceptualization of the illness. In earlier
times this role was inferred by clinical analogy with known frontal lobe disorders and findings in neuropsychology and nonhuman
primate studies. Substantial indirect but compelling evidence from these multiple domains conferred the status of best studied and
most implicated region in schizophrenic pathophysiology on the frontal lobes. Early functional neuroimaging studies substantiated
this role, beginning with Ingvar and Franzen’s 1974 seminal finding that patients with schizophrenia had relatively lower blood flow
to frontal regions (14 ). Changes in rCBF in response to cognitive activation were also first observed in these early studies; this
approach has been greatly refined and now has largely replaced the inherently ill-defined and variable resting state as the
cornerstone of functional brain imaging studies in schizophrenia. These data presaged the body of literature that emerged over the
ensuing 15 to 20 years, most of which had similar frontal lobe findings (Table 54.1 ).
 P.748

TABLE 54.1. FRONTAL LOBE FINDINGS WITH ACTIVATION PARADIGMS IN

SCHIZOPHRENIA SINCE 1985

Characterization of Dorsolateral Prefrontal Functional Alterations

The relatively subtle prefrontal functional alterations observed in schizophrenia have been increasingly brought into focus by recent
advances in the available neuroimaging armamentarium. Table 54.1 lists activation studies published
 P.749

over the past 15 years that report frontal lobe results. The overwhelming majority of these investigations have detected abnormal
prefrontal responses to a variety of cognitive activities designed to access and/or control frontal neural circuitry, particularly
working memory. The prefrontal site most commonly affected is the dorsolateral prefrontal cortex (DLPFC), and, until recently, the
physiologic abnormality in this brain region was consistently seen as hyporesponsivity. Indeed, from the time of Ingvar and Franzen’s
first study and throughout most of the last decade researchers were satisfied that the qualitative nature of the frontal lobe
abnormalities was known. However, the relative universality with which the schizophrenic prefrontal cortex had been reported to be
hypofunctional, in the past several years, has given way to the notion that the aberrant neural responses in prefrontal cortex are
more complex, including hyperfunction under some circumstances. It is noteworthy that abnormally increased prefrontal response
has been primarily seen in studies carried out with fMRI, rather than PET, and mainly when the cognitive paradigms take advantage
of the temporal properties of fMRI in order to employ shorter blocks of task performance, and/or require task switching. This fact
suggests that the anatomic and/or chemical perturbations of the schizophrenic prefrontal cortex can be manifest by inappropriate
over recruitment of prefrontal neural circuitry during relatively brief cognitive challenge and failure to sustain this recruitment over
longer periods; however, more work is required to understand the implications of these recent observations and to fully characterize
them. Regardless of the direction of prefrontal physiologic abnormality, the functional neuroimaging literature leaves little doubt
that such abnormality exists. A variety of potential epiphenomenological explanations for this pathophysiology have been considered,
and a number of neurobiologically plausible mechanisms have been proposed.

Effect of Neuroleptic Treatment

One potential epiphenomonological confound is the possibility of a causative role of antipsychotic medications, an important
consideration because the majority of functional neuroimaging studies have been carried out in patients who were either receiving
neuroleptics at the time of study or previously treated and then withdrawn for some prior period. However, taken as a whole, the
literature provides little evidence that neuroleptics generate the functional neuroimaging abnormalities. First, although additional
longitudinal investigations with newer techniques are necessary, an 18-year follow up (15 ) showed that prefrontal hypofunction in
chronic patients is remarkably stable over time and unaffected by long-term consistent neuroleptic treatment. Second, prefrontal
abnormalities like those observed in schizophrenia are not seen in other illnesses in which neuroleptics are used, such as
Huntington’s disease (16 ,17 ). Third, a study of monozygotic twins concordant for schizophrenia, but with differing lifetime
histories of neuroleptic intake, found that in most pairs the twin with less exposure to neuroleptics was actually the more
hypofrontal of the pair during a prefrontally linked task (18 ), a result opposite to that expected if neuroleptics caused
hypofrontality. Fourth, studies examining metabolic or rCBF changes occurring when patients go from the unmedicated to the
medicated state are quite inconsistent (4 ,8 ). Fifth, frontal lobe functional abnormalities have been found in first-degree relatives
of patients with schizophrenia who have not been treated with neuroleptics or hospitalized (19 ,20 and 21 ). Finally and perhaps
most conclusively, frontal lobe abnormalities during cognition have been found in a number of studies of young patients who have
never received neuroleptics (22 ,23 ,24 ,25 and 26 ). Thus, in summary, there are little convincing data that neuroleptic treatment
is a major factor in frontal lobe functional abnormalities.

The Link with Cognitive Performance

Most prominent among potential epiphenomena and confounds examined has been the effect of poor performance. It is undisputed
that patients with schizophrenia perform poorly on the very cognitive tasks that best show prefrontal pathophysiology—the
Wisconsin Card Sorting test (WCST), the N-back test, the Tower of London, or others. This is not surprising for the reason that the
tasks are chosen because they are thought to reflect core deficits of the illness and they access neural systems relevant to it;
however, considerable controversy has arisen around the possibility that the poor performance—or differences in attention or effort
and motivation—somehow cause the frontal pathophysiology, rather than the more neurobiologically plausible explanation that
underlying pathophysiology is responsible for the poor performance. It is clear that if patients are simply not engaged in a cognitive
task during scanning they will not activate relevant brain regions; such results are obviously artifactual. It is less clear what should
be predicted if patients (or healthy subjects) are manifestly working at a task, but performing it abnormally.

The relationship between performance level and the degree to which the brain neurophysiologically responds is complex, even in
the presumptive absence of pathology in healthy control subjects. Several studies have found significant correlations between
prefrontal neural activity and cognitive function, suggesting that these two variables are paradigmatically linked, but both positive
and negative (27 ) relationships have been described (8 ). The research challenge has been to (a) understand this relationship and (b)
tease apart abnormal cognitive performance and abnormal brain activity in patients to determine which is primary. This is a difficult
issue to investigate experimentally, and no single study alone can answer the question; however, convergent evidence derived from
several different research
 P.750

directions leads to the conclusion that prefrontal pathophysiology cannot be accounted for as an epiphenomenon (8 ).

First, studies have been carried out on patient populations who, like schizophrenics, perform poorly on frontal lobe tasks but who
have disorders other than schizophrenia. In principal, if the prefrontal physiologic deficit found in patients with schizophrenia is an
epiphenomenon of poor performance per se, then other subjects who perform as poorly should have similar prefrontal function.
Pathophysiology quite distinct from that characterizing schizophrenia has been reported in Huntington’s disease (28 ) and normal
aging (29 ,30 ) where performance is matched to that in schizophrenia, as well as in Down’s syndrome (31 ) in which performance is
worse. These findings indicate that poor performance per se does not necessarily produce the pathophysiologic picture seen in
schizophrenia.

A second way to experimentally attack this “chicken and egg” question, and at least on the face of it the most direct way, is to
match patients and normal controls for level of performance. However, ensuring good performance in patients (by using different
versions of the task for patients and controls or by making the task extremely easy) does not guarantee that the effort involved or
the cognitive operations used by the two groups are equated. Moreover, in the absence of neuropsychologic impairment, the neural
systems accessed and findings may have very little to do with the illness. Thus, the strategy of employing “easy” tasks that result in
some measures of performance being “normal” in patients is not as straightforward an approach to exploring neurophysiologic
abnormalities in schizophrenia as it may appear (4 ,8 ). An alternative to “matching” for good performance is to study normal
controls who perform as poorly on a given task as the patients. This strategy at least addresses the question of whether normals and
patients fail by the same pathophysiologic mechanisms. In a study designed on this premise, patients performing the WCST were
found to activate DLPFC less overall than performance-matched normals, but they activated a more anterior area of prefrontal
cortex that is not recruited in normals as a group. Moreover, the more a given normal subject activated this “schizophrenic card sort
area,” the more he or she perseverated (8 ).

This overactivation of an aberrant area (or of an appropriate area, for that matter) would be difficult to explain on the basis of
disengagement from the task and poor performance per se. In a similar example, affected members of monozygotic co-twins
discordant for schizophrenia showed hypofunction of prefrontal cortex accompanied by hippocampal hyperfunction during the WCST
(32 ). Analogous results have been described by Friston and colleagues using paced verbal production (33 ) and have recently been
demonstrated with eigenimage analysis in a study of medication-free patients performing the N-back working memory task (34 ).

Yet a third approach to inform the performance conundrum is to model the abnormal cognition in normal controls. Both
underactivation and overactivation have been described in the context of performance difficulties. Goldberg and associates (1998)
found that healthy subjects performing a dual task, working memory plus auditory and verbal shadowing, had significant decrements
in both performance and DLPFC blood flow (35 ). Callicott and co-workers (1999) demonstrated that normal controls pushed beyond
their working memory capacities also demonstrate reduced DLPFC responses (36 ). Electrophysiologic recordings in working memory-
related neurons of nonhuman primates provide a neurobiological framework for these observations, where failed working memory
trials are accompanied by decreased firing rates (37 ,38 ). Thus, findings of decreased prefrontal response in schizophrenia can be
seen as part of an expected curve between working memory load and neural response—a dose–response curve that is shifted in the
face of patients’ reduced prefrontal capacities. Given the fact that other poorly performing patient populations with pathology that
is different than schizophrenia do not show the same prefrontal response one (28 ,30 ,31 ), this particular shift in the dose–response
curve with poor performance is neither inevitable nor the only possible one.

The DLPFC overactivtion response in schizophrenia also has a precedent in the normal neurocognitive imaging literature. Rypma and
D’Esposito (1999) found in healthy subjects that the longer the reaction time (an indicator of difficulty with a task), the greater the
DLPFC neural response (27 ). Although this may simply represent increased “time on task,” it again provides a context in which to
view recent findings of overactivation in patients. Although some clues about the cellular mechanism underlying patients’ aberrant
prefrontal function are discussed in the following, it remains for future research to determine whether patients and controls share
the same source for abnormal prefrontal responses (both underactivation and overactivation) in the context of performance
difficulties.

The data summarized in the preceding leave little doubt that, although the patients’ poor performance is certainly an integral,
primary component of their illness, it is not the cause of the abnormal neural function, rather it is an effect of that pathophysiology.
Given this conclusion and the now extensive literature documenting it, the most efficacious use of functional neuroimaging in future
schizophrenia research will occur if the poor performance and prefrontal abnormalities are considered as inextricably linked and
studies are designed to elucidate the mechanism of these linked phenomena.

Putative Underlying Mechanisms

A number of clues about potential pathophysiologic mechanisms emerge when the neuroimaging findings are considered in light of
other neurobiological hallmarks of schizophrenia and findings from different clinical neuroscience research modalities. First, it is
likely that cellular pathology in DLPFC contributes to the cognitively linked dysfunction.
 P.751

The degree of both hyperfunction and hypofunction of DLPFC in patients are predicted by decreased n-acetyl-aspartate (NAA), an
MR spectroscopy measure of cellular integrity (39 ,40 ). Second, several compelling lines of evidence suggest a prominent role for
dopaminergic dysfunction. It is well documented in nonhuman primates that optimal dopamine function is necessary for maximal
working memory and DLPFC physiologic function (41 ). Similar, albeit less direct, evidence also exists in humans: Pharmacologically
altering dopaminergic tone with agents such as amphetamine affects DLPFC activity in both healthy subjects (42 ,43 ) and patients
(44 ); and a relationship between DLPFC rCBF during the WCST and CSF levels of the dopamine metabolite homovanillic acid has
been found in schizophrenia (45 ). Third, converging data increasingly point to developmental mechanisms. A considerable body of
literature now documents that disruption of corticolimbic connectivity in neonatal animals via hippocampal lesions models many
features of schizophrenia, including working memory impairment, reduced prefrontal NAA, and dopamine dysregulation (53 ).
Developmental pathology with a genetic basis also appears likely. A recent study links a genetic attribute that affects prefrontal
dopamine to both working memory performance and DLPFC activation in patients, their sibs, and unrelated healthy individuals (21 ).
Moreover, an association of schizophrenia with the allele that confers poor prefrontal function (a functional polymorphism in the
catechol-o-methyl transferase [COMT] gene that affects enzyme activity) is indicated by several family-based studies. Investigations
of this type, which explore the interaction of genetic and neurophysiologic characteristics, hold the greatest promise for elucidating
the etiology of the illness and effecting innovative treatments.

Other Frontal Lobe Subregions

Dysfunction, primarily hypofunction, of portions of the frontal lobes other than the DLPFC has also been described. Just as a
cognitive foundation for DLPFC dysfunction has been found in working memory and executive function, the dorsal anterior cingulate
is classically considered to play an important role in vigilance, attention, and effort. It may, thus, be especially prone to
epiphenomenologic effects. In normal subjects the region is activated by a variety of cognitively demanding tasks requiring stimulus–
response selection in the face of competing information, such as the Stroop test, complex motor control tasks, verbal fluency, and
working memory. These observations have led cognitive neuroscientists to propose more refined cognitive roles such as on line
monitoring, conflict monitoring, and error detection (46 ). Further research is necessary to clarify which of these putative cognitive
roles, or which epiphenomena, may be linked to the finding of anterior cingulate underactivation in schizophrenia (9 ,10 and 11 ).
Orbitofrontal cortex, along with the ventral portion of the anterior cingulate, has been most linked to emotion. Few studies have
been carried out that formally investigate the function of these regions in schizophrenia.

Lateral And Medial Temporal Lobe

The temporal lobe is of interest in schizophrenia for several reasons. Diseases of the medial temporal lobe can be associated with
psychotic symptoms, and some neuropsychological aspects of schizophrenia implicate both lateral and medial temporal lobe. A
number of neuroimaging studies have reported functional abnormalities in both lateral and medial temporal lobe structures (47 ).
The data as a whole, however, are less compelling than for frontal lobe, and confounds and potential mechanisms are less well
explored. Findings of both hyperfunction and hypofunction have been reported, but the bulk of the evidence leans toward
overactivity. Heckers and colleagues (48 ) reported reduced hippocampal activation during the effort to retrieve poorly encoded
material; however, it is of interest that, hippocampal activity appeared to be increased at baseline, again emphasizing the task-
dependence of neurofunctional findings in general (48 ). Several studies point to a role for lateral temporal cortex in hallucinations
and other positive symptoms—abnormal functional interactions between temporolimbic and prefrontal structures are also discussed
in the following.

Miscellaneous Regional Changes

Functional abnormalities, primarily hypofunction, of many other regions have been reported. Although most are unreplicated,
several are worth mentioning. Both increased and decreased basal ganglia activity have been found, but a role for neuroleptic
treatment in such findings must be considered. Several investigators have suggested that schizophrenia is characterized by increased
posterior cortical activity. In fact, Ingvar and Franzen’s seminal 1974 rCBF study of schizophrenia suggested that the hypofrontal
pattern represented a redistribution of flow with relatively lower flow to frontal areas as well as relatively higher flow to posterior
cortex (14 ).

Such changes in activity patterns or distributions have received considerable attention. In particular, the notion that schizophrenia
may involve disordered functional lateralization has been explored using a variety of methods. Left temporal overactivation was
seen in this light in early studies. More recent work suggests that apparent alterations in functional laterality in schizophrenia may
not actually reflect abnormal lateralization per se, but rather a failure to organize a lateralized response (6 ,49 ). For example,
Mattay and associates (1997) reported less lateralized and localized lateral premotor area activation in patients during a simple
finger movement paradigm (50 ). This may also be viewed
 P.752

within the more general context of nonfocalized, less efficient, neurophysiologic responses in schizophrenia.

Interregional Relationships and Functional Connectivity

As the foregoing indicates, a number of brain areas have been shown to function abnormally in schizophrenia, often depending on
the cognitive or other conditions under which the scanning is performed. It has been proposed that such multiple, seemingly local
changes may be indicators of more ubiquitous dysfunction throughout widely distributed and interactive brain networks (12 ,51 ), a
heuristically appealing pathophysiologic model for schizophrenia given the apparent subtlety of the neurophysiologic abnormalities
in the face of the devastating and complex nature of the illness. This conceptualization is consistent with recent trends in viewing
higher brain processes as parallel and distributed functions.

Because of the particularly extensive connectivity of the frontal lobe with other cortical and thalamic relay areas and its special role
in schizophrenia, it is not surprising that many putative aberrant networks in the illness also involve it. Although the specifics of this
network will obviously vary by task, a consistent finding in studies of working memory is a coactivation of prefrontal, anterior
cingulate and parietal structures. Consistent with this, Bertolino and colleagues found a tight correlation between DLPFC NAA
(indicative of neuronal integrity) and rCBF activation during the WCST, not only in DLPFC, but also with the other nodes in the
working memory pathway (39 ). Because this was not evident in healthy controls, these findings appear to reflect a rate-limiting
factor related to the disease process of schizophrenia.

Neurofunctional evidence of abnormal interactions between prefrontal and temporal/limbic areas has accrued for a number of years.
Weinberger and associates (52 ) found in monozygotic twins discordant for schizophrenia an inverse relationship between the volume
of the hippocampus (the structural variable that best differentiated well from ill twins) and the degree of dorsolateral prefrontal
activation during prefrontal cognition (the physiologic variable that best differentiated the co-twins). This suggests dysfunction of
neocortical–limbic connectivity in schizophrenia and is consistent with, if not confirmatory of, a neurodevelopmental mechanism
(53 ). It has been suggested that abnormal development or plasticity of hippocampal connectivity affects the development and
function of prefrontal cortex or, alternatively, that both regions are “put at risk” by the same pathologic mechanism (e.g., genetic
variation) (54 ).

The new analytic tools recently developed to search more incisively for evidence of subtle and multidimensional abnormalities
across the entire brain (see the foregoing) have provided results that are consistent with and extend the notion of
temporohippocampal and prefrontal circuitry failure (13 ,33 ). During working memory, Meyer-Lindenberg and colleagues (34 ), using
an eigenimage method (discussed in the preceding), uncovered differences between patients and controls in task-independent
functional connectivity patterns charcterized by hypofrontality coexisting with increased temporal/hippocampal and cerebellar
overactivity in the patients; another, task-related, pattern involving the working memory system (including DLPFC and inferior
parietal lobule) was found to be more variable (i.e., showed altered modulation) in the patients, specifically during the working
memory condition. Friston and Frith (12 ), using PET data from a verbal fluency experiment and a method that allowed them to
assess patterns of activation most different between normals and patients, found that the prefrontal and temporal coactivations in
normals were uncoupled (i.e., did not appear in the same pattern) in the patients. Fletcher and colleagues (55 ) reported similar
results, and Jennings and co-workers (56 ) using structural equation modeling found altered neural interactions among frontal
regions as well as between the frontal and temporal cortices in schizophrenics during a semantic processing task. Disruption of
frontal–temporal connectivity has also been found using an EEG coherence measure (57 ).

Other studies have focused on medial prefrontal and cortical–striatal–thalamic circuit abnormalities (49 ); Biver and colleagues (58 )
and Mallet and associates (59 ), calculating correlations between various regions of glucose metabolic rate in PET, found decreased
intrafrontal, as well as frontal–posterior connectivity. Andreasen (60 ) has advanced a hypothesis implicating compromised
connectivity among prefrontal regions, several thalamic nuclei, and the cerebellum as the cause of a fundamental cognitive deficit
in schizophrenia. She called the disruption in this circuitry “cognitive dysmetria,” signifying “poor coordination of mental activities”
that manifests itself in difficulty in prioritizing, processing, coordinating, and responding to information. This hypothesis is based on
a number of studies from her group (61 ,62 and 63 ) in which the structures enumerated above were found to differ in activation
between schizophrenics and controls during several unrelated tasks and in different cohorts, and on the fact that the circuit
described is anatomically connected.

In summary, taken together, the functional brain imaging evidence is consistent with the notion that schizophrenia involves
dissolution of neuronal interactions and that many features of schizophrenia may best be viewed as dysfunctional interregional
circuitry. The details of this circuitry dysfunction differ, depending on the distributed network called into play during the particular
behavior, but prefrontal cortex may play a special role.

The Relationship of the Neurofunctional Abnormalities to Clinical Hallmarks

The importance of linking pathophysiologic findings in schizophrenia to clinical aspects of the illness was recognized
 P.753

early on. Ingvar and Franzen (14 ) noted that “hypofrontality” was most prominently seen in the most withdrawn, inactive, socially
isolative, and “hypointentional” patients, whereas they related the hyperfunction in posterior areas that they observed to a
“hypergnostic” component of the illness. The attempt to delineate the clinical and neurobiological implications of the physiologic
abnormalities remains an important focus. Studies have primarily searched for neurophysiologic associations with cognitive deficits,
symptom clusters, and individual clinical features such as hallucinations. The small sample sizes of some studies and the necessarily
phenomenologic nature of research into the neurophysiologic underpinnings of clinical symptoms makes firm conclusions difficult,
but some consistent findings have emerged. Frontal lobe dysfunction is consistently linked to negative symptoms and cognitive
deficits, particularly working memory and executive function. For example, Goldberg and colleagues (64 ) used an intra-twin pair
difference method in which unaffected co-twins served as individual controls for each patient in the NIMH monozygotic twin sample.
Although left hippocampal size predicted a parameter of verbal memory, prefrontal blood flow and perseveration on the WCST were
related. These data are part of a growing literature implicating medial temporal and prefrontal regions in symptom expression and
some neurocognitive deficits of the illness.

In general, hallucinations are associated with sensory modality-specific activation in brain regions involved in normal sensory
processing (65 ). For example, auditory hallucinations appear linked to language-related regions such as Broca’s area (66 ) and left
superior temporal cortex. Silberswieg and co-workers (67 ) made similar findings using a technique that quantifies the relationship
between brain activity and density of hallucinations during the scanning period.

Specific cerebral blood flow patterns have been associated with distinct syndromes of schizophrenic symptoms (5 ): psychomotor
poverty with decreased activity in DLPFC; disorganization and impaired suppression of inappropriate responses with increased
activity in the right anterior cingulate gyrus; and reality distortion, which may arise from disordered internal monitoring, with
increased activity medial temporal lobe at a locus activated in normal subjects during internal monitoring of eye movements.
Thought disorder has been linked to temporal lobe overactivity. Kaplan and associates (68 ) found an association of marked
psychomotor poverty with superior parietal as well as prefrontal areas, hallucinations and delusions with abnormalities in left
temporal cortex, and disorganization with left inferior parietal lobule abnormalities. Further work undoubtedly will refine these
interesting clinical and pathophysiologic correlates.

QUESTIONS FOR THE FUTURE

Part of "54 - Functional Neuroimaging in Schizophrenia "

Further characterization of the abnormalities delineated by functional neuroimaging in schizophrenia is a clear goal for the future.
One important advance will come from temporal dissection of the abnormal neurophysiologic signals that have now been localized
with great anatomic precision. For example, the particularly high degree of both segregation and interaction of the frontal lobe
complex appears to be essential for regulating and monitoring the functions it supports via multisynaptic feedback loops modulating
posterior brain areas. It is likely that the functional disconnection in schizophrenia described in the preceding includes abnormalities
in these feedback loops, which operate on a time scale less than 200 msec. Progress in understanding the etiology of frontal lobe
dysfunction in schizophrenia, therefore, requires a methodology that has optimal resolution both spatially (in order to reliably
differentiate functionally segregated areas) and temporally (to tap into the time scale in which the feedback loop organization
operates). The simultaneous combination of PET or fMRI studies (which afford relatively high spatial resolution) with methods having
excellent temporal resolution such as EEG or MEG (which provide temporal resolution in the order of milliseconds) will allow explicit
investigation of specific hypotheses about prolongation of the feedback and feed-forward latencies and about disease-related
changes in the order in which components of distributed neural systems come into play in schizophrenia.

A second important way in which characterization of the abnormal neurophysiologic signals must advance is further investigation
into their relationship to other neurobiological features of the illness. One question that requires closer scrutiny is the relation of
the neurophysiologic abnormalities to dopaminergic and other neurochemical parameters. Hypotheses about neurochemical
mechanisms can be tested directly with functional brain imaging, both by examining the effects of pharmacologic interventions and
direct in vivo measurements of various components of neurochemical systems. Also, the relationship of the functional abnormalities
to the neurostructural and neurochemical findings described in other chapters must be further elucidated. Not only does such a
multimodal approach provide critical cross-validation of the information gleaned from the different technologies and help to rule
out epiphenomena, but it is also a means to more closely approach causality and mechanism. For example, links with dopaminergic
dysfunction can elucidate putative genetic mechanisms (21 ). Similarly, the fact that the prefrontal functional abnormalities may
relate to structural pathology in other (particularly limbic) areas lends credence to the notion of a neurodevelopmental mechanism,
although it does not provide proof; further work, perhaps expanding on insights from animal models (53 ), will be necessary to test
the roles of the temporal lobe and aberrant neural development in the genesis of schizophrenic psychopathology.

Clarifying the mechanism by which the pathophysiology and illness arise is the most important question that can be addressed with
functional brain imaging. Considerable
 P.754

work remains to be done, although some clues have emerged, particularly with regard to the frontal lobe. Longitudinal studies are necessary to differentiate trait from state
phenomena. Brain imaging undoubtedly will also continue to impact the current effort on many fronts to uncover the genetic foundations of schizophrenia, by offering new targets for
linkage and association studies and providing clues to direct hypothesis-driven genetic investigations, as discussed in this chapter. Such studies provide a unique perspective from which
to view brain function, one that offers the possibility of uncovering basic fundamental principals important in the genesis of schizophrenia and that has the potential to lead to direct
intervention.

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55
Structural Magnetic Resonance Imaging Studies in Schizophrenia

Robert W. McCarley

Robert W. McCarley: Harvard Department of Psychiatry, Brockton VAMC and VA Boston Healthcare System, Brockton,
Massachusetts

… We thus come to the conclusion that, in dementia praecox, partial damage to, or destruction of,
cells of the cerebral cortex must probably occur, which may be compensated for in some cases, but
which mostly brings in its wake a singular, permanent impairment of the inner life (1 ).

The window on the brain provided by structural imaging has transformed our view of schizophrenia to one that views the very
structure of the brain as altered, a view echoing Kraepelin’s prescient statement. Beginning with Johnstone’s CT findings of
enlarged ventricles (which actually confirmed earlier, less systematic pneumoencephalographic studies), subsequent reports using
magnetic resonance imaging (MRI) have provided key information detailing volume reductions in particular brain anatomic regions of
interest (ROI). These data have provided the major evidence in support of our current view that schizophrenia is a brain disorder
with altered brain structure, and consequently involving more than a simple disturbance in neurotransmission.

Section Organization
The next section is a nontechnical introduction to some of the basic concepts of MRI, and may be read independently of the other
sections or skipped by those who wish to concentrate on the clinical data. Subsequent sections discuss the application of structural
MRI to questions in schizophrenia research, standards for technical quality and reviews of studies, current MRI findings in
schizophrenia (limited to studies with defined ROI), and newer technologies.

 THE BASIS OF STRUCTURAL MRI AND PULSE SEQUENCES

 STRUCTURAL MRI: WHAT CAN IT TELL US ABOUT SCHIZOPHRENIA?
 WHAT ARE THE DESIRABLE FEATURES OF A STRUCTURAL MRI STUDY?
 HOW SHOULD LITERATURE FINDINGS BE REPORTED IN REVIEWS? EXPERT OPINION VERSUS COUNTING VERSUS METAANALYSIS
 CURRENT STRUCTURAL FINDINGS IN SCHIZOPHRENIA
 AUTOMATING STRUCTURAL MRI ANALYSIS: BRAIN WARPING AND VOXEL-BASED ANALYSIS
 DIFFUSION TENSOR MR IMAGING
 CONCLUSION

THE BASIS OF STRUCTURAL MRI AND PULSE SEQUENCES

Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

Addressing the physics of structural MRI is a major topic on its own, and Brown and Smeleka’s book (2 ) is recommended as a good
introduction that demands only a limited background in mathematics and physics. The reader is warned that the following brief
exposition is highly (over)simplified. Essentially, the tissue characteristics sensed by MRI depend on disruptions of a strong external
magnetic field. This external field has aligned the orientation of atomic nuclei by aligning the magnetic field of each nucleus that is
associated with its spin direction. Because of its biological ubiquity and good magnetic properties, the most commonly used basis
element for MRI is hydrogen, which has a single proton in its nucleus. The reader may want to think of hydrogen nuclei as analogous
to a large set of spinning tops or gyroscopes. In a state without an external magnetic field, their direction of spin is random, and so
is the net magnetization (a vector), because each rotating proton has a magnetic field that is parallel to its axis of rotation.
Applying a strong external magnetic field can be thought of as aligning this set of spinning tops in a uniform direction of spin,
snapping them to attention, as it were. The resultant population net magnetization can be thought of as vector aligned with the z-
axis (vertical axis in our example), perpendicular to the x-y plane, and in the direction of the external magnetic field. The magnetic
field strength is described in units of tesla (T) and most current clinical imagers use an external field of 1.5 T.

This vertically aligned population of protons (vertical magnetization vector) is then perturbed by applied radiofrequency (rf) pulses,
which can be thought of as having the effect of moving the magnetization vector away from the vertical axis (z-axis); for example,
in our analogy, moving the tops from their average vertical orientation to a “tilt.” This applied change of the magnetization vector
then decays, and, during the course of the decay, give off energy in the form of radiofrequency emissions. It is this emitted energy
that provides the key information for MRI scans.

There are two main kinds of information about tissue characteristics derived from this perturbation decay, often referred to as a
“relaxation.” The T1 relaxation time is the
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time constant describing the time course followed by the “tilted” magnetization vector in returning to its original orientation. The
T1 relaxation time is the time required for this vector to return to 63% of its original vertical orientation value following an rf
excitation pulse. Again, in our analogy to spinning tops subjected to a tilt, this T1 is the time required to return to about two-thirds
of its original vertical orientation.

The second measure, T2 relaxation time, requires us to think about each nucleus (spinning top) individually. Again and again crudely,
one can visualize a group of spinning tops oriented upward (z-axis) and then simultaneously tipped away from this vertical
orientation. As everyone who has spun tops or played with a gyroscope knows, if one tilts a top from a vertical orientation, the top
will not only tilt but will rotate about the vertical (z-axis), a wobble technically called precessing. Figure 55.1 illustrates this
process. In the beginning all the individual tops will wobble (precess) about the z-axis with the same frequency, but gradually they
will lose their coherence and wobble at different frequencies, leading to progressively less net magnetization in the x-y plane. T2 is
the time required for the coherence to decrease to 37% of its original value. This “dephasing time” or T2 relaxation is always less
than or equal to T1. Often the term T2* is used to take into account the observed variations in relaxation time owing to
inhomogeneities in the tissue being imaged and in the applied magnetic field. The web site, http://ej.rsna.org/ej3/0095-
98.fin/index.htm has a nice animated illustration of T2 relaxation (on the menu page, select Diffusion and Magnetic Resonance). Of
relevance to this description, Pfefferbaum and colleagues (3 ) found T2 relaxation times were longer in schizophrenic patients than
in controls in both gray and white matter, suggesting possible differences in fundamental tissue organization in schizophrenia.

FIGURE 55.1. Illustration of effects of applied magnetic field on hydrogen nuclei (protons, images 1 to 4). With only a static
magnetic field (left arrow) present, all nuclei have the same vertically aligned spin directions parallel to the static magnetic
field and the z axis (this state is not illustrated). Application of an rf pulse “tilts” the orientation so there is a transverse plane
component (broken line) in Image 1. Initially all protons precess uniformly, so that images 1, 2, 3, and 4 can be thought of as
successive snapshots (successive moments in time) of the counterclockwise precession (rotation) of the net magnetization
vector about the z-axis. Over time the protons dephase and show different precession frequencies; as an illustration of this
case, images 1 to 4 should be thought of as a single snapshot of individual protons at the same instant in time. There is no net
transverse plane magnetic vector because the individual protons show no uniformity of phase.

In terms of T1, an rf pulse may “tilt” the net magnetization (spin) vector, but usually a second pulse is applied before there is a full
return to the vertical orientation, and subsequent rf pulse repetitions lead to a steady-state orientation prior to each new pulse.
This new vector depends on a number of values; two are of particular relevance: the T1 relaxation time (how efficiently the protons
give up their energy) and number of protons per unit of tissue, proton density.

Spatial Localization
The resonance frequency of protons, the frequency at which energy is maximally absorbed by protons, is dependent on the strength
of the magnetic field. By applying small magnetic field gradients (typically less than 1% of the total field strength) for short periods
of time it is possible to spatially localize the signals resulting from the applied rf pulses. In the presence of a magnetic gradient field
each proton will resonate at a unique frequency that depends on its exact position within the gradient field. The MR image is a
frequency and phase map of the protons at each point or picture element (pixel) throughout the image. The pixel intensity is
proportional to: the number of protons present in the volume represented by the pixel weighted by the T1 and T2 relaxation times.
Different sequences of rf pulses will produce images that mainly reflect one of these variables, and these images are often
referred to as proton density-, T1-, or T2-weighted images. Operationally, the initial step in spatial localization is localization of
the rf excitation to a region of space (slice) by the slice selection gradient. When images are viewed, the slice selection direction is
always perpendicular to the surface. A second spatial direction is determined by a phase encoding gradient, which differentially
alters the precessional frequency of protons at different positions in
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the phase encoding direction, thereby enabling spatial localization. In MRI the signal is always detected in the presence of a readout gradient, perpendicular to the slice selection and
phase encoding gradient, and producing the third dimension of the image. The readout gradient detects differences arising from both the slice selection gradient and the phase
encoding gradient, with the latter varying in amplitude with each repetition of the slice selection and readout gradient.

Pulse Sequences
Appendix A describes commonly used pulse sequences in terms of our knowledge about the relaxation processes for the reader wishing insight into the terminology and rationale of
pulse sequences.

STRUCTURAL MRI: WHAT CAN IT TELL US ABOUT SCHIZOPHRENIA?

Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

Structural MRI
In 1984 Smith and co-workers (4 ) performed the first MRI study of schizophrenia. The capability of structural MRI to provide information about gray and white matter parenchyma of
the brain and CSF-filled spaces is new with MRI studies; it represents an important advance over CT studies that poorly visualize parenchyma and can not differentiate gray and white
matter. This gray–white differentiation is important for schizophrenia studies, because abnormal tissue classes (tumors, infarcted areas, etc.), which may be detected by CT, have not
been found to characterize schizophrenia. The term “structural MRI” is used to differentiate it from “functional MRI,” where indices of short-duration change (e.g., blood oxygenation)
are used; this topic is treated in the second part of this chapter.

Our use of the term schizophrenia is in the sense of a syndrome and not a single disease entity. The current major questions about the schizophrenia syndrome include:

1. What are the brain changes in this disorder? Which areas of the brain are affected?
2. What is the cause of the brain changes?
3. At what life stage do brain abnormalities occur and are they static or progressive? Are they developmental (prenatal and perinatal) and/or progressive?
4. How are brain abnormalities related to clinical symptom abnormalities?
5. Are brain findings in schizophrenia distinct from those in affective psychosis?
6. What are the most effective treatments? Is treatment neuroprotective?
7. Are there structural endophenotypes that will help us in the genetic analysis of the disorder?

Structural MRI studies of schizophrenia have the potential of addressing all of these questions, although space restrictions confine our focus primarily on the first question, which has
also been the major focus of empirical studies.

WHAT ARE THE DESIRABLE FEATURES OF A STRUCTURAL MRI STUDY?

Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

We here briefly summarize the features.

1. Thinner is better. Smaller units of volume analysis (called voxels, for volume element) allow for more precise determination of the irregular contours of brain regions, by
reducing the voxel mixing of the desired region with neighboring structures in the voxel. This mixing is called partial voluming. Many earlier studies used MRI acquisitions
with “gaps” between slices, with interpolation used to estimate the volume in the “gap”; this obviously limits precision of measurement. Thus studies with thinner slices
(1.5 mm is the current standard), and no gaps between slices, will likely lead to more precise MR morphometric volume measures.
2. Quantitative versus qualitative analysis. Early studies relied on subjective, visual ratings of abnormalities. There is now general agreement that computation of volumes
of the ROI examined is essential. When raters are used, as is generally the case, inter-rater reliability is important, and should be r ≥ 0.85. Moreover, the ROI should be
objectively and clearly defined, so that others can measure the same entity. Such objectively defined criteria should include detailed specification of the internal
landmarks used to define each ROI.
3. Segmentation. Segmentation involves sorting the tissue classes into gray matter, white matter, or cerebrospinal fluid (CSF). It seems to us that all studies of cortical gyri
should, whenever possible, separate gray and white matter in the analysis, because this is a fundamental distinction in brain tissue; however, not all studies distinguish
between gray and white matter, making comparisons with studies that do segment gray and white matter problematic. Finally, segmentation is often automated or
semiautomated; unfortunately, there is no agreed-on gold standard for the quality of segmentation, because “phantoms” with known composition do not reflect the
complexity of the outlines of brain gray or white matter, and postmortem estimates of tissue and fluid volumes may not exactly parallel those in vivo. (See ref. 5 for
discussion.) Figure 55.2 provides an example of a segmented image with ROI tracing and three-dimensional (3D) reconstruction.

FIGURE 55.2. A: Coronal slice (1.5 mm) through the temporal lobe of a normal
control subject. This is a SPGR proton density-weighted image. The regions of
interest for the structures are outlined: the gray matter of Heschl’s gyrus (HG) is
red on subject left and green on subject right. The gray matter of planum
temporale (PT) is labeled yellow on subject left and blue on subject right. B: Top-
down view of the three-dimensional (3D) reconstruction of HG and PT placed atop
an axial magnetic resonance slice. This axial slice has been constructed by
reformatting the coronal images. Anterior is top. HG is red on subject left and
green on subject right, and PT is blue on subject left and yellow on subject right.
C: 3D reconstruction of the left and right ROI (color-coded as in B but from a
slightly different angle of rotation than B). Note the tubular structure of the gray
matter of the STG, most clearly seen anteriorly, where gray codes non-HG, non-PT
portions of STG. D: 3D reconstructions viewed from a different angle than C. (D1 is
subject right and D2 is subject left). (Reproduced from Hirayasu Y, McCarley RW,
Salisbury DF, et al. Planum temporale and Heschl’s gyrus volume reduction in
schizophrenia: an MRI study of first-episode patients. Arch Gen Psychiatry
2000;57:692–699.)

4. Quality of imager and postacquisition processing. The quality of the MR scanner is also important and should include technical assessments such as the homogeneity of
the magnetic field, which greatly influences the postprocessing segmentation of tissue into different tissue components. Day-to-day assessment of inhomogeneities in the
magnetic field is thus a critical quality assurance feature for the quality
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of the MR scans, and consequently also the quality of the postprocessing of MR images. Most modern imagers have
magnetic fields of 1.5 T or greater, which is important for signal-to-noise ratio. Additionally, postacquisition filtering may
improve signal-to-noise ratio (6 ).

5. Variability and reliability of MRI findings. An important question is the variability in MRI findings that is to be expected,
not only from variation in measurement techniques, but also from physiologic changes. This becomes increasingly
important because MR imaging is done at different time points in the disorder. Unfortunately, there are currently very few
studies that have examined the extent of changes in MR over multiple measurements, and more careful controlled studies
would be useful. Our laboratory found less than 1% variation over gray–white–CSF segmentation values in one subject who
had an MR scan on two different days (5 ). Kikinis and colleagues (7 ) presented data from a female subject who received
23 MR scans during 1 year. The variance of the intracranial cavity was only 1.2% over the course of the 23 MR scans. Also,
there is little evidence of the idea of a physiologic variation in gray or white matter volume throughout the brain in
studies with high-resolution MRI techniques. For example, Gur and associates (8 ) found changes in volume over 2.5 years
in whole frontal lobe in schizophrenia without finding changes in volumes of whole-brain and CSF, a finding difficult to
reconcile with the idea
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of “whole brain variability” in gray or white matter, or CSF caused by hydration or other factors affecting all brain tissue, for
example.

HOW SHOULD LITERATURE FINDINGS BE REPORTED IN REVIEWS?

EXPERT OPINION VERSUS COUNTING VERSUS METAANALYSIS
Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

The reader trying to shape an informed opinion on the current state of the field (not only structural MRI but any field) relies on
reviews with essentially three main approaches, which can be combined.

Expert Opinion
One approach for reviewers is to survey the literature, and then provide an informed opinion as to the summary trends and findings,
with specific citations to drive home the points. Many reviews, in fact, adopt this approach of “trust the reviewer.” The
disadvantage is that the reader cannot form a judgment of the accuracy of the review’s conclusions based on the data presented
without reading the literature.

Counting
This approach tabulates the number of studies with findings supporting or not supporting an abnormality in a particular region. The
disadvantage of this approach is that the subject N of each study and the effect size are not taken into account.

Metaanalysis
Metaanalysis essentially involves weighting each individual study by a function of its N and effect size, and then using this
information to produce an estimate of the combined effect size (9 ,10 and 11 ). If all studies used equivalent technology and
subject populations, this would be the method of choice. However, they do not, and the reader should realize that: (a) MR scanner
technology in the past decade has been changing rapidly; therefore, studies are not quantitatively comparable; (b) the extent of
detail varies in anatomically based ROI information used in the measurement of images; (c) there is a wide difference in moderator
variables of subject gender, chronicity (age of onset), medication, parental SES, etc.; and (d) metaanalysis, especially of MRI studies,
is beset with the difficulty of estimating the number of studies with negative findings that did not get published—Rosenthal’s “file
drawer” problem (11 ). Practically, this means one cannot do a review that is both comprehensive and metaanalytically valid, unless
the items (a) through (c) had remained constant. Another disadvantage is that many studies must be rejected for reasons of subject
or methodologic consistency; this omission has a potential effect of distorting results by omitting technically good studies that could
not be included; for example, the metaanalytic study of Nelson and colleagues (12 ) omitted 45% of the studies on medial temporal
lobe found in the literature.

Our summary relies on a combination of these approaches. The conclusions are congruent with our subjective opinions and we
provide tabulation of more than a decade’s results of MRI studies; however, we are sympathetic to the need to provide more than a
simple “box score” of positive and negative results. Accordingly, we have followed a suggestion of Rosenthal (11 ), and computed
the probability of the observed number of positive and negative findings for each region. This is simply done by using a two-tailed
alpha level of p < .05 for each study finding positive results, and then using the binomial theorem to calculate the overall probability
of finding the observed number of positive studies. The resulting overall probability does not assume a normal distribution, but does
assume comparability of the studies. (Caution: This assumption does not necessarily hold.) Whatever the degree of comparability,
this statistic does have the distinct advantage of providing the reader with a sense of the weight of current evidence for each region
of interest beyond that of a simple “majority vote,” which neglects the odds against an individual study’s finding positive results at
the p < .05 level. We see this as an improvement on simply saying “some studies find… but others…” and providing the reader a
backdrop of estimating the weight of evidence from peer-reviewed studies based on a probability of .05, assuming the prerequisites
of metaanalysis hold, and then allowing a subjective “dilution factor” for all of the problems of a metaanalytic analysis.

CURRENT STRUCTURAL FINDINGS IN SCHIZOPHRENIA

Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

A complete review is not appropriate here because the explicit focus of this volume is on summary of main trends and new
developments. However, because it seemed essential to us that the reader have at least an overview of structural results in
schizophrenia (with references), we have drawn on data from a recent comprehensive review (13 ). This covered all peer-reviewed
schizophrenia studies with control groups during the time period 1987 to May 1998, and to our knowledge is the most recently
published comprehensive review.

We summarize here the results from this review, whereas Table 55.1 presents the results and references in tabular form. (Table 2 in
the original article summarized the subject N and characteristics, and should be consulted for further detail [13 ].)
 P.762

TABLE 55.1. SUMMARY OF MRI STUDIES REPORTING POSITIVE AND NEGATIVE

FINDINGS IN SCHIZOPHRENIA FROM 1987 TO MAY 1998 (FROM McCARLEY ET AL.,
1999)

Most studies (81% of 31) did not find abnormalities

P.763
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of whole brain/intracranial contents, but lateral ventricle enlargement was reported in 77% of 43 studies, third ventricle
enlargement in 67% of 24 studies, whereas none of the three studies evaluating the fourth ventricle found abnormalities.

The temporal lobe was the brain parenchymal region with the most consistently documented abnormalities, with 62% of 37 studies
finding whole lobe volume decreases. Of all cortical areas surveyed, the superior temporal gyrus most consistently showed volume
reduction (81% of 16 studies) and, if the gray matter of this structure was evaluated separately from white matter, all seven studies
showed a volume reduction. Fully 77% of the 30 studies of the medial temporal lobe reported abnormalities in one or more of its
constituent structures (hippocampus, amygdala, or parahippocampal gyrus). Neuropathologic studies in general support the presence
of temporal lobe limbic system abnormalities in schizophrenia (14 ,15 ), although some do not (16 ). Unfortunately, there is a lack of
quantitative postmortem studies of temporal lobe neocortex.

Despite the presence of functional abnormalities, frontal lobe structural MRI investigations did not consistently find abnormalities,
with 55% of the 33 studies describing volume reduction. In a postmortem quantitative study, Selemon and associates (17 ) found only
a small (8%) reduction in prefrontal cortical thickness, a reduction that was not statistically significant, although noteworthy
abnormalities in density of various cell types were present in schizophrenia. This and the MRI findings suggest that frontal lobe
volume reductions may be small, and near the threshold for MRI detection. The parietal and occipital lobes have been much less
studied, and there are about the same percentage of positive and negative findings in each. Most of the seven studies of cortical
gray matter (86%) find that volume reductions are not diffuse, but are more pronounced in certain areas, as might be anticipated
from the preceding statistics on individual regions of interest.

About two-thirds of the studies of subcortical structures report positive findings, including the six studies of the thalamus, 18 studies
of the corpus callosum, and 17 studies of the basal ganglia. Basal ganglia tended to show increased volume when patients were on
typical but not on atypical neuroleptics. The cerebellum had mainly negative findings (in four of the six studies), but was not
studied with the same volumetric precision as other ROI. Almost all (91%) of the 11 studies of cavum septi pellucidi (CSP) showed
that schizophrenics have less fusion of the septum, a developmental abnormality probably linked to limbic system pathology.

Statistics
The binomial theorem computation (using p < .05 for a positive study) shows that all ROI surveyed in Table 55.1 show a two-tailed p
< .05 for the number of positive studies, except for the fourth ventricle and cerebellum (p = .66), and all ROI had p’s ≤ .002 except
for the occipital lobe (.004). Again, caution should be exercised on the strength of these probability estimates because
comparability is not strict and an (unknown) percentage of studies with negative results may not have been published.

Clinical Symptom Implications

Clinical scale data suggest that positive symptoms (and disorganization in a three-factor model) may be most closely related to
temporal lobe volumetric abnormalities, whereas more limited evidence supports, in some studies, a relationship between negative
symptoms and frontal lobe MRI abnormalities. Finally, there now appears to be growing evidence that MRI abnormalities differ in
affective (bipolar) psychosis and schizophrenia, with reductions in neocortical gray matter, especially in temporal and prefrontal
neocortex being especially prominent in schizophrenia. (See McCarley and co-workers 1999 for a more complete review and Sheline
[this volume] for a review of structural MRI in affective disorder [13 ].)

The presence of CSP and sulco-gyral abnormalities (for the latter, see Kikinis and colleagues ) (18 ) and abnormalities in first-
episode patients all suggest a possible developmental origin. However, there are growing (although still limited) data pointing to
progression of volumetric abnormalities over time. This suggests that both developmental and progressive features may be present
in schizophrenia; these are consistent with, we hypothesize, a “two-hit” model of schizophrenia.

AUTOMATING STRUCTURAL MRI ANALYSIS: BRAIN WARPING AND

VOXEL-BASED ANALYSIS
Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

The tedious task of manual definition of regions of interest by tracing outlines—even if assisted by automated segmentation—has
prompted interest in using automated methods of MRI analysis. The two (closely related) major classes of methods are: (a) brain
warping, using a standard or “atlas brain” to compare and define features on subject brains, and (b) voxel-based analyses. Because
these techniques, although promising, are new and thus far have limited data on validity, we have not included the studies in the
summary table of ROI findings. This section concludes with a brief discussion of shape analysis, often based on the brain warping
techniques described in the first part of this section.

Brain Warping
In one use of this technique the ROI definitions and anatomic features of an index template (atlas brain image) are warped (mapped)
onto a new case (target image). In this
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context, the atlas brain can be compared to a rubber brain, which is stretched and compressed nonlinearly in order to match the
contours of the new brain. At the end of the registration, all the structures previously defined for the atlas brain are also defined for
the new brain image. In general, the first step in matching the atlas brain and subject (patient or object brain) is linear registration
to correct for the differences in size, rotation, and translation between the two brain images. This step is illustrated in the first
panel of Fig. 55.3 . (Parenthetically, linear transforms use scaling, translation and rotation uniformly for each element [voxel],
whereas nonlinear transforms use different and more complicated transforms for different voxels.)

FIGURE 55.3. Schematic of brain image warping. An “atlas” image (red hexagon) is warped (mapped) onto a new “patient”
image (yellow oval). Top: A simple uniform linear transformation (translation, rotation or scaling) does not work. Instead,
nonlinear transformations are used to “warp” the “atlas” image onto the “patient” image (middle and bottom panels). This
warping resulted in a “vector field” (blue arrows). The nonuniform displacement of each pixel is then represented in the right
field of Fig. 55.1, by means of the deformation of the rectangular grid (the elastic membrane). Examples are presented for
atlas contraction (middle panel) and dilatation (bottom panel).

Nonlinear Elastic Matching

The atlas information can then be projected into other MRI scans by applying an elastic match (i.e., warping the atlas into the shape
of the new brain image). The global registration technique used by our lab (19 ) to match an anatomic MR atlas with defined ROI
onto new-segmented MR images, was based on the theory of elastic membranes (20 ,21 ), and was similar to Grenander and Miller’s
(22 ) approach. The elastic membrane model can be intuitively understood as the deformations occurring when a set of points on the
membrane is stretched. The goal of the elastic matching algorithm was to find a 3D vector deformation field that transformed the
source data set (atlas) so that it matched the target data set (patient) with the greatest fidelity, that is, maximized the local
similarity between the two data sets under the constraint of using a restricted set of “stretches” of the membrane. The middle and
bottom panels of Fig. 55.3 show, in a cartoon, how contraction and expansion of particular voxels lead to a match between the atlas
brain and the target brain. The right-hand part of these panels graphically illustrate the voxel-by-voxel deformation (stretching or
compression) resulting from the vector deformation field used to transform the atlas image. We note that other choices of an
“index brain” include the probabilistic atlas used by the Montreal Group (23 ) and interpolated data from an anatomic atlas, as used
by Gee and colleagues (24 ).

Brain Warping: How Good Is It?

Any new technique must be validated, and brain warping is no exception. The current gold standard is manual ROI definition. The
technique used by Iosifescu and co-workers (19 and unpublished data) was compared with manual ROI definition for volumes.
Agreement on volumes over 28 subjects (half schizophrenia patients and half controls) was 97% for whole brain volume, 97% for
whole white matter, 91% for whole gray matter, 96% for thalamus (both sides), 93% for putamen, 91% for caudate nucleus, and 76%
for globus pallidus. A more rigorous and better method of comparison is the extent of overlap of voxels in the manually defined ROI
with those in the automated definitions. In measurements of 20 brain cortical and subcortical brain structures on one brain image
the extent of voxel-by-voxel correspondence, was defined as:
 P.766

This averaged 90% for subcortical structures and 98% for total gray and white matter volumes; however, for cortical gyri the overlap
averaged only 60%. The automated computer algorithm assumed the neuroanatomic variability among subjects to be a topologic
invariant. However, cerebral gyri frequently split in two in some subjects, whereas they remain one single structure in others. These
differences could not be taken into account by the automated registration in its present form. Taken together, these data suggest:

1. Each automated warping procedure should be compared with the results using manual ROI definitions.
2. Accuracy may be good for subcortical structures (because of their relatively small variability in shape) and total brain
gray and white matter.
3. Accuracy is questionable for the neocortex, because of the irregularity of sulco-gyral patterns.

A recent use of the technique of warping is to use the vector deformation field to provide a statistical test of whether each voxel is
significantly displaced or not (25 ). (See Fig. 55.3 for a description of extent and direction of warp.) This methodology does not
attempt to map gyrally defined ROI, but rather looks at changes in gray matter on a global or regional basis, often using Talairach
space.

Gaser and colleagues (26 ) compared the 3D vector deformation fields required to warp each voxel of an index brain (source not
specified, presumably that of the Talairach atlas) onto spatially normalized brains of a large group of schizophrenic patients (n = 85)
and controls (n = 75). They then computed the statistical significance of the difference between the schizophrenic patients’ and
controls’ deformation fields, finding volume reduction bilaterally in Talairach spatial locations corresponding to thalamus and
superior temporal gyrus and unilateral reductions in the superior and middle frontal gyrus, precentral gyrus, lingual gyrus, and
cerebellum. This study forms a good transition to the next section because the spatial normalization techniques and the nonlinear
registration method, those of SPM99 and Ashburner and Friston (27 ), respectively, are described in the following section.

Voxel-Based Morphometry
Ashburner and Friston (27 ) define this technique as “a voxel-wise comparison of the local concentration of gray matter between
two groups of subjects,” and have provided a detailed description of this methodology, closely related to that of SPM99. As a first
step, this method takes all subject images and normalizes them to the same stereotaxic space, using procedures similar to those
used in SPM for fMRI and PET data. This procedure involves an initial linear (affine) match (similar to that described for brain
warping) followed by a nonlinear registration using smooth spatial basis functions. These authors emphasize that this spatial
normalization “does not attempt to match every cortical feature exactly, but merely corrects for global brain shape differences,”
thereby differentiating it from more exact attempts at a match, as discussed in the brain warping section. The second step involves
segmentation of the normalized images into gray matter, white matter, and CSF. The third step is smoothing using a convolution
with a Gaussian kernel, which leads to each voxel being the mean of gray matter density for it and, to a spatially progressively
lesser degree, its neighbors. The last step is statistical analysis using the general linear model to identify regions of gray matter
concentration that are significantly related to the variable under study (if normality is not present a nonparametric statistical
analysis is used).

Compared with manually drawn ROI, this technique has the following clear advantages: (a) enabling of regional comparisons
throughout the whole brain without the restrictions of a few selected areas used in the typical manually drawn region of interest
methodology; (b) the reduction of labor; and (c) the ability to use large samples with an attendant increase in statistical power as a
corollary to (b).

Unfortunately, however, there as been an absence of work comparing the spatial specificity and sensitivity of voxel-based analysis
with manual ROI analysis, the current standard, and thus the question of validity has been incompletely addressed.

Wright and co-workers (28 ) have undertaken some comparisons with manual ROI. They performed manual area measurements of the
head of the caudate in the transverse slice 12 mm superior to the intercommissural plane in the untransformed data. They then
compared these with voxel values in the transformed data at coordinates corresponding to the center of the caudate in Talairach
space for each of 20 the subjects. They found Pearson product–moment correlations between the area measurement and the voxel
gray matter values for the transformed data for the 20 subjects to be about r = 0.8. These data do not, unfortunately, provide
information on spatial specificity in terms of a measurement of the boundaries of the caudate in the untransformed data for the 20
subjects and the transformed data. Nor do these data, taken from the center point of a regular structure, provide any clear
information on how well the transformation would work on the much more irregular cerebral cortex. Because one of the findings
with transformed data was decreased gray matter in the schizophrenic group in the voxels corresponding to the right amygdala, one
would have liked to see a comparison with manually drawn ROI in this structure as a way of validating the voxel analysis (and/or a
comparison in the other regions found to be abnormal, the temporal pole/insula, and left dorsolateral prefrontal cortex). Wright
and associates did find that voxel analysis could detect artificial “lesions,” created by setting gray matter content to zero in a group
of voxels, including a 4- × 4-mm bar and a 12- × 25-mm grid. They did not try more realistic “lesions” with parametric variation of
degrees of lesser gray matter content; nor did they quantify
 P.767

the spatial specificity. In concluding the discussion of this technique, Wright and colleagues voiced the important caveats that voxel
based morphometry may not detect “very small gray matter reductions, gray matter reduction in areas of high variability in gray
matter volume or gray matter reductions with an inconsistent location.”

A direct comparison of manual ROI and voxel-based analysis would seem to be a high priority, because some estimate of the
specificity and sensitivity of voxel analysis for various brain regions and ROI could be formed. Until such validation procedures are
done, any results with voxel-based morphometry (VBM) will, of necessity, be viewed by many workers in the field as tentative.
Because of the importance of the validity question, our laboratory has recently begun to compare SPM99 VBM results with traditional
ROI analysis (Kubicki and colleagues, unpublished data). For VBM applied to whole brain, only the left posterior superior temporal
gyrus region was significantly different between schizophrenic and control groups, a finding consistent with our ROI analysis. In a
less statistically less stringent analysis (taking into account peak z values and voxel cluster extent), there was significance bilaterally
in the anterior cingulate gyri and insula (regions not examined with ROI), but not in medial temporal lobe where ROI analysis showed
differences.

Taken together, these data suggest the following methodologic conclusions:

1. Each VBM study should be compared with manual ROI definitions until validity is established.
2. VBM may be useful for generating hypotheses to be validated with traditional ROI analyses.
3. Much work remains to be done in comparing the validity of VBM and ROI analysis, and formulating reasons for any
differences.

Shape Analysis
It is readily apparent that ROI shape as well as volume may carry information about pathology. Casanova and colleagues (29 ) used
3D Fourier techniques to characterize shape of temporal lobe regions, finding schizophrenics and controls differed. However, Fourier
techniques cannot pinpoint where in the shape the abnormality occurs, as can brain warping and other methodologies. Csernansky
and associates (30 ), using a variant (based on Grenander’s work) of the brain warping techniques described in the preceding, found
that maximal differences between controls and the schizophrenia subjects were localized to the lateral aspect of the head of the
hippocampus and medial aspect of the body, where the subiculum is found. The study of shape using a number of different
algorithms is a current area of very active interest in MR schizophrenia research, especially in the study of the corpus callosum.

DIFFUSION TENSOR MR IMAGING

Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

This is a new MRI technology that is able to provide information on the orientation and integrity of fiber tracts. In diffusion tensor
imaging (DTI), a tensor describing local water diffusion is acquired for each voxel; crudely, this tensor can be thought of as a
mathematical description of the direction and velocity component of diffusion relative to the orientation of the chosen coordinate
system, or “basis.” Diffusion may be “isotropic,” equal in all directions, as occurs in CSF, and the diffusion volume (3D
representation of diffusion pathways) has a spherical geometry in this case. Or diffusion may be “anisotropic” (e.g., not isotropic)
and greater in one direction, in which case there is an ellipsoid shape. The limiting case for maximal anisotropy is an infinitely long
and thin cylinder. In white matter fiber tracts diffusion is mainly in the direction of the fibers. Factors that affect the shape of the
apparent diffusion tensor (shape of the diffusion ellipsoid) in the white matter include the density of fibers, degree of myelination,
average fiber diameter, and directional similarity of the fibers in the voxel. For example, the DTI-measured diffusion coefficients
are larger when measured along (parallel to) white matter fibers (in the range of 1.0 × 10-3 mm2/sec) than across the fibers (in the
range of 0.6 × 10-3 mm2/sec). The geometric nature of the measured diffusion tensor within a voxel is thus a meaningful measure of
fiber tract organization.

The degree of anisotropy in schizophrenia has been investigated in two recent studies. Using DTI, Buchsbaum and associates (31 )
reported evidence of lower diffusion anisotropy in some inferior portions of prefrontal white matter in patients with schizophrenia
than in controls. Lim and co-workers (32 ) found that abnormally low white matter anisotropy in patients with schizophrenia was
present in both hemispheres and was widespread, extending from frontal to occipital brain regions. For group statistics, Lim and co-
workers used the median value of voxel anisotropy (measured as fractional anisotropy; 1 is maximal and 0 minimal) in each slice
within the white matter regions of interest in the control and schizophrenia groups. These studies raised the important question of
whether white matter connectivity is disturbed in schizophrenia, although Lim and colleagues caution that the proper statistical
measures for DTI are still being worked out.

A recent technical advance in DTI has been line scan diffusion imaging (LSDI). This method, in contrast to the commonly used
diffusion-sensitized, ultrafast, echo-planar imaging (EPI) technique, is less sensitive to gross motion and cardiovascular pulsations.
LSDI also has higher resolution, exhibits minimal image distortion, and does not require cardiac gating, head restraints, or post-
processing image correction. It also can be implemented without specialized hardware on all standard MRI scanners.

Recent work has focused on measurements extending beyond the scalar measurement of the degree of anisotropy
 P.768

in a voxel to characterizing the spatial trajectory and orientation of fiber tracts. Although the individual axons and the surrounding
myelin sheaths cannot be revealed with the limited spatial resolution of in vivo imaging, distinct bands of white matter fibers with
parallel orientation may be distinguished from others running in different directions if MRI techniques are sensitized to water
diffusion and the preferred direction of diffusion is determined. Figure 55.4 shows the degree to which orientation of fiber tracts
can be quantified and displayed using color-coding. An important point for summarizing data made by Westin and associates (33 ) is
that remaining within the tensor domain when processing is useful, as contrasted with operating on scalars and vectors to produce
summary statistics. In processing DTI images, it is important to note that averaging of a diffusion tensor field and then deriving a
scalar measure from the averaged field is not the same as averaging a scalar field derived from the original field. By using
geometrically defined diffusion measures on locally averaged tensors local directionality consistency can be determined (e.g.,
existence of larger fiber tracts). This averaging approach can be used to derive a tensor field that will describe macrostructural
features in the tensor diffusion data. For example, a measure of linearity derived from the averaged tensor field can be used for
quantitative evaluation of fiber tract organization.

FIGURE 55.4. A: Sagittal schematic of brain fiber tracks. The vertical line shows the approximate plane of the coronal
diffusion tensor image to the right. (Adapted from Gray H, Bannister LH, eds. Gray’s anatomy: the anatomical basis of
medicine and surgery, thirty-eighth ed. London: Churchill Livingstone, 1995.) B: In this diffusion tensor imaging (DTI) image,
white matter tracts that are within the coronal plane are color-coded blue. Note the corpus callosum (top blue arrow) and
anterior commissure (bottom blue arrow). White matter tracts perpendicular to the plane are coded red-orange. Note the
cingulum bundle (top arrows), the white matter tract within the cingulate gyrus, and the uncinate fasiculus (bottom arrows),
the tract connecting anterior temporal lobe with inferior frontal lobe. (Unpublished image from our laboratory, Shenton et al.,
2000; technique and application discussed in Kubicki M, Maier SE, McCarley RW, et al. Uncinate fasciculus in schizophrenia: a
diffusion tensor study. American Psychiatric Association New Research Abstracts, 2000.) C: Parasagittal image showing
anterior–posterior course of the cingulate bundle as constructed from DTI. (Unpublished image courtesy of Stephan Maier and
Carl-Fredrik Westin, Surgical Planning Laboratory, Brigham and Women’s Hospital).

Still another promising application of DTI is tracking white matter tracts. The operation begins with a seed point in a voxel element
and then generates a tracking sequence
 P.769

if the adjacent elements have similar linear orientation. This similarity is at the voxel level, and does not, of course, permit tracking
of individual fibers; rather, it tracks groups of fibers.

CONCLUSION
Part of "55 - Structural Magnetic Resonance Imaging Studies in Schizophrenia "

A clear current and positive trend is to use as much automation as possible in structural MRI analysis because of the labor involved in
traditional ROI analysis. Currently, however, the field is still in a state of flux with respect to the validity of the new techniques,
such as VBM and brain warping, because of the absence of detailed comparisons with ROI analysis and formulation of the reasons for
differences. Validity evaluation for new technologies is thus a high priority item.

Another clear and positive trend is employ new technologies, and to use multimodal imaging, with diffusion tensor imaging as the
prime example within the structural field. Similarly, as discussed in another chapter in this volume by Dr. Berman, “functional”
imaging is becoming increasingly multimodal and a desideratum is the combination of structural and functional approaches, just as
anatomy and physiology are inextricably linked in basic neuroscience studies. The reader will likely notice a certain “mismatch” in
the brain regions emphasized in the functional imaging chapter (frontal lobe) and in this structural imaging chapter (temporal lobe).
It is clear that functional studies have defined more prominent abnormalities in frontal lobe than in temporal lobe, whereas
structural studies have tended to show a greater degree of abnormality in temporal lobe. The mismatch may arise, in part, because
the frontal lobe receives input from and communicates with virtually all cortical (and many subcortical) areas. Functional
neuroimaging “activation” in a region primarily represents postsynaptic potentials; these and not action potentials constitute the
major metabolic and energetic load and hence the main signals used in functional analysis. It is consequently often very difficult to
disambiguate abnormalities in input to frontal lobe from intrinsic abnormalities. Similarly, although temporal lobe gray matter
volume changes appear quantitatively larger than those in frontal cortex, no brain region acts on its own and interconnections and
abnormalities of interconnections, as well as intrinsic volume changes must be considered in the explanation of the features of
schizophrenia. This task of seamlessly integrating information from multiple technologies is both one of the most exciting and also
the most challenging for work in the next few years.

APPENDIX A: STRUCTURAL MRI PULSE SEQUENCES

STRUCTURAL MRI PULSE SEQUENCES
Spin echo pulse sequences use at least two pulses. The first is an initial excitation pulse (tilting the magnetization vector 90 degrees
from the steady-state field orientation), followed by one or more refocusing pulses, and directed 180 degrees from the orientation
of the steady-state field. These refocusing pulses reintroduce phase coherence (again, the web site http://ej.rsna.org/ej3/0095-
98.fin/index.htm provides a useful animated illustration of this). The reformation of phase coherence induces another signal known
as a “spin echo,” which does not have the potential confounds of magnet and tissue inhomogeneity (they remain constant over
pulses), and thus this signal provides a better measure of T2. In spin echo pulse sequences the repetition time (TR) is the time
between excitation pulses, whereas the echo time (TE) is the time from the excitation pulse to the echo maximum.

Relatively short TR and TE standard single echo sequences produce T1-weighted images. Multiecho sequences produce proton
density weighted images at short TE (less than 30 ms) and T2-weighted images at long TE (more than 80 ms) when TR is long enough
to allow for nearly compete T1 relaxation (more than 2,000 ms for most tissues). Fast spin echo sequences are a variant of
multiecho sequences that maximize efficiency of data collection and shorten acquisition time. They are commonly used to produce
T2-weighted images. Inversion recovery pulse sequences are still another variation of the spin echo sequence, in which an additional
180-degree pulse is applied before the excitation pulse, thereby increasing T1 weighting (commonly used for improving contrast
between different tissues).

Gradient Echo Pulse Sequences

These sequences do not use 180-degree refocusing pulses. The most commonly used pulse sequence in volumetric work is a spoiled
gradient echo sequence, called spoiled GRASS (SPGR) in GE imagers and FLASH in Siemens imagers. This pulse sequence uses a
“spoiling scheme” to dephase the transverse (x-y plane) magnetization following signal detection, commonly using “spoiler” (also
called “crusher”) gradient pulses that have the same duration and magnitude as the first excitation pulse, but the opposite polarity.
This has as a consequence that, at the time of the next excitation, only the longitudinal direction (vertical direction in our analogy)
has any remaining coherence. If the first pulse has a low excitation angle (small “tilt” of the tops in our analogy) this allows shorter
repetition times to be used, speeding acquisition.

Signal Intensity of Tissue Elements and T1 and T2 Weighting

SPGR pulses lead to proton density-weighted images, because the small “tilt” and short TR diminishes any T1 or T2 effects. In a
proton density image produced by the SPGR sequence most commonly used in schizophrenia research, CSF appears dark, gray matter
is gray, and white matter
 P.770

has the most signal (is brightest). Spin-echo sequences can produce proton density, T2- or T1-weighted images. The normal CSF T1
relaxation time is 3,000 ms at 1.5 T, whereas that of fat is 200 to 250 ms; gray matter has a longer T1 relaxation time than white
matter and thus shows a brighter signal with sequences allowing longer T1 relaxation times. Because the ability to capture relatively
complete T1 relaxation depends on longer TRs, longer TRs thus give brighter CSF and gray matter brighter than white matter. The
tissue intensity in T2-weighted images depends on the TE in spin echo sequences. CSF has longer T2 values than other brain tissues
and shows up as a bright signal in T2-weighted acquisitions with the long TE values commonly used in schizophrenia volumetric
studies. In general, a long TR allows more time for T1 relaxation and produces more signal from tissues with long T1 values, whereas
a long TE allows more time for T2 relaxation and produces more signal from tissues with long T2 values.

ACKNOWLEDGMENTS
Supported in part by VA Medical Research Service, Department of Veterans Affairs Center for Clinical and Basic Neuroscience Studies
of Schizophrenia, NIMH 40977 and 52807 (RWM). Parts of the introduction are adapted from a previous review: McCarley RW, Wible C,
Frumin M, et al. MRI anatomy of schizophrenia. Biol Psychiatry 1999;45:1099–1119.

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56
Therapeutics of Schizophrenia

Seiya Miyamoto

Gary E. Duncan

Donald C. Goff

Jeffrey A. Lieberman

Seiya Miyamoto, Gary E. Duncan, and Jeffrey A. Lieberman: Departments of Psychiatry and Pharmacology, School of Medicine,
The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Donald C. Goff: Psychotic Disorders Program of the Massachusetts General Hospital, and Consolidated Department of Psychiatry,
Harvard Medical School, Boston, Massachusetts.

The introduction of the first antipsychotic medication in the early 1950s revolutionized mental health care strategies and led to the
era of deinstitutionalization, a period in which patients with schizophrenia and related psychotic disorders were released from state
hospitals in large numbers to be cared for in the community (1 ). Nonetheless, with the growing understanding that a significant
percentage of patients responds poorly to conventional antipsychotics, as well as the recognition of discouraging long-term
outcomes for schizophrenia, the need to develop new therapeutic agents that work rapidly, potently, broadly, and with fewer side
effects has become increasingly appreciated. The reintroduction of clozapine heralded the second generation of atypical
antipsychotic drugs and a new pharmacotherapy of schizophrenia. To date, the greater benefits of the atypical antipsychotic drugs
in many outcome domains have been demonstrated (2 ), and novel medications are replacing the conventional antipsychotics as
treatments of choice. The development of additional novel strategies to obtain potentially new antipsychotic compounds possessing
unique pharmacologic profiles with few side effects is being pursued based on specific hypotheses (3 ). This chapter provides a
review and critique of currently available pharmacologic and psychosocial treatments in schizophrenia, and focuses on
investigational treatments and potential strategies for future pharmacotherapy.

 HISTORY OF ANTIPSYCHOTIC DRUG DEVELOPMENT

 REVIEW AND CRITIQUE OF CURRENT SCHIZOPHRENIA PHARMACOTHERAPY
 EXPERIMENTAL TREATMENTS AND STRATEGIES
 CONCLUSION
 ACKNOWLEDGMENTS

HISTORY OF ANTIPSYCHOTIC DRUG DEVELOPMENT

Part of "56 - Therapeutics of Schizophrenia "

Since the discovery of the prototypical antipsychotic chlorpromazine in the early 1950s, a number of neuroleptics were developed
based on the hypothesis that schizophrenia reflected a disorder of hyperdopaminergic activity, with the dopamine D2 receptor most
strongly associated with antipsychotic response (4 ). In many patients with schizophrenia, the widely used conventional
antipsychotic drugs (e.g., chlorpromazine and haloperidol) are effective in the treatment of the positive symptoms of schizophrenia,
and also in preventing psychotic relapse (5 ); however, there are crucial limitations in the use of these agents. As many as 25% to
60% of patients treated with conventional antipsychotics remain symptomatic and are labeled either treatment-refractory, or
partially responsive (3 ). In addition, these drugs at best only modestly improve negative symptoms of the deficit syndrome and a
range of cognitive impairments, which may be fundamental to the disease (6 ). Further, conventional antipsychotics cause a variety
of side effects both acutely (e.g., extrapyramidal side effects [EPS]) and with long-term exposure (e.g., tardive dyskinesia [TD])
(7 ,8 ). Such adverse effects may reduce compliance and represent a major drawback of these drugs.

For a number of years, there was a widely held view that any compound that was an effective antipsychotic agent must also induce
EPS. The availability of clozapine and other newer atypical antipsychotic agents, however, have disproved this notion. The
development of atypical antipsychotic drugs was aimed at increasing the ratio between doses that produce therapeutic effects and
those that produce side effects, as well as improving efficacy (e.g., against a broader spectrum of psychopathologic symptoms and
the treatment-resistant aspects of the disorder) (1 ). Although there is currently no uniform definition of the term “atypical,” in its
broadest sense it is used to refer to drugs that have at least equal antipsychotic efficacy compared to conventional drugs, without
producing EPS or prolactin elevation (1 ). A more restrictive definition would require that atypical drugs also have superior
antipsychotic efficacy (i.e., they are effective in treatment resistant schizophrenic patients, and against negative symptoms and/or
neurocognitive deficits).

Although agents like thioridazine were first suggested to

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have atypical characteristics, it now is generally accepted that clozapine, first synthesized in 1958, is the prototypical “atypical”
antipsychotic (9 ). Clozapine underwent extensive clinical testing in the 1970s, but its development was halted in the United States,
and limited in other countries, because of a relatively high incidence of a potential fatal side effect, agranulocytosis. Nevertheless,
its superior outcomes ultimately led to further development and eventual reintroduction beginning in 1990 (10 ). The renaissance of
clozapine was based on several advantages: It appears to be more effective than typical neuroleptic drugs (e.g., chlorpromazine and
haloperidol) in treatment refractory schizophrenia (11 ); it can ameliorate some of the negative as well as positive symptoms of
schizophrenia (12 ); it can reduce relapse; it may improve certain cognitive functions; it may alleviate mood symptoms associated
with schizophrenia and reduce the likelihood of suicidal behavior; it has very low liability for EPS and TD; and it does not induce
sustained hyperprolactinemia (10 ). The reintroduction of clozapine represented a breakthrough in the treatment of schizophrenia.
In recent years, concerted research and development efforts have been made to produce a second generation of “atypical”
antipsychotic drugs, including risperidone, olanzapine, quetiapine, and ziprasidone, with the therapeutic advantages of clozapine,
without the properties contributing to its serious side effects (13 ). Ongoing clinical evaluation of the new “atypical” antipsychotic
drugs will eventually allow comprehensive assessment of their efficacy and safety.

REVIEW AND CRITIQUE OF CURRENT SCHIZOPHRENIA

PHARMACOTHERAPY
Part of "56 - Therapeutics of Schizophrenia "

Conventional Antipsychotic Drugs

Pharmacology
Conventional or typical antipsychotic drugs can be classified as high, intermediate, or low potency based on their affinity for
dopamine D2 receptors and the average therapeutic dose, compared with a 100-mg dose of chlorpromazine (14 ). Haloperidol, the
prototypical high-potency typical antipsychotic, has relatively high affinity for D2 receptors and a dose of 2 to 4 mg of haloperidol is
equivalent to approximately 100 mg of chlorpromazine. Low-potency drugs (e.g., thioridazine) have a chlorpromazine equivalent
dose of more than 40 mg. There is a good correlation between antipsychotic potency and D2 affinity for conventional antipsychotics
of several chemical classes (4 ). Conventional drugs have various interactions with serotonin receptors, ranging from slight (e.g.,
haloperidol) to moderate (e.g., chlorpromazine).

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies have further elucidated the
importance of dopamine receptor occupancy as a predictor of antipsychotic response and adverse effects. Prospective studies have
demonstrated that antipsychotic effects require a striatal D2 receptor occupancy of 65% to 70% (15 ,16 ,17 and 18 ), and D2
occupancy greater than 80% significantly increases the risk of EPS (15 ). Thus, a threshold between 65% and 80% D2 occupancy
appears to represent the optimal therapeutic range to minimize the risk of EPS for typical antipsychotic drugs (18 ,19 and 20 ). It
should be noted, however, that despite adequate D2 occupancy, many patients do not respond to medication (17 ). Moreover, results
of studies with atypical drugs such as olanzapine indicate that receptor occupancy levels above 80% are not invariably associated
with the occurrence of EPS, thus casting some doubt over the generalizability of the D2 occupancy model with regard to atypical
antipsychotics (21 ).

In preclinical studies, acute treatment with conventional antipsychotics (e.g., haloperidol and fluphenazine) increases the
expression of c-fos mRNA or Fos protein in the dorsolateral striatum, as well as the shell of nucleus accumbens in rats (22 ,23 ,24
and 25 ). Neuroleptic-induced expression of Fos in the nucleus accumbens has been postulated to relate to the antipsychotic activity
of both conventional and atypical drugs (26 ,27 ). The Fos expression in the dorsolateral striatum, which is not induced by clozapine,
has been proposed to be predictive of a liability to induce EPS (23 ,27 ). More recently, it has been reported that haloperidol, but
not clozapine, increased the immediate-early gene, arc (activity-regulated cytoskeleton-associated gene) mRNA levels in the rat
striatum (28 ). After chronic treatment, haloperidol also induces an increase in D2 receptor density and D2L receptor mRNA in the
striatum (29 ,30 and 31 ). Interestingly, several investigators have reported striatal enlargement after chronic treatment with
conventional antipsychotics, but not atypical drugs, in both schizophrenic patients (32 ,33 ) and rats (34 ). Thus, available data
suggest that conventional antipsychotic drugs may induce long-term plastic changes that lead to morphologic alterations in the
striatum, and that the efficacy and side-effect profile of typical antipsychotics relate to antagonistic actions at D2 dopamine
receptors.

Efficacy
Although typical neuroleptics vary in side-effect profile and hence tolerability, there is little evidence for differences in efficacy
between these drugs (3 ). However, in rare cases, patients failing a trial of one class may respond to the other. Although
conventional neuroleptic drugs are effective for alleviating positive symptoms of schizophrenia, and preventing their recurrence in
many patients, they have serious limitations. Approximately 30% of patients with acutely exacerbated psychotic symptoms have
little or no response to conventional antipsychotics, and up to 50% of patients have only partial response to medication (5 ,7 ).
Negative symptoms, mood symptoms, and cognitive deficits are marginally responsive to conventional neuroleptics. In particular,
primary negative symptoms are very resistant to the
 P.777

typical drugs (7 ,35 ). The presence of negative symptoms and cognitive impairment often leads to poor social and vocational
function (36 ,37 ). Thus, in the absence of a clinical response at acute phase of the illness, clinicians often switch to a newer
atypical agent (38 ).

Safety
Most conventional antipsychotics are associated with a wide range and a variable degree of undesirable acute and long-term adverse
effects, including EPS; sedation; anticholinergic, autonomic, and cardiovascular effects; weight gain; sexual dysfunction;
hyperprolactinemia; and neuroleptic malignant syndrome, a condition that is potentially life threatening (7 ,39 ). Up to 70% of
patients given recommended therapeutic dosages of conventional antipsychotics develop acute EPS (40 ). The most troublesome
neurologic side effect, tardive dyskinesia (TD), can be irreversible, and incidence rates have been estimated at about 5% per year in
the nonelderly and as high as 30% per year in the elderly (41 ). Further, the anticholinergic drugs that are often used to reduce EPS,
can also produce serious side effects (e.g., dry mouth, constipation, delirium and memory deficits) (42 ). All these adverse effects
can contribute to treatment noncompliance, and hence increase rates of relapse and rehospitalization during the course of the
chronic illness (7 ,39 ).

Effectiveness
Treatment with typical antipsychotics may result in poorer clinical and quality of life outcomes than with atypical antipsychotics (6 ).
The mean first-year relapse rate during continuing maintenance treatment with conventional antipsychotics is approximately 26% in
schizophrenic patients with first or multiple episodes (43 ). Even under the best conditions, when patients are maintained on
therapeutic doses of depot conventional antipsychotics, approximately 30% of discharged patients with schizophrenia will be
rehospitalized within 1 year (44 ). Hospital readmission rates are higher for conventional antipsychotics than for atypical
antipsychotics (45 ). The monthly relapse rate of compliant patients taking optimal doses of a depot neuroleptic is estimated to be
3.5% per month, and the rate for patients who have discontinued their medication is 11.0% per month (44 ).

In terms of relapse prevention, higher doses of conventional antipsychotics may help stability, yet the patient's quality of life will be
reduced because of increased side effects. Often, when considering the best dose of a conventional antipsychotic, there is a trade-
off between maximizing relapse prevention and optimizing comfort (46 ). Although there has been substantial progress in
understanding maintenance dosing, for most patients with schizophrenia, this unfortunate trade-off is inevitable with conventional
antipsychotic treatment (46 ).

Atypical Antipsychotic Drugs

A series of atypical compounds has been developed since the introduction of clozapine. These include risperidone, olanzapine,
quetiapine, and ziprasidone, which were approved by the FDA in 2000, and aripiprazole and iloperidone, which are in late Phase III
development.

Pharmacology
The pharmacologic properties that confer the unique therapeutic properties of atypical antipsychotic drugs are poorly understood
despite intensive research efforts. Defining the role of the individual complex actions of clozapine responsible for its unique
therapeutic profile (Table 56.1 ) is necessary for the rational design of new and improved atypical (clozapine-like) antipsychotics
because this drug is the prototype atypical drug.

TABLE 56.1. AFFINITY OF

ANTIPSYCHOTIC DRUGS FOR
HUMAN NEUROTRANSMITTER
RECEPTORS (Ki, nM)a

A distinguishing feature of clozapine in comparison to conventional antipsychotics is the relatively high affinity of clozapine for the
5-HT2A receptor. Meltzer and associates (47 ) provided evidence that combined 5-HT2A/D2 antagonistic actions, with greater relative
potency at the 5-HT2A receptor, may be critical to atypicality, in terms of enhanced efficacy and reduced EPS liability. Based on this
theoretic model, risperidone was developed to mimic the relative 5-HT2A/D2 affinities of clozapine, although risperidone has
substantially higher affinity for both receptors than clozapine (Table 56.1 ). The reduced EPS side effects associated with low-dose
risperidone treatment (4 to 6 mg per day), even at high levels of D2 receptor occupancy, may be owing to the 5-HT2A antagonistic
properties of the drug (47 ,48 ). However, at higher doses, risperidone produces EPS, indicating that 5-HT2A receptor antagonism
alone cannot completely eliminate EPS associated with high D2 receptor blockade. The potential role of 5-HT2A receptor antagonism
in therapeutic responses to atypical antipsychotic drugs may become more apparent when data from clinical trials are available for
the selective 5-HT2A antagonist M-100907. However, the results to date support the hypothesis that some degree of D2 antagonism is
still required to achieve antipsychotic effects. Moreover, at this point it is unclear what clinical effects 5-HT2A antagonism confers, in
addition to mitigating the adverse effect of striatal D2 antagonism, and propensity to cause EPS (21 ).

Risperidone, like clozapine, has relatively high affinity for α1- and α2-adrenergic receptors (Table 56.1 ), but the potential
therapeutic significance of the adrenergic receptor blocking properties of clozapine and risperidone is uncertain. Addition of the α2-
antagonist idazoxan to the regime of patients treated with the typical neuroleptic fluphenazine resulted in improved treatment
responses in patients refractory
 P.778

to treatment with fluphenazine alone (49 ). However, there has been no subsequent confirmation of the effects of α2 antagonists as
adjuncts to typical neuroleptic treatment, and it has been suggested that α2 agonists may actually be useful for treating cognitive
deficits of the disease (50 ).

Olanzapine is a closely related in chemical structure to clozapine, and the two drugs have many common receptor binding
characteristics. Primary considerations in selection of olanzapine for development were the drug's relatively potent antagonistic
effects at both D2 and 5-HT2Areceptors (51 ,52 ). Olanzapine is more potent at 5-HT2A than D2 receptors (Table 56.1 ), similar to
clozapine and risperidone. In addition, receptor binding characteristics of olanzapine in regard to other dopaminergic, serotonergic,
cholinergic, and adrenergic receptor subtypes are similar to clozapine, but there are also some notable distinctions between the two
drugs. For example, clozapine has substantially higher affinity for 5-HT1A and 5-HT7 receptors in comparison to olanzapine (Table
56.1 ).

Quetiapine is another drug with greater relative affinity for 5-HT2A than for D2 receptors, but also some affinity for α1-adrenergic and
H1 receptors (53 ) (Table 56.1 ). Interestingly, quetiapine produces only transiently high striatal D2 occupancy in schizophrenic
patients, although the study has clinical and technical limitations (54 ). Ziprasidone has potent 5-HT2A and D2 affinities, and like
clozapine, it shows 5-HT1A agonist properties that could potentially act as protective effects on the development of EPS. Ziprasidone
also has significant affinity for 5-HT1D and 5-HT2C, as well as H1 and α1-adrenergic receptors (55 ) (Table 56.1 ). Iloperidone has in
addition to affinity for 5-HT2A and 1A and D2,3 receptors, also affinity for the α1- and α2c-adrenergic receptors. Aripiprazole is distinct
from the other atypical antipsychotic drugs because it is selective for the dopamine system and acts through partial agonism.

PET studies showing that therapeutic doses of risperidone and olanzapine produce greater than 70% occupancy of D2 receptors
suggest that D2 receptor antagonism could be a predominant mechanism of action of these atypical drugs (56 ,57 ). Clozapine,
however, does not exhibit high levels of D2 receptor occupancy at therapeutically effective dose (15 ,57 ,58 ), suggesting that D2
receptor antagonism alone cannot explain the greater therapeutic efficacy of clozapine (13 ). The low occupancy of striatal D2
receptors by clozapine could account for its low EPS liability (20 ,58 ,59 ).

Clozapine, risperidone, and olanzapine occupy more than 80% of 5-HT2A receptors in the therapeutic dose rage in humans (15 ,56 ,57
and 58 ,60 ). Although 5-HT2A receptor antagonism is likely to be associated with the low EPS liability of risperidone and olanzapine,
the role of this molecular action in the superior therapeutic responses to clozapine is unclear (13 ).

Efficacy
Although the proportion of patients who improve and the magnitude of therapeutic effects vary greatly, atypical antipsychotics
 P.779

are at least as effective for psychotic symptoms as conventional drugs (3 ). Well-controlled double-blind studies of atypical
antipsychotics suggest that clozapine, risperidone, and olanzapine may be superior to haloperidol for controlling psychotic symptoms
(61 ). At selected doses, risperidone appears to be more effective than haloperidol in treating positive and negative symptoms (53 ).
Olanzapine has been demonstrated to be effective for positive, negative, and depressive symptoms (62 ), and in some studies the
drug was superior to haloperidol and risperidone in terms of negative symptoms and long-term efficacy (63 ,64 ). However, in a
recent large double-blind study (that has only been preliminarily reported), risperidone demonstrated significantly greater efficacy
than olanzapine in reducing anxiety/depression and positive symptoms (65 ). Quetiapine appears to be comparable to
chlorpromazine and haloperidol in treating both positive and secondary negative symptoms (61 ). Similarly, ziprasidone appears to
be as effective as haloperidol in alleviating positive and negative signs in an acute treatment study (66 ), whereas a 52-week
placebo-controlled maintenance study found primary and secondary negative symptom efficacy for ziprasidone (67 ).

To date, clozapine is the only drug that has proven efficacy in treatment-refractory schizophrenia (68 ,69 ). The efficacy rates for
clozapine in treatment-refractory patients vary from 20% to more than 70% (11 ,70 ,71 ). In some studies, risperidone does not
appear to be as effective as clozapine in treatment-resistant schizophrenic patients (72 ,73 and 74 ); however, Bondolfi and
associates (75 ) found no difference between risperidone and clozapine in treatment-resistant patients. In this latter study, certain
methodologic issues may have led to an overestimation of the efficacy of both clozapine and risperidone, and there are questions as
to whether the patient population studied represented “truly resistant” patients (69 ). Further investigation is necessary to
adequately compare the relative efficacy of risperidone and clozapine in treatment-resistant patients. Olanzapine was found to be
more effective than haloperidol (74 ,76 ), but not chlorpromazine (77 ), in treatment-refractory patients. In a recent randomized
double-blind study of treatment-resistant schizophrenia, olanzapine and clozapine had similar antipsychotic efficacy (74 ).
Additional studies are needed to reach definitive conclusions regarding efficacy of the newer atypical antipsychotics in treatment-
resistant schizophrenia. Results of studies investigating the effects of atypical antipsychotics in treatment-resistant patients are
discussed elsewhere in this chapter.

The efficacy of atypical antipsychotics in treating primary negative symptoms has not been clearly demonstrated (61 ). Thus, the
choice of atypical drugs for patients with predominantly negative symptoms is less clear (8 ). In addition, the effects of atypical
antipsychotics on cognitive impairment have not yet been clearly proved. A metaanalysis of 15 studies (only three of which were
double-blind) of atypical antipsychotics and cognitive impairment in patients with schizophrenia suggests that they may improve
attention and executive function (37 ). Available results, however, are relatively inconsistent and modest in effect size.
Furthermore, there are statistical limitations and a lack of standard conventions in the studies of cognition (78 ). It appears that
there could be significant differences among the atypical drugs in terms of what types of cognition they improve.

Atypical antipsychotics have been associated with a reduction in the incidence of suicidality, which may be relevant to
antidepressant effects of these agents, at least in part (6 ). Clozapine, risperidone, and olanzapine, in particular, appear to have
beneficial effects on the depressive component of schizophrenia (6 ,65 ) (Table 56.2 ).

TABLE 56.2. CLINICAL AND SIDE-EFFECT PROFILE OF ATYPICAL ANTIPSYCHOTIC

DRUGS
Although atypical drugs have shown some instances of superior efficacy in comparison with conventional drugs, they are not
effective in all patients and against all symptom dimensions of psychotic disorders (Table 56.2 ). It is clear
 P.780

that the atypical drugs are unable to fully reverse already-established impairment in cognition, negative symptoms, and social
disability in many patients (79 ). Thus, the possible use of these agents in the prodromal period of schizophrenia, before the
emergence of psychosis, is an important issue to address in the next decade (79 ).

Safety
Although atypical antipsychotics were developed to improve on the shortcomings of conventional drugs it has already become
apparent that they also have significant limitations in terms of side effects in the relatively brief period that they have been in
general clinical use (3 ). As a class, and with some variation between the individual drugs (Table 56.2 ), they have a much more
favorable side-effect profile, particularly in terms of EPS and TD. They do, however, produce side effects, including sedation,
hypotension, dry mouth, constipation, sedation, and some types of sexual dysfunction (3 ). Neuroleptic malignant syndrome has also
been reported with atypical antipsychotics such as clozapine, risperidone, and olanzapine (80 ). Weight gain is the most worrisome
and potentially serious side effect that appears to be class wide, except perhaps for ziprasidone and drugs that have not yet been
approved for marketing by the FDA, including aripiprazole and iloperidone (81 ). In particular, weight gain and sedation are common
reasons for drug discontinuation for adolescent patients (78 ). In addition, the atypical antipsychotics have been associated with new
onset type II diabetes mellitus (82 ). It is unclear whether these effects are secondary to weight gain, independent, or causative.
Atypical drugs are also associated with increases in cholesterol and lipids, the long-term medical consequences of which are largely
unknown (78 ). It appears prudent to monitor fasting blood sugar and lipid levels in patients treated with these agents. The new
atypical drugs also have their own individual and idiosyncratic side-effect profiles (Table 56.2 ). Thus, each new drug should be
evaluated individually in terms of side effects and safety (39 ).

Clozapine is associated with a very low propensity for EPS and little or no incidence of TD; thus, it is a valuable option for patients
who experience EPS (11 ). However, clozapine can cause serious side effects that impose substantial limitations on its use. Not only
must initial dose titration be quite gradual, but also there is a significant occurrence (around 0.9%) of agranulocytosis (83 ) and
seizures, as well as sedation, hypotension, hypersalivation, and weight gain (8 ). The frequency of agranulocytosis with clozapine is
such that regular white blood count monitoring is required (8 ).

Risperidone has a favorable side effect profile in comparison to haloperidol (84 ). Risperidone can produce dose-related EPS (≥6 mg
per day), but the rate of TD is low (0.6%) for dose currently used (2 to 8 mg per day) (84 ,85 ). Risperidone is associated with
prolactin elevation, hypotension, somnolence, insomnia, and agitation (39 ,86 ).

The incidence of EPS with olanzapine is not significantly different from that with placebo, and the incidence of olanzapine-related
TD is low (1%) (87 ). There is a risk of mild sedation and mild anticholinergic side effects, and the risk of weight gain appears greater
than with risperidone, but comparable to clozapine (78 ).

Quetiapine is associated with very low levels of EPS and its prolactin level elevation is indistinguishable from that of placebo (88 ).
The incidence of TD with quetiapine is reportedly low or virtually nonexistent, although this remains to be demonstrated
prospectively. There is a potential risk of lenticular opacities that were associated in one preclinical study in beagles (89 ), but have
not been found in nonhuman primates or patients, yet monitoring is recommended until additional data are available. The risk of
weight gain with quetiapine appears to be less than that with olanzapine and clozapine (78 ). Although quetiapine has virtually no
cholinergic activity, tachycardia is a possible side effect, perhaps secondary to its adrenergic effects on blood pressure (39 ). There
are several other side effects with quetiapine such as decrease in T3 and T4, orthostatic hypotension, and sedation, necessitating
gradual dose titration (39 ).

Ziprasidone has a risk of EPS that is not significantly different from that with placebo (90 ). The risk of TD is not known. Ziprasidone
is associated with mild dyspepsia, nausea, dizziness, and transient somnolence (90 ). Ziprasidone treatment has been associated
with minimal weight gain, which could distinguish it among other atypical agents (80 ). The FDA delayed ziprasidone approval
because of concern about its ability to prolong the Q-T interval (90 ), but an FDA Advisory Committee recommended its approval for
the treatment of schizophrenia in July 2000, and the FDA issued an approval letter in September 2000.

Effectiveness
Considerable evidence indicates that relapse and rehospitalized rates are substantially better with the group of atypical
antipsychotics than with conventional antipsychotics for patients who are compliant with their maintenance antipsychotic regimen
(46 ). The decreased EPS liability of the atypical drugs will make it easier to prescribe more effective doses of antipsychotic that can
maximize relapse prevention, without simultaneously interfering with the patient's quality of life or motor functioning (46 ). Patient-
based measures of quality of life show improvement with the atypical drugs over the conventional neuroleptics (45 ).

In one randomized controlled trial comparing clozapine with standard neuroleptic therapy for treatment-resistant schizophrenic
inpatients, the actual hospital discharge rates at 1 year were 27% for clozapine and 29% for standard care (91 ). The clozapine group,
however, had decreased readmission rates within the first 6 months compared with the neuroleptic group (3% versus 29%) (91 ).
 P.781

In another randomized double-blind comparative study of clozapine and haloperidol in patients with refractory schizophrenia over 1
year, clozapine-treated patients showed significant quality-of-life improvements when compared with haloperidol-treated patients
(53% versus 37%) (92 ). The patients assigned to clozapine had significantly fewer mean days of hospitalization for psychiatric
reasons than patients assigned to haloperidol (144 versus 168 days) and used more outpatient services (134 versus 98 units of service)
(92 ).

Several studies have examined the impact of risperidone on health care utilization in the 2 years before and after risperidone
treatment in small groups of schizophrenic patients. Decreases of 20% to 31% in the number of hospitalization days were reported
(93 ,94 ), but Viale and colleagues (95 ) observed an increase of 12% in hospitalization days in the first year of risperidone therapy.

Extensive controlled studies have proven olanzapine to be significantly superior to haloperidol in long-term maintenance of response
(62 ,96 ). The estimated 1-year risk of relapse was 19.7% with olanzapine and 28% with haloperidol (97 ). Furthermore, a
significantly greater proportion of the olanzapine- than risperidone-treated responders maintained their improvement in the
extended follow-up after 28 weeks of therapy (63 ). It is not clear whether the lower relapse rates are owing to increased
prophylactic efficacy or better treatment compliance because of better tolerability. To date, there have been no definitive
prospective random-assignment studies on compliance rates for atypical antipsychotics (46 ).

Cost-Effectiveness in Comparison with Conventional Drugs

Atypical antipsychotic drugs are approximately 10 to 40 times more expensive than conventional drugs (98 ). In the past few years, a
number of studies comparing the cost-effectiveness of the atypical antipsychotics with that of the typical drugs have been published.
However, many of these studies have frequently been criticized because of limitations in experimental design; thus, the cost-
effectiveness of atypical antipsychotics has not yet been fully established (98 ,99 ). Most of the available cost-effectiveness
evidence is from retrospective studies or economic computer models, which have considerable methodologic limitations (98 ).

Perhaps the best study of the cost-effectiveness of clozapine published to date in terms of its methodology is a randomized
controlled trial conducted by Rosenheck and associates (92 ), that compared clozapine with haloperidol in patients with treatment-
refractory schizophrenia over 1 year. After 1 year of treatment, the clozapine group had lower inpatient but higher outpatient costs.
The total medical costs (including inpatient hospital costs, outpatient medical costs, and medication costs) of the clozapine group
($58,000) were not significantly lower than the haloperidol group ($61,000). Overall, clozapine was concluded to be cost neutral,
although it demonstrated improved clinical outcomes, suggesting that it may be cost-effective (92 ).

The higher price of olanzapine compared with classic neuroleptics may be offset by reductions in the use of inpatient and outpatient
services (45 ,100 ). For example, Hamilton and colleagues (100 ) compared the cost-effectiveness of olanzapine to those of
haloperidol for the treatment of schizophrenia, in a randomized clinical trial, for 6 weeks (acute phase) and up to 1 year
(maintenance phase). The medication costs for olanzapine were about 22 times larger than those for haloperidol after 6 weeks of
treatment; however, patients treated with olanzapine had significantly lower inpatient and outpatient medical expenses than
patients treated with haloperidol. Overall, mean total medical costs during the acute phase for the olanzapine patients were
significantly lower (US$388/6 weeks) than those for the haloperidol patients. As was seen in the acute phase, these total medical
cost differences were sustained (US$636 lower per patient for olanzapine over 46 weeks) during the maintenance phase (100 ).
Glazer and Johnstone (99 ) also reported that the total health care costs for olanzapine treatment for 6 weeks and up to 1 year were
lower than those for haloperidol treatment ($431/month lower and $345/month lower, respectively).

Palmer and associates (101 ) used a decision analytic model to estimate the total medical costs and effectiveness outcomes of
olanzapine, haloperidol, and risperidone over 5 years for schizophrenia treatment in the United States. The estimated 5-year total
medical cost of olanzapine, haloperidol, and risperidone was US$92,593, $94,132, and $94,468, respectively. The estimated
disability-free years of these agents were 3.19 (olanzapine), 2.62 (haloperidol), and 3.15 (risperidone). The quality-adjusted life
years (QALYs) were 3.15 (olanzapine), 2.95 (haloperidol), and 3.12 (risperidone). These data suggest a modest cost-effectiveness
advantage for olanzapine over haloperidol and risperidone (101 ), whereas the decision-modeling approach appears to be subjective
to imprecision and possible bias (45 ). There have been no published randomized controlled studies of the cost-effectiveness of
risperidone. In addition, so far, no prospective randomized studies have been completed that compare the cost-effectiveness of the
atypical antipsychotics to each other for the treatment of schizophrenia. Furthermore, the other atypical drugs are too new to have
had their cost-effectiveness evaluated to any significant extent. Additional prospective randomized clinical trials with larger sample
sizes and long-term assessment should be conducted in order to evaluate the cost-effectiveness of atypical antipsychotics
adequately (45 ).

First-Episode Patients

Pharmacotherapy
First-episode patients as a group may differ from chronic patients in several aspects of pharmacologic responsiveness.
 P.782

Relatively high response rates of positive and negative symptoms have been reported in first-episode samples; for example,
Lieberman and colleagues (102 ) reported remission rates of 83% after 1 year of treatment with conventional antipsychotic agents in
70 first-episode patients. Surprisingly, remission did not occur until a median of 11 and mean of 36 weeks of treatment. Despite the
apparent heightened responsiveness of first-episode patients, residual cognitive deficits and poor psychosocial adjustment are
common (103 ,104 ). First-episode patients may also require a lower mean dose of antipsychotic medication and may be more
sensitive to drug side effects compared to more chronic patients (105 ). Kopala and colleagues (106 ) treated 22 first-episode
patients openly with risperidone for a mean of 7 weeks and observed a 91% response rate in patients who received risperidone 2 to 4
mg per day compared to a 27% response rate in patients who received a dose of 5 to 8 mg per day. The lower-dose group exhibited
no EPS, whereas 32% of the higher-dose group developed akathisia or parkinsonism. However, because this was not a fixed-dose
design, conclusions regarding dose–response relationships must be considered preliminary. In a different approach, Sanger and
colleagues (107 ) analyzed results from the 83 first-episode patients (out of a total of 1,996 subjects) who participated in a double-
blind, 6-week comparison of olanzapine and haloperidol. First-episode patients who received olanzapine had significantly better
clinical response and fewer EPS than the haloperidol group. Of particular interest, first-episode patients treated with olanzapine
achieved a significantly higher response rate than chronic patients treated with olanzapine. In addition, chronic patients treated
with haloperidol developed significantly fewer EPS than first-episode patients treated with haloperidol. Mean doses of haloperidol
and olanzapine were similar between first-episode and chronic patient groups (10.8 versus 11.0 mg per day and 11.6 versus 12.0 mg
per day, respectively). Although these findings suggest that the relative benefits of olanzapine (and perhaps of other atypical agents)
compared to conventionals may be greater in first-episode patients than chronic patients, issues of nonequivalent dosing between
drugs may be of particular concern in light of recent work indicating that optimal D2 receptor blockade may be achieved in first-
episode patients with haloperidol 0.25 to 2 mg per day (18 ). Two other double-blind controlled studies have been preliminarily
reported that address the question of whether first-episode patients respond better to atypical antipsychotic drugs. The first is a 52-
week study of clozapine versus chlorpromazine in 164 first-episode treatment naive schizophrenia patients in China (108 ). The
cumulative response rates of patients at 12 and 52 weeks, respectively, were 81.2% and 96.3% for clozapine (mean dose 292 mg per
day), and 68.3% and 97.7% for chlorpromazine (mean dose 319 mg per day). The first-episode patients treated with clozapine had
more rapid response, fewer EPS, and higher treatment retention and relapse prevention than the chlorpromazine group (108 ). The
second is a comparison between olanzapine and haloperidol in 262 patients with first-episode psychotic disorder (109 ). At 12 weeks,
the patients treated with olanzapine (mean dose 9.1 mg per day) demonstrated a higher response rate (55% versus 46%) and greater
cognitive improvement than the patients treated with haloperidol (mean dose 4.4 mg per day).

Response of first-episode patients has also received renewed attention because of the widely held belief that early intervention may
favorably affect the course of the illness. This hypothesis, which often invokes “neurotoxicity of untreated psychosis” as a
mechanism, is largely based on one naturalistic study reported by Loebel and colleagues (110 ). Other naturalistic studies have
failed to find a relationship between duration of initial untreated illness and outcome (111 ,112 and 113 ). Prospective controlled
trials are needed to determine whether early intervention with specific antipsychotic agents improves the early course of the illness.

Psychosocial Interventions
Psychosocial interventions potentially may have the greatest impact on first-episode patients and their families. Preliminary studies
have looked at stress-reduction approaches for patients identified as “premorbid” or at risk for schizophrenia, combining cognitive
therapy or stress reduction interventions alone or in combination with medication (114 ,115 and 116 ). Preliminary studies have
indicated that cognitive-behavioral therapy (CBT) approaches that have been developed for patients with treatment-resistant
psychosis can be successfully modified for first-episode patients (117 ). Psychoeducation, family support, and interventions to
enhance compliance are also expected to play important roles early in the course of the illness. However, two studies of first-
episode patients in Norway failed to find benefit from the addition of behavioral family management (BFM), which emphasizes
communications skills, to a basic psychoeducation program (118 ,119 ). The authors concluded that families of first-episode patients
may be in greatest need of information and support, rather than the intensive communication skills training offered by BFM.

Maintenance Treatment

Pharmacotherapy
Maintenance treatment with conventional and atypical antipsychotic medications has consistently demonstrated prophylactic
efficacy against relapse. Hogarty (120 ) reviewed the literature on maintenance treatment with conventional antipsychotic agents
and found that the average relapse rate during the first year after hospitalization was 41% with active medication compared to 68%
with placebo. Among patients who survived the first year, annual relapse rates with medication dropped to 15%, whereas relapse
rates on
 P.783

placebo remained constant at 65%. This pattern suggests that maintenance treatment is relatively ineffective for a substantial
proportion of patients; only after this poorly responsive subgroup is removed from the sample does the benefit of medication
become fully apparent. Consistent with this view are the results of a low-dose maintenance treatment trial with depot fluphenazine
in which a dose–response relationship only emerged during the second year of follow-up (121 ,122 ). Depot preparations have
significantly lowered relapse rates by an average of 15% compared to oral neuroleptics in six double-blind, randomized trials (123 ).
The advantage of depot administration may be understated in these trials, however, because research subjects were probably poorly
representative of typical clinical samples and most trials did not extend beyond 1 year. Research comparing low and standard-dose
maintenance with depot neuroleptics has demonstrated a trade-off between adverse effects with higher doses, including neurologic
side effects and dysphoria, versus increased relapse rates with lower doses (122 ,124 ). “Intermittent” maintenance treatment was
associated with an unacceptable rate of hospitalizations, whereas relapses associated with low-dose depot medication generally
were responsive to rescue with brief augmentation with oral neuroleptic or benzodiazepine; hospitalization rates were not elevated
with low compared to standard doses (122 ,124 ). Carpenter and colleagues (125 ) reported that administration of diazepam at the
earliest sign of exacerbation in medication-free patients was more effective than placebo and comparable to fluphenazine in
preventing relapse. This work suggests that lower doses of depot neuroleptic may provide acceptable protection against relapse if
accompanied by close monitoring and rapid psychosocial and pharmacologic intervention at the first sign of relapse. These measures
presumably will also enhance maintenance treatment with atypical agents, although dose-limiting side effects are not as
problematic.

Growing evidence suggests that maintenance treatment with atypical agents provides greater protection against relapse compared
to conventional oral agents. In a large, open trial, Essock and colleagues (126 ) found that chronically hospitalized patients
randomized to clozapine were not more likely to be discharged than patients receiving treatment as usual, but once discharged,
relapse rates were significantly lower with clozapine. Pooled results from three double-blind extension studies revealed that relapse
rates were significantly lower with olanzapine (20%) compared to haloperidol (28%) in patients with schizophrenia and related
psychoses (97 ). Until depot preparations of atypical agents are available for study, it will be difficult to determine whether the
advantage of certain atypical agents is primarily the result of enhanced compliance versus a direct modulatory effect on symptom
exacerbation. It is clear from depot neuroleptic studies that large numbers of patients relapse despite adequate compliance; relapse
in medication-compliant patients is often associated with depression and resolves spontaneously without change in medication (127 ).
Whether all atypical agents are equally effective in preventing relapse is also unknown. In a naturalistic study, Conley and
colleagues (128 ) found that relapse rates were quite similar during the first year after discharge in patients treated with clozapine
versus risperidone. During the second year, no additional relapses occurred on clozapine, whereas the rate of relapse on risperidone
increased from roughly 13% to 34%. In the only published comparison between risperidone and olanzapine, rates of exacerbation
(increase in PANSS score by 20%) were significantly higher at 28 weeks in patients who had responded to risperidone (mean dose 7
mg per day) compared to olanzapine (mean dose 17 mg per day) (63 ). It will be important to determine whether specific drugs
differ in prophylactic efficacy against relapse when compliance is controlled and issues of dosing equivalence are addressed. It is
possible that clozapine and perhaps other atypical agents are more effective in suppressing relapse; this effect may be relatively
independent of antipsychotic efficacy and mediated by different neurotransmitter systems. Continued development of psychosocial
interventions to improve compliance and monitor and respond to early signs of relapse will be equally important.

Psychosocial Interventions
A diverse range of psychosocial interventions has been shown to reduce relapse rates. In over 20 controlled trials, family therapies
emphasizing psychoeducation and support have reduced relapse rates for schizophrenia patients who have regular contact with
family members (129 ,130 ). Although differences in theoretical orientations and intensity of treatment have not produced
consistent differences in efficacy, recent evidence has suggested that multiple-family psychoeducation groups may be particularly
effective (131 ). Several controlled trials have also indicated that relapse rates can be reduced by assertive community treatment
programs (PACT) or similar outreach programs that provide intensive monitoring, skills training, and case management in the
community, usually with continuous availability of staff (132 ,133 ). Social skills training improves role functioning of patients with
schizophrenia, but has not substantially reduced symptoms or reduced relapse rates compared to control conditions in most studies
(134 ). In an illuminating study, Herz and colleagues (135 ) found that a relatively simple, weekly monitoring of schizophrenia
patients in psychoeducation groups in conjunction with the availability of rapid pharmacologic and psychosocial interventions at the
first sign of decompensation substantially reduced relapse rates, by approximately fourfold, compared to treatment as usual.

Noncompliance

Pharmacotherapy
Cramer and Rosenheck (136 ) surveyed the literature on antipsychotic medication and found that compliance rates
 P.784

averaged 42%. Similar surveys have not been conducted looking specifically at atypical agents, although it is generally believed that
reduced relapse rates reported with olanzapine and clozapine may reflect, in part, improved compliance (97 ,126 ). Factors
contributing to noncompliance are complex and probably involve the patient's perception of benefits and side effects of medication,
as well as the patient's level of insight. Compliance can be compromised by psychosis, agitation, and comorbid substance abuse
(137 ,138 ). Van Putten (139 ) studied compliance in 85 schizophrenia patients chronically treated with conventional neuroleptics
and determined that 46% took less antipsychotic medication than prescribed. Medication refusal was associated with an early
dysphoric response, which Van Putten attributed to subtle akathisia. Analysis of responses by 150 schizophrenia patients to a “Drug
Attitude Inventory” revealed that, based on responses to 10 items, 89% of patients could be correctly assigned to compliant versus
noncompliant categories as determined by clinician assessment of compliance (140 ). The strongest predictor of compliance was a
positive experience with medication—this factor accounted for 60% of the total variance, whereas the factor representing a negative
subjective experience accounted for 12%. Factors representing attitudes and beliefs about medication had minimal predictive power.
Other studies have also found that a patient's perception of benefit from medication is the strongest predictor of compliance (141 ).
Whereas many clinicians expect atypical agents to achieve higher levels of compliance by virtue of reduced or absent EPS, this view
may seriously underestimate the impact of other side effects. Two studies have found that clinicians tend to misjudge the relative
distress produced by different medication side effects (142 ,143 ). Side effects associated with certain atypical agents, such as
sedation, patients rated weight gain, drooling, and sexual dysfunction as more distressing than EPS in these surveys (142 ,143 and
144 ). The advantage of atypical agents in terms of compliance may stem less from their reduced EPS and more from their improved
efficacy for symptoms of anxiety, depression, and tension. Whether targeting cognitive deficits and impairment in insight will
improve compliance remains to be seen.

Psychosocial Interventions
Most approaches to noncompliance involve psychoeducation, supervision, and supportive therapy in which the benefits of treatment
are emphasized, whereas barriers to adherence and medication side effects are minimized (145 ). Family therapy and social skills
training may also exert a positive impact on compliance. Cognitive behavioral approaches have recently been applied to
noncompliance by Kemp and colleagues (146 ,147 ), who developed “compliance therapy,” a four- to six-session intervention based
on motivational interviewing techniques that targets attitudes towards medication and discharge planning during acute
hospitalizations. In a randomized, controlled trial, compliance therapy was found to improve insight and observer-rated adherence
to treatment over an 18-month treatment period (147 ). Patients in the compliance therapy group also displayed significantly
greater improvement in social functioning and lower relapse rates than the control group (147 ). In addition to educational and skills
training approaches, Cramer and Rosenheck (148 ) demonstrated that interventions that assist patients in remembering to take
medications, such as placing microchip schedulers on pill bottles, can also substantially improve compliance.

Treatment Resistance
Estimates of the incidence of treatment resistance have varied with changes in the diagnostic classification of schizophrenia and
definitions of treatment response (149 ), which have tended to obscure potential improvements in outcome associated with
advances in pharmacologic and psychosocial treatments. For example, Hegarty and colleagues (150 ) reviewed results of 320 clinical
trials and found that, since the introduction of modern antipsychotics in the mid-twentieth century, about 50% of patients were
improved at follow-up, whereas the rate of improvement dropped to 35% in the decade ending in 1994. A narrowing of the
diagnostic criteria is believed to account for this decline in response rates. Rates of response have tended to be higher in first-
episode psychosis, although dropout rates have been high in this population, particularly with conventional agents (102 ,107 ).
Persistence of psychotic symptoms is more common in drug trials involving chronic patients, presumably reflecting progression of the
illness as well as a possible selection bias favoring participation by more refractory patients. If the definition of treatment
resistance is broadened to include persistence of negative symptoms, cognitive deficits, or failure to achieve premorbid levels of
functioning, treatment resistance can be considered the rule rather than the exception.

Psychotic Symptoms

Antipsychotic Monotherapy
Response of psychotic symptoms to conventional antipsychotics, risperidone, and olanzapine has been associated with D2 receptor
occupancy in excess of 65% (18 ,57 ), although persistence of psychotic symptoms has been shown to occur despite adequate D2
blockade in a subgroup of refractory patients (151 ). As noted, only clozapine has consistently demonstrated efficacy for psychotic
symptoms in treatment of refractory patients; the mechanism responsible for this therapeutic advantage remains uncertain. In a
sample of 268 patients prospectively established to be neuroleptic resistant, 30% in the clozapine group met criteria for response at
6 weeks compared to 7% treated with chlorpromazine (11 ). Response rates as high as 60% have been reported
 P.785

after 6 months in open trials with clozapine in patients less rigorously defined as treatment refractory (152 ). The extent to which a
prolonged trial is necessary to determine efficacy of clozapine and other atypical agents is the subject of debate (153 ,154 ).

The relative efficacy of atypical agents other than clozapine in patients who have failed conventional neuroleptic therapy is less
clear. Marder and colleagues (155 ) found that schizophrenia patients presumed to be treatment-resistant on the basis of having
been hospitalized for 6 months or longer at the time of study entry did not respond to haloperidol 20 mg per day but significantly
improved with risperidone 6 mg per day or 16 mg per day compared to placebo. Similarly, analysis of a subgroup of 526 patients
from a larger trial identified retrospectively as having had a poor response to at least one prior antipsychotic, revealed greater
response of psychotic symptoms to olanzapine (mean dose 11 mg per day) than haloperidol (mean dose 10 mg per day); this
difference was significant in the intent-to-treat analysis but not in a comparison of completers (76 ). Trials specifically designed to
study treatment-resistant patients have provided less consistent support for efficacy of risperidone and olanzapine. In 67
schizophrenia patients with histories of neuroleptic resistance, risperidone 6 mg per day significantly improved total BPRS scores
compared to haloperidol 15 mg per day at 4 weeks, but response did not differ between groups at 8 weeks (156 ). In contrast,
risperidone produced significantly higher response rates than haloperidol in a large, randomized open trial involving 184
schizophrenia patients with a history of poor response (157 ). Relative response of psychotic symptoms to risperidone increased over
time and reached a maximum improvement compared to haloperidol at the final 12-month assessment. In a 6-week trial designed to
mirror the landmark Clozapine Collaborative Trial (11 ), only 7% of patients prospectively determined to be treatment resistant to
haloperidol responded to olanzapine 25 mg per day, a response rate that did not differ from chlorpromazine (77 ). The same group
reported that 41% of 44 patients identified as unresponsive to olanzapine in the preceding study or in an open trial subsequently
exhibited a response to clozapine (158 ). In addition, open trials in which patients have been switched from clozapine to olanzapine
or risperidone have reported a high incidence of clinical deterioration, casting doubt on claims for therapeutic equivalence between
clozapine and the second-generation agents, at least at the doses tested (159 ,160 ). Of interest, two controlled trials have found
comparable efficacy for risperidone and clozapine. However, in one 4-week trial, the 59 participants were not screened for
treatment resistance at baseline and, despite equivalence in outcomes between groups using an LOCF analysis, 25% of the
risperidone group dropped out owing to lack of efficacy compared to only 5% in the clozapine group (161 ).

The evidence is strongest in support of clozapine monotherapy as an intervention for neuroleptic-resistant patients; serum levels of
350 ng/mL or greater have been associated with maximal likelihood of response (162 ). Given the risk of agranulocytosis, the burden
of side effects, and the requirement of white blood cell monitoring, the second-generation agents (risperidone, olanzapine, and
quetiapine) are commonly tried before proceeding to clozapine. The appropriate first choice among these agents is unclear; two
controlled studies that compared olanzapine and risperidone have produced divergent results, probably reflecting differences in
dosing of the two agents and the use of intent-to-treat versus completer analyses (63 ,163 ). The focus of this research has been on
comparisons of mean responses between groups; predictors of response have not been identified, nor have subgroups of patients
that may exhibit preferential response to one agent of the class. Many clinicians express the impression that certain patients do
respond preferentially to a single agent of this class. Sequential controlled trials of the newer agents in treatment-resistant patients
will be necessary to fully examine this issue.

Combinations of Antipsychotics
The practice of combination therapy is gaining widespread popularity in the absence of controlled data in its support (164 ). In part
based on empirical experience and the demonstration that clozapine at optimal doses achieves relatively low degrees of D2
occupancy, European clinicians commonly add low-doses of neuroleptics to clozapine in partially responsive patients (165 ).
Uncontrolled trials and case reports have described benefits associated with the addition of risperidone (4 mg per day) (159 ,166 )
and pimozide (167 ) to clozapine in partially responsive patients. In a small, placebo-controlled trial, addition of sulpiride 600 mg
per day to clozapine significantly improved positive and negative symptoms at the end of 10 weeks in 28 subjects (168 ). Other
combinations, most notably olanzapine plus risperidone, are also increasingly employed, often because clinicians perceive improved
response during the cross-tapering phase of switching from one to the other. A theoretical rationale for this combination is less
apparent, given that each agent produces maximal D2 and 5-HT2 occupancy when appropriately dosed (57 ). If combined treatment
with olanzapine and risperidone is found in suitably controlled study designs to offer advantages over optimal monotherapy with
either agent, such a finding would argue in favor of the existence of additional contributory receptor actions unique to each drug.

Adjunctive Treatments
A diverse range of adjunctive treatments has been proposed for antipsychotic-resistant schizophrenia, although therapeutic effects
generally have been small or inconsistent in controlled trials. Very little data are available from controlled trials augmenting
clozapine in partial responders (169 ). Lithium augmentation frequently has been cited as
 P.786

the best-established intervention based on positive results from three small studies (170 ,171 and 172 ); however, two recent
placebo-controlled studies found no benefit when well-characterized neuroleptic-resistant patients were treated with lithium
(approximately 1.0 mEq/L) added to haloperidol or fluphenazine decanoate (173 ,174 ). Augmentation with lithium may enhance
response of some patients, particularly in the presence of affective symptoms or excitement (175 ,176 ). Carbamazepine
augmentation of conventional neuroleptics has been associated with modest reductions in persistent symptoms, including tension
and paranoia, in several controlled trials (177 ,178 and 179 ), particularly in patients with abnormal EEGs or violence. However,
induction of hepatic microsomal enzymes by carbamazepine can substantially lower blood levels of certain antipsychotic agents
(180 ) and in one report, resulted in clinical deterioration (181 ). Valproate does not significantly affect serum concentrations of
most antipsychotic drugs, but results from two small controlled augmentation trials have been inconsistent. Wassef and colleagues
(182 ) reported efficacy for negative symptoms and global psychopathology associated with addition of divalproex to haloperidol in a
placebo-controlled 12-week trial in 12 schizophrenia patients hospitalized for acute exacerbation. In contrast, Ko and colleagues
(183 ) found no effect when valproic acid was added to conventional neuroleptics in six treatment-resistant patients in a placebo-
controlled crossover design. Augmentation with benzodiazepines also has been advocated, in part, because of the potential role of
GABAergic agents in modulating dopamine transmission, although the evidence for efficacy is not compelling (184 ). Short-term,
acute treatment with high-dose benzodiazepines may reduce agitation and psychotic symptoms in as many as 50% of patients
(185 ,186 ), but early reports of benefit of longer-term treatment with benzodiazepines have not been replicated consistently by
controlled trials (186 ,187 ).

Electroconvulsant Therapy and Transcranial Magnetic Stimulation

The most consistent evidence for efficacy in neuroleptic-resistant patients can be found in the literature describing
electroconvulsant therapy (ECT) (188 ). Response rates between 50% and 80% were observed when ECT or the convulsant, Metrozole,
were administered unblinded in neuroleptic-naive patients prior to the introduction of antipsychotic medication (189 ,190 and 191 ).
Three double-blind randomized trials comparing neuroleptic plus ECT versus neuroleptic plus sham-ECT have demonstrated a
significantly greater and more rapid reduction in psychotic symptoms (delusions) with the combination treatment during 2- to 4-
week trials (192 ,193 and 194 ). Benefits of ECT were lost, however, at follow-up 10 to 28 weeks after treatment. Predictors of a
positive response to ECT include acute onset and brief duration of illness (188 ,195 ,196 ,197 and 198 ). Mood symptoms in
schizophrenia patients have tended to be relatively unresponsive to ECT and a diagnosis of schizoaffective disorder did not predict a
favorable response (192 ,193 ,194 and 195 ). Cases describing the successful combination of ECT with clozapine in refractory
patients have also been reported, suggesting that augmentation of atypical agents with ECT warrants further investigation
(199 ,200 ). Recently, interest has focused on the potential use of transcranial magnetic stimulation (TMS) as an alternative to ECT
in schizophrenia. TMS has shown promising efficacy in depression (201 ,202 and 203 ). In a preliminary, sham-TMS controlled
crossover study in 12 medication-resistant schizophrenia patients, the frequency and severity of auditory hallucinations were
significantly reduced following 12 to 16 minutes of stimulation (204 ). Improvement of auditory hallucinations persisted for a mean
of 14 days (range 1 to 60 days). This is an intriguing area for future research, both as a tool to explore the neural circuits underlying
symptoms of schizophrenia as well as a potential treatment option in medication-resistant cases.

Psychosocial Interventions
A particularly promising psychosocial approach to medication-resistant psychotic symptoms is cognitive-behavioral therapy (CBT)
(205 ). CBT for psychosis generally consists of alliance formation, examination, and challenge of psychotic beliefs, and the teaching
of self-monitoring and coping skills. Four randomized trials, all performed in the United Kingdom, demonstrated superior efficacy for
CBT compared to active control treatments on measures of global psychopathology and positive symptoms among chronic,
medicated patients (206 ,207 ,208 and 209 ). A recent metaanalysis determined that the between-groups effect size was .65,
favoring CBT over comparison treatments for the response of psychotic symptoms; delusions were generally more responsive than
hallucinations (210 ). Improvements in ratings of psychotic symptoms have been found to persist at follow-up, 1 year after
completion of CBT (209 ). Although therapeutic effects have been impressive, only about half of subjects have displayed
improvement in controlled trials (205 ). Preliminary evidence suggests that patients who exhibit a capacity to entertain alternative
explanations for psychotic beliefs at baseline are more likely to respond to CBT (205 ).

Negative Symptoms

Antipsychotic Monotherapy
Although atypical antipsychotics have generally demonstrated superior efficacy for negative symptoms compared to high-potency
conventional agents, the degree of improvement is usually quite modest, leaving substantial levels of residual negative symptoms.
For example, across several studies, the effect size of risperidone 6 mg per day compared to placebo on negative symptoms was
small (.27) (211 ). Path analysis has suggested that both risperidone and olanzapine exert direct effects on negative symptoms
independent of differences in psychotic, depressive, or extrapyramidal
 P.787

symptoms (212 ,213 ). Recently, Volavka and colleagues (74 ) preliminarily reported a prospective double-blind randomized study,
comparing the effects of clozapine, olanzapine, risperidone, and haloperidol, for 14 weeks in 157 treatment-resistant inpatients.
Clozapine (mean dose 527 mg per day) and olanzapine (mean dose 30 mg per day), but not risperidone (mean dose 12 mg per day),
demonstrated significantly greater efficacy than haloperidol (mean dose 26 mg per day) in reducing negative symptoms (74 ).
However, it is debated whether clozapine's established efficacy for negative symptoms extends to the treatment of primary negative
symptoms of the deficit syndrome (153 ,154 ,214 ). Few data are available from controlled trials to guide treatment of negative
symptoms that persist despite optimal treatment with atypical agents (215 ). Clinicians commonly employ augmentation strategies,
but evidence supporting this practice is derived mostly from an older literature describing combinations of augmenting agents added
to conventional agents.

Adjunctive Agents
Following clozapine's example as an antagonist of D2 and 5-HT2 receptors, investigators combined haloperidol with ritanserin, a
relatively selective 5-HT2A and 5-HT1C antagonist (216 ). In a 6-week, placebo-controlled trial, addition of ritanserin to haloperidol
produced significant reductions in negative symptoms (primarily affective expression and social withdrawal) and depressed mood.
Addition of 5-HT2 blockade may improve negative symptoms by enhancing mesocortical dopamine release. Svensson and colleagues
demonstrated that 5-HT2 blockade increases firing of midbrain dopamine neurons and reverses the effects of N-methyl-D-aspartate
(NMDA) antagonism (217 ) and hypofrontality (218 ) on A10 dopamine neuronal firing. Because the available atypical agents achieve
maximal occupation of 5-HT2 receptors at usual therapeutic doses (57 ), it is unlikely that augmentation with 5-HT2 antagonists (e.g.,
nefazodone) will further improve response of negative symptoms.

Another serotonergic augmentation strategy has involved addition of selective serotonin reuptake inhibitors (SSRIs) to conventional
neuroleptics, based largely on early empirical observation (219 ). Fluoxetine and fluvoxamine significantly improved negative
symptoms when added to conventional neuroleptics in three of four controlled trials, producing generally modest effects (220 ). In
one study, fluoxetine 20 mg per day added to depot neuroleptics decreased ratings of negative symptoms by 23% compared to a 12%
reduction with placebo; this improvement occurred despite a mean 20% elevation in haloperidol serum concentrations and a 65%
increase in fluphenazine levels (221 ). However, addition of sertraline 50 mg per day to haloperidol produced no symptomatic
change in an 8-week, placebo-controlled trial in 36 chronic inpatients with schizophrenia (222 ). In the only reported controlled trial
of SSRI augmentation of an atypical agent, fluoxetine at a mean dose of 49 mg per day produced no improvement in negative
symptoms when added to clozapine in 33 patients (223 ).

Anticholinergic agents are commonly added to conventional antipsychotics for control of EPS (224 ). The atypical agents vary
substantially in their muscarinic anticholinergic activity; clozapine is strongly anticholinergic, whereas quetiapine and risperidone
exhibit very low affinity for muscarinic receptors (Table 56.1 ). Addition of anticholinergic agents to conventional agents was
associated with reductions in negative symptoms in one study (225 ) but not others (176 ,226 ,227 and 228 ). Whether primary
negative symptoms are improved by anticholinergics, as suggested by Tandon and colleagues (229 ), cannot be answered by studies
in which subjects are treated with conventional agents; by attenuating psychomotor side effects of the neuroleptic, the
anticholinergic may be improving secondary negative symptoms only. To address this issue, two small placebo-controlled trials have
administered anticholinergic agents to medication-free patients. Negative symptoms were improved by biperiden in one study (230 )
and were unchanged with trihexyphenidyl in the other (231 ). Although the efficacy of augmentation with muscarinic anticholinergic
agents for negative symptoms remains poorly established, the potential cognitive impairment that these agents can produce is well
described (232 ,233 ).

Dopamine agonists have also been studied as augmenting agents for negative symptoms. Three of four placebo-controlled trials
demonstrated improvement of negative symptoms following a single dose of amphetamine given orally or intravenously
(234 ,235 ,236 and 237 ); in one study efficacy for negative symptoms was not affected by coadministration with pimozide (236 ).
However, Casey and colleagues (238 ) found no clinical benefit in an extended, 20-week placebo-controlled trial of amphetamine
augmentation of chlorpromazine. Augmentation trials of psychostimulants added to atypical agents have not been reported.

As discussed elsewhere in this chapter, augmentation strategies for negative symptoms have recently targeted glutamatergic
receptors, in part based on the NMDA antagonist model for schizophrenia and the observation that clozapine differs from
conventional agents in its effects on NMDA receptor activity (239 ). Significant improvements in negative symptoms consistently
have been produced in placebo-controlled trials by the addition to conventional antipsychotics of agonists at the glycine site of the
NMDA receptor. D-cycloserine, a partial agonist at the glycine site, produced a selective, 23% mean improvement of negative
symptoms at 6 weeks that, compared to placebo (7% reduction), represented a large effect size (.80) (240 ). The full agonist, glycine,
at a dose of 60 g per day produced a 30% mean reduction in negative symptoms and also improved a qualitative measure of cognitive
functioning (241 ). Augmentation with another endogenous full agonist, D-serine 30 mg per kg per day, was associated with
significant improvements
 P.788

in negative, positive, and cognitive symptoms when added to conventional agents and to risperidone in an 8-week trial (242 ).
Consistent with evidence that clozapine differs from conventional agents in its effects on NMDA receptor responsiveness, glycine, D-
cycloserine, and D-serine did not improve negative symptoms when added to clozapine (242 ,243 ,244 and 245 ). Whether strategies
that enhance NMDA receptor activation will improve response to other atypical agents remains uncertain, although both olanzapine
and quetiapine resemble clozapine in certain models of NMDA receptor responsivity.

Psychosocial Treatments
Existing psychosocial approaches have not achieved notable success in the treatment of negative symptoms. Negative symptoms are
substantially less responsive to CBT than are psychotic symptoms and patients with prominent negative symptoms are generally poor
candidates for CBT (205 ). Similarly, in a pilot study, Kopelowicz and colleagues (246 ) found that patients meeting criteria for the
deficit syndrome were relatively less likely to benefit from a program of psychoeducation and social skills training than patients
without prominent negative symptoms. The presence of negative symptoms also predicts poor outcome in vocational rehabilitation
programs for patients with schizophrenia (247 ). Although most forms of outreach and involvement of deficit syndrome patients in
psychosocial programs may improve their quality of life by reducing social isolation and countering apathy, negative symptoms
constitute a serious obstacle to participation in such programs and are unlikely to improve with psychosocial treatment.

Mood Symptoms

Antipsychotic Monotherapy
Depressive symptoms are common during all stages of schizophrenia and are associated with poor outcome, including relapse and
suicide (248 ,249 and 250 ). It is not uncommon for patients to present initially with depression during the prodromal stage, prior to
the appearance of psychotic symptoms (251 ). Approximately 25% of first-episode patients exhibit depression, although estimates of
the incidence of comorbid depression vary widely according to choice of diagnostic criteria (251 ,252 and 253 ). The prevalence of
depression as defined by moderate scores on depression rating scales ranges between 25% and 50% in chronic patients (252 ,254 ).
Although considerable overlap exists between symptoms of depression and certain negative symptoms (e.g., anhedonia, poor
concentration, psychomotor retardation), dysphoria appears to discriminate between the two (255 ,256 ).

Conventional antipsychotics tend to have little effect on comorbid depression, although anxiety and depression associated with
acute psychotic exacerbation frequently respond to neuroleptic monotherapy (257 ,258 ). However, dysphoric reactions to high-
potency conventional agents, although generally not meeting criteria for major depression, can closely resemble the depressive
symptoms often associated with the illness (254 ,259 ,260 ). Clozapine, olanzapine, and risperidone have all demonstrated
significantly greater efficacy for depressive symptoms compared to conventional neuroleptics in large, double-blind trials
(64 ,211 ,261 ). Path analysis suggested that 57% of the superior response of depressive symptoms to olanzapine compared to
haloperidol was a direct effect, whereas effects on negative symptoms accounted for only 21% and reductions in EPS accounted for
13% of the difference in depressive symptom response (64 ). Antidepressant activity of the atypical agents may have important
clinical consequences because perceived improvement in anxiety and depression is a strong predictor of compliance and emergence
of depressive symptoms often accompanies relapse.

Adjunctive Agents
In a placebo-controlled trial reported in 1989, Kramer (258 ) found that addition of desipramine or amitriptyline 5 weeks after
initiating haloperidol to acutely decompensated patients with schizophrenia and depression was associated with poorer
antipsychotic response and did not improve depressive symptoms. Subsequently, Siris and colleagues (262 ,263 ) demonstrated that
imipramine added to conventional agents in stable outpatients significantly improved depression without adversely affecting
psychotic symptoms. In a carefully controlled trial, imipramine 200 mg per day was associated with substantial improvement in
depressive symptoms in 42% of patients compared to 12% with placebo. Hogarty and colleagues (176 ) found that desipramine
improved symptoms of depression, anxiety, and psychosis when added to fluphenazine decanoate in a placebo-controlled trial.
Benefits of desipramine were only significant in female patients and did not achieve significance until week 12. The investigators
noted that improvement of psychotic symptoms might have resulted from successful prophylaxis against depressive episodes, which
were associated with worsening of psychosis. Several trials of tricyclic antidepressants added to conventional agents have been
reported; this literature generally supports their use for acute and maintenance treatment of depressive symptoms in stable
patients (264 ,265 ). Augmentation with selective serotonin reuptake inhibitors has been studied primarily as a treatment for
negative symptoms—use of these agents in schizophrenia patients with depression is not well studied. Similarly, addition of
antidepressants to atypical agents has not been reported in schizophrenia patients with comorbid depression.

Cognitive Symptoms

Antipsychotic Monotherapy
A wide range of cognitive deficits are usually present at the time of the first psychotic episode (266 ) and remain stable
 P.789

or only slowly progressive during the course of the illness, independent of psychotic symptoms (267 ,268 and 269 ). Cognitive
deficits are particularly prominent in patients meeting criteria for the deficit syndrome (270 ) and in patients with tardive dyskinesia
(271 ). The latter association may indicate that cognitive deficits are a risk factor for tardive dyskinesia, or alternatively, that the
neurotoxic mechanism responsible for irreversible motoric deficits also compromises cognitive functioning. Targeting cognitive
impairments is now a major focus of drug development because cognitive deficits are powerful determinants of vocational and social
functioning and may influence quality of life (36 ) more than psychotic symptoms.

The conventional neuroleptics produce small and inconsistent effects on cognitive functioning; sustained attention improved in some
studies, whereas motor control (finger tapping) worsened and memory and executive functioning were minimally affected (272 ).
Recent evidence in monkeys indicates that chronic neuroleptic exposure results in decreased prefrontal cortical D1 receptor density
after 6 months (273 ); treatment with a D1 agonist reversed neuroleptic-associated deficits in working memory (274 ). In normal
subjects, clozapine administered as a single 50-mg dose worsened attention, concentration, and motor functioning (275 ),
presumably reflecting sedative and anticholinergic properties. Studies in patients with schizophrenia have found either no effect
following a switch to clozapine (276 ), or improvements in a wide range of cognitive functions, including verbal fluency, attention,
and reaction time (37 ,277 ). In general, clozapine, olanzapine, and risperidone have demonstrated superior efficacy compared to
conventional agents on tests of verbal fluency, digit-symbol substitution, fine motor function, and executive function (37 ,277 ).
Atypical agents least affected measures of learning and memory (37 ). Enhanced performance with atypical agents could result, in
part, from reduced parkinsonian side effects because these tests all measure performance during a timed trial (37 ). Methodologic
issues limit comparisons between atypical agents, however, preliminary evidence suggests that risperidone may be more effective
for visual and working memory than clozapine (277 ). In a 12-month, double-blind trial involving 55 schizophrenia patients randomly
assigned to olanzapine (mean dose 11 mg per day), risperidone (mean dose 6 mg per day), or haloperidol (mean dose 10 mg per day),
risperidone and olanzapine produced significantly greater improvement in verbal fluency compared to haloperidol, and olanzapine
was superior to both haloperidol and risperidone in effects on motor skills, nonverbal fluency, and immediate recall (278 ). However,
this finding is complicated by the high incidence of anticholinergic administration prior to the final cognitive assessment;
anticholinergics were prescribed to 73% in the haloperidol group, 45% in the risperidone group, and 15% in the olanzapine group. As
in efficacy studies for negative symptoms, dose equivalency is an important factor in trials comparing cognitive effects of atypical
agents, particularly because excessive dosing can impair performance on time-sensitive tasks and increase anticholinergic exposure.

Adjunctive Agents
Augmentation with glutamatergic agents has shown promise for cognitive deficits in schizophrenia (279 ). As noted, glycine and D-
serine improved ratings of cognitive functioning when added to conventional neuroleptics (241 ,280 ). Both agents improved the
“cognitive subscale” of the PANSS compared to placebo, and D-serine was also associated with improved performance on the
Wisconsin Card Sort. These findings are of interest given that NMDA antagonists produce in normal subjects deficits in attention and
memory similar to those found in schizophrenia (281 ,282 ). The partial agonist, D-cycloserine, did not improve cognitive functioning
when added to conventional agents in a study that utilized formal cognitive testing, however (240 ). Positive modulators of the
glutamatergic AMPA receptor are also under investigation, as these agents improve performance in tests of learning and memory in
animal studies (283 ). In a preliminary 4-week, placebo-controlled trial involving 19 schizophrenia patients, CX-516, a positive
modulator of the glutamatergic AMPA receptor, improved performance on tests of memory and attention when added to clozapine
(284 ). Effect sizes favoring CX-516 over placebo were moderate to large (.5 to 1.2) on tests of cognitive performance.

Psychosocial Treatments
Although cognitive remediation treatments have long been used for brain-injured individuals, similar treatment approaches
targeting cognitive deficits in schizophrenia are relatively recent. In small studies in which schizophrenia patients practiced
graduated cognitive exercises, performance on laboratory measures of attention and memory function improved, although the
functional benefits of these gains are not clear (285 ,286 ). Brenner and colleagues (287 ) developed integrated psychological
therapy (IPT), a cognitive remediation program in which cognitive exercises are provided in a group format stressing the integration
of cognitive skills with social functioning. In a 6-month randomized trial in which patients received IPT or supportive treatment in
addition to comprehensive psychiatric rehabilitation, the IPT group displayed greater improvement on the primary outcome measure
of interpersonal problem solving and on a laboratory measure of attentional processing (288 ). This study was conducted prior to the
introduction of atypical antipsychotics. Following another approach, Hogarty and Flesher (289 ) recently developed cognitive
enhancement therapy (CET), which combines interactive software and social group exercises to improve socially and behaviorally
relevant cognitive functioning. This approach is based on a neurodevelopmental model for cognitive deficits in schizophrenia (290 ).
Preliminary results
 P.790

from a controlled 1-year trial of CET have also been encouraging (289 ).

EXPERIMENTAL TREATMENTS AND STRATEGIES

Part of "56 - Therapeutics of Schizophrenia "

Selective Dopamine Antagonists

There are several lines of evidence suggesting that selective dopamine D4 receptor antagonists may be potential novel antipsychotic
drugs. Clozapine has a relatively higher affinity for the D4 versus D2 or D3 receptors (291 ) (Table 56.1 ). Not only clozapine, but also
a number of clinically efficacious antipsychotics have relatively high affinity for this receptor site (Table 56.1 ). In addition, an
increase in D4 receptors has been reported in the brains of patients with schizophrenia (292 ). Furthermore, the D4 receptor,
enriched in the prefrontal cortex and hippocampus, is located in dopamine terminal fields potentially associated with emotion and
cognition, but not with movement, underscoring the potential of this receptor as a target. The selective D4 antagonist,
sonepiprazole (U-101387) increases dopamine release in the frontal cortex, but decreases dopamine release in the nucleus
accumbens in rats (293 ). Sonepiprazole attenuates apomorphine-induced impairment of prepulse inhibition in rats (294 ). It also
antagonized the decrease in c-fos expression in the medial prefrontal cortex and neurotensin mRNA in the nucleus accumbens
produced by repetitive amphetamine administration in rats, suggesting possible antipsychotic action of the agent (295 ).
Sonepiprazole is currently in Phase II clinical trials in patients with schizophrenia (293 ). An initial clinical trial with another highly
selective D4 antagonist, L-745,870, failed to demonstrate any antipsychotic activity in the treatment of schizophrenia (296 ,297 ).
Although the single dose tested makes it difficult to draw firm conclusions regarding the potential efficacy of D4 antagonists as
antipsychotic agents (298 ), this drug actually caused a worsening of symptoms (297 ). Similarly, NGD-94-1 also did not show clinical
efficacy in limited trials in schizophrenics (293 ). More extensive testing of D4 antagonists in patients with schizophrenia will be
necessary to adequately assess the therapeutic potential of such drugs.

Dopamine Partial Agonists

Partial dopamine agonists are agents with good affinity for one or more dopamine receptors, but with intrinsic activity less than
dopamine (3 ). Thus, such drugs may antagonize the actions of dopamine, yet by agonistic actions, activate other dopamine-related
functions (299 ). It has been proposed that some D2-like dopamine agonists have a greater affinity for autoreceptors than for
heteroreceptors. The action of these agonists at autoreceptors would induce a receptor-mediated inhibition of both the synthesis
and release of dopamine from nerve terminals, without producing significant activation of heteroreceptors on target cells (300 ).
Such partial dopamine agonists are therefore proposed to act as dopaminergic “buffers,” reducing dopaminergic transmission
without completely blocking it when dopaminergic activity is excessive, or conversely, stimulating it when it is reduced (7 ,299 ).

Despite the numerous compounds that were developed as partial agonists, none has proved to be sufficiently effective to warrant its
full development and introduction for clinical use. The first of this class to show consistent and robust efficacy comparable to
clinically used antipsychotic drugs, both conventional and atypical, is aripiprazole (301 ). Aripiprazole (OPC-14597) is a dual
dopamine autoreceptor partial agonist and postsynaptic D2 receptor antagonist (302 ,303 ). It has a modest affinity for 5-HT2
receptors, but no appreciable affinity for D1 receptors (304 ) (Table 56.1 ). Aripiprazole decreased striatal dopamine release (303 ),
and inhibited the activity of dopamine neurons when applied locally to the ventral tegmental area in rats (305 ). Animal behavioral
studies showed that the compound exhibited weak cataleptogenic effects compared to haloperidol and chlorpromazine despite the
fact it has almost identical D2 receptor antagonistic activity (302 ). The potency of aripiprazole to up-regulate striatal D2 receptors in
response to chronic treatment was much smaller than that of haloperidol, suggesting lower potential for EPS, including tardive
dyskinesia (31 ). Aripiprazole is currently going through worldwide Phase III development. Preliminary clinical studies have shown its
efficacy in alleviating both positive and negative symptoms of schizophrenia. Although current dogma suggests that such a D2-
selective agent would cause profound EPS and high sustained prolactin elevation, neither side effect has been seen clinically
(306 ,307 and 308 ). Based on available data, it would appear that aripiprazole is the first compound with partial D2 agonist
properties to be a clinically effective antipsychotic agent. It has been proposed that aripiprazole induces “functionally selective”
activation of D2 receptors coupled to diverse G proteins (and hence different functions), thereby explaining its unique clinical
effects (304 ).

CI-1007 is a new dopamine autoreceptor agonist and partial dopamine D2/D3 receptor agonist that is currently under development for
the treatment of schizophrenia (309 ,310 ). In preclinical studies, CI-1007 demonstrated that it inhibited the firing of dopamine
neurons and reduced the synthesis, metabolism, utilization, and release of dopamine in the brain (310 ). In addition, it produced
behavioral effects predictive of antipsychotic efficacy and indicated a low liability for EPS and TD (311 ).

5-HT Agents
The 5-HT2A receptor subtype has received considerable attention because of its potential roles in the therapeutic action of atypical
antipsychotic drugs (21 ,312 ); it is involved
 P.791

in perception, mood regulation, and motor control (313 ). Available evidence indicates that 5-HT2A receptor stimulation plays a role
in promoting the synthesis and release of dopamine, either by effects on firing rates of dopamine neurons, or via heteroreceptors on
dopamine nerve terminals, or both (312 ,313 ,314 and 315 ). 5-HT2A receptor blockade may therefore contribute to “normalizing”
levels of dopamine release (316 ) and theoretically possess antipsychotic activity.

M-100907 (formerly MDL-100,907) is a selective 5-HT2A receptor antagonist devoid of affinity to dopamine receptors (21 ). Like the
atypical antipsychotics, it decreases the firing rate of A10, but not A9, neurons after chronic treatment (317 ). M-100907 inhibited
the behavioral response not only to amphetamine and cocaine (316 ,317 and 318 ), but also to NMDA receptor antagonists at doses
that did not affect spontaneous activity given alone in rodents (319 ,320 and 321 ). M-100907, like clozapine, markedly increases
dopamine release in the medial prefrontal cortex in rats (322 ), suggesting that the agent may have efficacy for negative symptoms.
In contrast, it attenuates dopamine release in the nucleus accumbens induced by the NMDA receptor antagonist MK-801 (323 ). M-
100907 also antagonized MK-801-induced prepulse inhibition deficit in rats (324 ). Further, in electrophysiologic studies, it prevented
phencyclidine (PCP)-induced blockade of NMDA responses (325 ). These preclinical results suggest that M-100907 can attenuate
variable responses to NMDA receptor antagonists in vivo and modulate NMDA receptor-mediated neurotransmission. M-100907,
however, exhibited lower antipsychotic efficacy compared with haloperidol in Phase III clinical trials (326 ). Insufficient data are
currently published to adequately judge the efficacy of the drug.

It has been suggested that the partial agonist activity of clozapine at 5-HT1A receptors may contribute to its therapeutic action
(313 ,327 ). Preclinical studies have suggested that serotonin 5-HT1A agonists may potentiate the antipsychotic activity of
dopaminergic antagonists (328 ). Activation of inhibitory 5-HT1A autoreceptors may also counteract the induction of EPS owing to
striatal D2 receptor blockade (329 ). Further, in schizophrenic patients, increased 5-HT1A receptor binding was seen in the prefrontal
cortex (330 ,331 ). Based on these preclinical data, compounds that act as serotonin 5-HT1A agonists are being developed as potential
antipsychotic compounds.

S-16924 is a novel, potential antipsychotic agent with high affinity for dopamine D2/4, α1-adrenergic, and serotonin 5-HT2A receptors,
similar to that of clozapine, in addition to being a potent partial 5-HT1A agonist (332 ). Reflecting its partial agonist actions at 5-HT1A
receptors, it attenuates cerebral serotonergic transmission, and preferentially facilitates dopaminergic transmission in mesocortical
as compared to mesolimbic and nigrostriatal pathways (333 ,334 ). S-16924 exhibited a profile of potential antipsychotic activity and
low EPS liability in animal behavioral models, similar to clozapine (332 ).

Muscarinic Agents
In patients with Alzheimer's disease (AD), cholinesterase inhibitors (e.g., physostigmine) have been shown to not only improve
cognition, but also reduce hallucinations, delusions, suspiciousness, and other behavioral disturbances sometime associated with the
illness (335 ,336 ,337 and 338 ). Similar positive effects on cognitive and psychotic-like symptoms in AD have been observed after
treatment with the direct muscarinic agonist, xanomeline (339 ). In addition, high doses of some muscarinic antagonists produce
psychotic-like symptoms and memory loss (340 ). Thus, it has been proposed that muscarinic agonists could be novel potential
treatments for positive and cognitive symptoms of schizophrenia (341 ).

Recent findings that partial agonists of m2/m4 muscarinic receptors are active in animal models that predict antipsychotic activity
suggest potential usefulness of muscarinic agonists in the treatment of schizophrenia (342 ). The drug (5R,6R) 6-(3-propylthio-1,2,5-
thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (PTAC) is a muscarinic partial agonist at muscarinic m2 and m4 receptor subtypes (342 ).
PTAC acts as a functional dopamine antagonist in many paradigms (consistent with known dopamine-acetylcholine interactions),
although it has minimal or no affinity for central dopamine receptors. PTAC attenuates apomorphine induced climbing (341 ),
inhibits the effects of D1 and D2 dopamine receptors agonists in 6-hydroxydopaminelesioned rats, and antagonizes amphetamine-
induced Fos induction and hyperactivity (343 ). In addition, after chronic administration, PTAC reduced the number of spontaneously
active dopamine cells in the ventral tegmental area, but not the substantia nigra (343 ). Such selective effects on the
mesocorticolimbic dopamine projection neurons are similar to those observed for clozapine and olanzapine (344 ,345 ). The notable
preclinical data of the effects of PTAC provide strong encouragement to examine the potential therapeutic effects of M2/M4
muscarinic agonists in schizophrenic patients. Among the agents that have been developed for the treatment of AD that are being
examined in schizophrenia are donepezil, metrifonate, galantamine, and xanomeline.

Glutamatergic Agents

The NMDA Receptor Hypofunction Hypothesis of Schizophrenia

Since the late 1950s, the anesthetics phencyclidine (PCP) and ketamine have been known to induce “emergence reactions” in 40% to
50% of individuals during the recovery from anesthesia, that resembles some features of schizophrenia (346 ). Recent work has
confirmed and extended the early clinical studies and has demonstrated that subanesthetic doses of ketamine can induce positive,
negative, and cognitive schizophrenia-like symptoms in normal humans (281 ,347 ).
 P.792

In chronic stabilized schizophrenic patients, subanesthetic doses of ketamine can also exacerbate cognitive impairment and in some
cases reproduce specific hallucinations and delusional ideation remarkably similar to those experienced during active phases of the
patients' illness (282 ,348 ,349 ). Both ketamine and PCP are potent noncompetitive NMDA receptor antagonists. These drugs bind to
a site within the calcium channel of the NMDA receptor complex, and thereby interfere with calcium flux through the channel.
Competitive NMDA receptor antagonists (i.e., drugs that inhibit binding to the glutamate recognition site) are also psychotomimetic
(350 ). The ability of NMDA antagonists to induce a spectrum of schizophrenia-like symptoms has led to the hypothesis that
hypofunction of NMDA receptors is involved in the pathophysiology of schizophrenia (346 ,351 ,352 and 353 ).

Antipsychotic Drug Actions in Relation to the NMDA Receptor Hypofunction

The well-documented psychotomimetic effects of NMDA antagonist in human suggest that effects of the drugs in experimental
animals could present useful pharmacologic models of schizophrenia. In our recent studies, striking effects of subanesthetic doses of
ketamine were observed on regional brain patterns of 14C-2-deoxyglucose (2-DG) uptake in both rats (354 ,355 ) and mice (356 ).
Ketamine induces robust and neuroanatomically selective patterns of brain metabolic activation, with especially large effects
observed in the hippocampus, nucleus accumbens, and medial prefrontal cortex (354 ,355 ). Pretreatment of rats with clozapine or
olanzapine can completely block these effects of ketamine (357 ,358 ). However, the typical antipsychotic haloperidol failed to
antagonize the brain metabolic activation induced by ketamine (357 ). Similarly, clozapine and olanzapine, but not haloperidol,
effectively block NMDA antagonist-induced electrophysiologic responses (325 ), deficits in prepulse inhibition (359 ,360 ), and
deficits in social interactions (361 ). Thus, in a wide range of experimental paradigms, atypical antipsychotic drugs selectively
antagonize the consequences of experimentally induced NMDA receptor hypofunction, raising the possibility that the therapeutic
effects of these agents may be associated with a similar neurochemical action (362 ).

Therapeutic Potential of Glycine Site Agonists

If reduced NMDA receptor function is involved in the pathophysiology of schizophrenia, then drugs that enhance NMDA receptor
function could be therapeutic agents and potentially improve upon, or supplement, current antipsychotic treatments (13 ). Direct
agonists of the NMDA receptor may not be feasible candidates in this regard, because of the propensity of such drugs to produce
excessive excitation and seizures.

Glycine is a positive allosteric modulator and obligatory coagonist at the NMDA receptor (363 ) and this allosteric regulatory site
represents a potential target for drugs to augment NMDA-mediated neurotransmission. Preclinical studies have demonstrated that
glycine-site agonists reverse the effects of noncompetitive NMDA receptor antagonists (364 ). There have been several clinical
studies to test effects of different glycine site agonists in patients with schizophrenia. The earliest studies in this regard used
glycine in doses of 5 to 15 g per day and obtained inconsistent results (365 ,366 ). In more recent work with glycine, higher doses
were administered (30 to 60 g per day) and more robust and consistent effects were found, primarily in the improvement of
negative symptoms (241 ,367 ,368 ).

D-cycloserine is a partial agonist at the glycine regulatory site on the NMDA receptor. Thus, at low dose of the amino acid,
stimulatory responses are observed, but at higher doses, D-cycloserine blocks the effects of endogenous glycine. D-cycloserine has
been tested in patients with schizophrenia, and in a very narrow dose range, the agent was shown to improve negative symptoms
when administered alone (369 ), and when added to conventional antipsychotic treatment regimes (240 , 370 ). The “inverted U”-
shaped dose response may result from the partial agonist properties of D-cycloserine, because antagonism of the actions of
endogenous glycine would be predicted at higher doses of the drug. Interestingly, when D-cycloserine was administered in
conjunction with clozapine, the negative symptoms of the patients worsened (244 ,371 ). A ready explanation for these effects is
not available, but understanding the mechanisms involved in the worsening of negative symptoms after administration of D-
cycloserine to clozapine-treated patients may be an important clue in understanding the actions of both of these drugs. The poor
penetration of the blood–brain barrier by glycine, and the partial agonistic properties of D-cycloserine, appear to make these agents
less than optimal for providing pharmacologic agonism of the glycine regulatory site on the NMDA receptor (13 ).

D-serine is a full agonist on the strychnine-insensitive glycine site of NMDA receptor (372 ) and is more permeable than glycine at the
blood–brain barrier, thus requiring a lower dosage. In a recent clinical trial, D-serine (30 mg per kg per day) added to neuroleptic
treatment in treatment-resistant patients with schizophrenia demonstrated significant improvements not only in negative and
cognitive symptoms but also positive symptoms, which is different from glycine (280 ). These data, together with the results of the
clinical investigations with glycine and D-cycloserine (346 ), offer promise for the therapeutic potential of enhancing NMDA receptor
function as a strategy for the pharmacotherapy of schizophrenia. Recently, Wolosker and colleagues (373 ) purified an enzyme from
Type II astrocytes that converts L-serine to D-serine. It may be that effectors of this enzyme (directly or through possible receptor-
mediated regulation) can provide a mechanism to modulate NMDA
 P.793

function. Examining the effects of synthetic compounds with greater potency and full agonistic activity at the glycine regulatory site
could be an intriguing line of future research. There are, however, no such compounds available for testing at present.

Potentiation of NMDA Receptor Function by Inhibition of Glycine Uptake

Glycine transporters have been identified on both neuronal and glial cells in the central nervous system. A function of these
transporters has been suggested to control the extracellular glycine concentration (374 ). Although there is some controversy as to
whether the glycine regulatory site on the NMDA receptor is saturated under physiologic conditions, recent data demonstrate that
inhibition of glycine transport by glycine transporter type 1 antagonist can potentiate electrophysiologic effects of NMDA (374 ,375 ).
Furthermore, the glycine uptake inhibitor glycyldodecylamide attenuated PCP-induced hyperactivity more potently than glycine
(364 ,376 ). These preclinical data suggest that inhibition of glycine uptake could represent a feasible approach to potentiate NMDA
receptor-mediated neurotransmission and, possibly, treat schizophrenic patients.

Glutamate Release-Inhibiting Drugs

A number of studies have indicated that administration of relatively low (subanesthetic) doses of NMDA antagonists induces
behavioral and brain metabolic activation in experimental animals and humans (362 ). Consistent with these data, NMDA antagonists
increase glutamate release in rats (377 ). In contrast to the increase in glutamate release by subanesthetic doses of ketamine,
anesthetic doses of the drug decreased glutamate levels (377 ). The effect of different doses of ketamine on glutamate levels is
consistent with our observations of increased 2-DG uptake in response to a subanesthetic dose, and reduction in 2-DG uptake in
response to an anesthetic dose of ketamine (354 ).

The stimulatory effect of NMDA receptor antagonism presumably results from disinhibitory actions, perhaps by reducing excitatory
input to inhibitory interneurons (362 ). In hippocampal formation, GABAergic interneurons are more sensitive to the effects of NMDA
antagonists than the glutamate-containing pyramidal cells (378 ), providing support for the hypothesis that NMDA antagonism could
result in excitatory effects by disrupting recurrent inhibitory circuits (362 ).

If behavioral activation induced by NMDA antagonists is related to increased glutamate release, pharmacologic agents that decrease
glutamate release should block the effects of the drugs. Glutamate release can be inhibited by Na+-channel blockers, Ca2+-channel
blockers, K+-decreasing agents, toxins that prevent fusion of vesicles with the presynaptic membrane, and presynaptic group II
metabotropic glutamate autoreceptor agonists (379 ,380 and 381 ).

Administration of LY-354740, a group II metabotropic glutamate receptor agonist, blocked both behavioral activation and increased
glutamate release induced by PCP in rats (382 ). In humans, Anand and co-workers (381 ) found that lamotrigine, a new
anticonvulsant agent that inhibits glutamate release, can reduce the ketamine-induced neuropsychiatric effects. These data suggest
the possibility that glutamate release-inhibiting drugs (e.g., LY-354740 and lamotrigine) could be useful in the treatment of
schizophrenia.

AMPA/Kainate Receptor Antagonists

The increased release of glutamate observed in response to NMDA antagonist could mediate some of the behavioral actions of the
drugs by activation of non-NMDA receptors, including α-amino-3-hydroxy-5-methy-isoxazole-4-propionic acid (AMPA) and kainate
receptors (377 ). In support of the hypothesis that behavioral effects of NMDA antagonists relate to increased glutamate release,
administration of an AMPA/kainate receptor antagonist, LY-293558, partially reversed impairment of working memory induced by
subanesthetic doses of ketamine in rats (377 ). Furthermore, AMPA/kainate receptor antagonists reduce NMDA antagonist-induced
hyperlocomotion (383 ,384 and 385 ) and neurodegeneration (386 ). These data suggest that AMPA/kainate receptor antagonists
may have utility for treatment of cognitive deficits in which NMDA receptor hypofunction is suspect (377 ).

Potential of Positive Modulators of AMPA Receptors

In apparent contrast to the postulated utility of AMPA/kainate receptor antagonists as antipsychotics, ampakines, a class of
compounds that allosterically enhance AMPA receptor function, have also been suggested to represent potential adjunctive
treatments for schizophrenia. Ampakines enhance excitatory (glutamatergic) transmission, facilitate long-term potentiation,
learning, and memory in rodents (387 ,388 ), and have synergistic effects with typical and atypical antipsychotics on blocking
behavioral effects of methamphetamine (389 ). In addition, preliminary results suggest that chronic administration of an ampakine
(CX-516) can improve negative and cognitive symptoms in schizophrenia patients that also receive clozapine (284 ). Thus, such
findings are paradoxical with regard to the foregoing discussion of the hypothesis that excessive glutamate release may be involved
in behavioral effects of reduced NMDA receptor function. Further clinical experience with the effects of positive and negative
modulators of non-NMDA glutamate receptors will be needed to clarify the potential of these compounds for treatment of
schizophrenia (3 ).
 P.794

Protein Kinase C Inhibitors

Accumulating evidence from Manji and colleagues has identified the family of protein kinase C (PKC) isozymes as a common target in
the brain for the long-term action of the two structurally highly dissimilar antimanic agents, lithium and valproate (390 ). Chronic
treatment of rats with lithium or valproate induces a reduction in the levels of two PKC isozymes, α and ε, in the frontal cortex and
hippocampus, as well as a reduction in the expression of a major PKC substrate, myristoylated alanine-rich C kinase substrate
(MARCKS), which has been implicated in long-term neuroplastic events in the developing and adult brain (391 ). In view of the
critical role of the PKC signaling pathway in the regulation of neuronal excitability, neurotransmitter release, and long-term
synaptic events, Manji and associates postulated that the attenuation of PKC activity might have antimanic efficacy. In a pilot study,
they found marked antimanic efficacy of a potent PKC inhibitor tamoxifen, which is also a synthetic nonsteroidal antiestrogen, in
the treatment of acute mania (392 ). Their heuristic preliminary data suggest that PKC inhibitors may represent a novel class of
antimanic agents for the treatment of bipolar disorder, and deserve further study in psychotic syndromes.

Steroidal Agents

Estrogen
The gender effect of delayed onset (by approximately 2 to 5 years) and relatively reduced symptom severity in females has been
consistently observed in schizophrenia (393 ,394 and 395 ). Some, but not all, researchers have found an additional smaller peak of
onset of schizophrenia for women at age 40 to 45 years, which is a time of decreasing levels of estrogen associated with menopause
(395 ,396 ). The inverse relationship between estradiol levels and specific psychopathology, especially positive symptoms, was also
observed over the menstrual cycle in premenopausal women with schizophrenia (397 ,398 ). The indirect clinical evidence suggests a
potential role for estrogen in delaying the onset or attenuating the severity of psychotic symptoms associated with schizophrenia
(393 ,395 ). In animal behavioral studies, estrogen reduces amphetamine- and apomorphine-induced stereotypy, as well as enhances
neuroleptic-induced catalepsy (399 ). In addition, preclinical biochemical studies have shown that estrogen can alter dopamine D2
receptor density and affinity in the brain (399 ), whereas the effect is dependent on the time course of the administration (395 ).
These findings suggest a neuroleptic-like effect of estrogen, and may have important implications for the prevention and therapy of
schizophrenia. To date, there have been few treatment studies examining the effect of estrogen in patients with schizophrenia.
Lindamer and associates (395 ) presented a case report of a postmenopausal woman with schizophrenia who had an improvement in
positive symptoms with estrogen augmentation of neuroleptic medication. Long-term larger double-blind trials are crucially needed
to evaluate the efficacy of estrogen in conjunction with neuroleptic treatment on psychotic symptoms in women with schizophrenia.

Dehydroepiandrosterone
Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEA-S) are neuroactive neurosteroids that represent steroid hormones
synthesized de novo in the brain and acting locally on nerve cells (400 ). Although DHEA and DHEA-S are the most abundant
circulating steroid hormones in humans, their precise physiologic roles remain to be elucidated. In humans, DHEA levels in blood rise
dramatically at puberty and sustain a monotonic decline with age, reaching very low levels in late life. In vitro data suggest that
DHEA and DHEA-S enhance neuronal and glial survival and differentiation in mouse embryonic brain tissue cultures (401 ,402 and
403 ). In addition, DHEA-S shows marked neuroprotective ability against the glutamate-induced toxicity (404 ) and oxidative stress
(405 ). In rodents, DHEA has been demonstrated to be a positive modulator of the NMDA receptor. In both the adult rat brain and
developing mouse brain, DHEA-S was shown to potentiate substantially physiologic responses to NMDA (403 ,406 ,407 ). The
enhancement of physiologic response to NMDA by DHEA has been suggested to result from agonistic actions at s1 receptors in the
brain (407 ). Consistent with a positive modulatory action of DHEA at the NMDA receptor, the neurosteroid has been demonstrated
to enhance memory in mice (408 ,409 ,410 and 411 ). Moreover, DHEA-S attenuates NMDA receptor antagonist MK-801-induced
learning impairment via an interaction with s1-receptors in mice (412 ). These preclinical studies provide the neurobiological
rationale for the clinical studies to explore the potential utility of DHEA to treat the NMDA receptor hypofunction postulated to
occur in schizophrenia. In chronic schizophrenics, significantly lower morning levels of plasma DHEA were observed (413 ). Further,
there are a number of earlier case reports suggesting that DHEA may be useful in the treatment of schizophrenia, especially for
negative symptoms (414 ,415 and 416 ), although these trials were not well controlled. A recent double-blind study of patients with
major depression suggests that DHEA has antidepressant effects (417 ). Although the mechanism of action of DHEA and DHEA-S has
to be further characterized, the possibility that these compounds may have efficiency in schizophrenia should be explored.

Phospholipid Compounds

Membrane Phospholipid Hypothesis of Schizophrenia

The membrane phospholipid hypothesis of schizophrenia originated with suggestion by Horrobin (418 ) that schizophrenia
 P.795

might be caused by a prostaglandin (PG) deficiency. The proposal was based on several clinical observations of a relationship
between pyrexia and the transient dramatic remission of psychosis, the relative resistance to PG-mediated pain and inflammation
and reduced rate of rheumatoid arthritis in patients with schizophrenia, and the observation that PGE1 injected into the CSF of
mammals could produce catalepsy (419 ). Because PGs are derived from membrane essential fatty acid (EFA), Horrobin and
colleagues (420 ) hypothesized that schizophrenia involves a failure to produce PGE1 from EFA precursors. Interestingly, over two
decades ago, it was suggested that the structure and pharmacologic actions of clozapine are consistent with its being a PGE
analogue (420 ). PGEs are potent stimulators of cAMP formation, and cAMP inhibits phospholipase A2 (PLA2). In fact, clozapine
treatment induced a dramatic rise in erythrocyte membrane concentrations of the major cerebral fatty acids, arachidonic acid (AA)
and docosahexaenoic acid (DHA) (421 ). Thus, a generally unrecognized mechanism of action of clozapine may be on membrane
phospholipid composition, in addition to its receptor-blocking profile (421 ).

The specific EFA content of synaptic membrane plays a significant role in modifying neuronal function. The changes in membrane
EFA concentrations alter the biophysical microenvironment and hence, structure and function of membrane proteins, including
neurotransmitter receptors, ion channels, and enzymes (419 ). EFAs also contribute to cellular regulation by acting as a source of
precursors for second messengers in intracellular and intercellular signal transduction (419 ).

In rat models, changes in brain fatty acid concentrations produced by chronic dietary omega-3 fatty acid deficiency alter
dopaminergic and serotonergic neurotransmission (422 ) and induce a decrease in D2 and increase in 5-HT2 receptor density in the
frontal cortex (423 ). Impaired behavioral performance and learning are observed in omega-3 deficient rats (424 ) and have been
hypothesized to reflect changes in attention, motivation and reactivity consistent with a deficit in the function of prefrontal
dopamine pathways (419 ).

The phospholipid hypothesis of schizophrenia has been supported by the accumulating consistent clinical findings in schizophrenic
patients that indicate reduced levels of erythrocyte membrane EFA, elevated serum and platelet PLA2 activity (probably owing to
accelerated breakdown of membrane phospholipids), and 31-phosphorus cerebral magnetic resonance spectroscopy (MRS) evidence
of decreased synthesis and increased breakdown of phospholipids in the prefrontal cortex (419 ). Furthermore, phospholipid
hypotheses are consistent with both dysfunction of multiple neurotransmitter systems and neurodevelopmental abnormalities
associated with aberrant cell remodeling, apoptosis, or migration (425 ).

Omega-3 Fatty Acid

The membrane EFA or PG deficiency hypotheses have provided the rationale for attempts to treat symptoms of schizophrenia with
supplementation of PG precursors, including omega-6 and omega-3 fatty acids and PGE1. Among the studies of these compounds
conducted to date, omega-3 EFA treatment has consistently yielded positive results. Two small open trials and a single double-blind
trial suggest supplementation with omega-3 eicosapentaenoic acid (EPA) may improve residual symptoms and tardive dyskinesia
when added to standard neuroleptic treatment in schizophrenic patients (419 ). Surprisingly, the more recent case report by Puri
and colleagues (426 ) demonstrates a dramatic and sustained efficacy of omega-3 EPA on both positive and negative symptoms of
schizophrenia in a drug-naive patient without any adverse side effects. In addition, cerebral atrophy, observed before omega-3 EPA
treatment, was reversed by 6 months of EPA treatment; however, small trials and a single case report make it difficult to draw firm
conclusions regarding the potential efficacy of omega-3 EPA. A recent double-blind placebo controlled study of omega-3 EPA as an
adjunctive treatment to antipsychotic drugs found no difference between placebo and omega-3 EPA (427 ).

Trophic Factors
There is converging evidence that an abnormal neurodevelopmental process is accountable for at least a proportion of the
pathophysiology of schizophrenia (428 ). The neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), and neurotrophin (NT)-3/4/5 play a decisive role in a neurodevelopmental process, including neuronal and glial
differentiation, migration, proliferation, and regeneration (429 ). They are not only active during embryogenesis and organogenesis,
but also influence the synaptic organization and synthesis of neurotransmitters in the adult brain, and are therefore involved in the
maintenance of neural plasticity (429 ). Thus, pathologic alterations of the neurotrophic factor system may lead to neural
maldevelopment, migration deficits, and disconnections, which are proposed to be the characteristic pathogenetic features of the
maldevelopmental hypothesis of schizophrenia (429 ). A more recent pathophysiologic theory of schizophrenia suggests that it is
involved in a limited neurodegenerative process reflected by the progressive and deteriorating clinical course of the illness (430 ). If
neurotrophic factors salvage degenerating neurons, facilitate desirable synaptic connections, and hence, halt the progression of
neurodegenerative process of schizophrenia, drugs that selectively stimulate the production of neurotrophic factors could represent
a new approach to forestall the progression of schizophrenia and prevent morbidity from increasing (431 ). However, the lack of
consistent evidence supportive
 P.796

of pathophysiologic progression in schizophrenia has been a weakness of this hypothesis (430 ). Recently, Riva and associates (432 )
found that acute or chronic administration of clozapine increased basic fibroblast growth factor (FGF-2) mRNA and protein in the rat
striatum, suggesting neuroprotective activity of clozapine. It has been proposed that small molecules that boost the endogenous
levels of BDNF or NT-3 might be useful for treating temporally protracted and severe forms of neurodegenerative disease, such as AD
or Parkinson's disease (433 ). Although neurotrophic factors are unable to cross the blood–brain barrier, potential alterations to
administration of these factors are transplantation of neurotrophic factor-producing cells, direct transfection of neurotrophic factor
gene, and development of compounds that modulate endogenous neurotrophic factor homeostasis and/or the influence their signal
transduction mechanisms (429 ). The augmentation therapy with neurotrophic factors suggests novel and innovative
pharmacotherapeutic, but as yet unproved strategies for schizophrenia.

CONCLUSION
Part of "56 - Therapeutics of Schizophrenia "
The therapeutic armamentarium for the treatment of schizophrenia has become rich and varied in the half century since the
inception of the pharmacologic era marked by the introduction of chlorpromazine. We now have the capacity to control many of the
symptoms of the disorder and restore the lives of patients. Much remains to be done in terms of drug discovery of new and novel
agents and the determination of their optimal use in conjunction with psychosocial and adjunctive therapies; however, there is
reason to be optimistic that future progress will be relatively swift.

ACKNOWLEDGMENTS
Part of "56 - Therapeutics of Schizophrenia "
Dr. Goff received research support from Cortex Pharmaceuticals, Eli Lilly & Company, Janssen Pharmaceuticals, and Pfizer, Inc. In
addition, he has received honoraria and/or served on an advisory board for Eli Lilly, Janssen, and Pfizer, Inc. Dr. Lieberman has
served as a consultant for a number of companies including: Janssen, Lilly, Astra Zeneca, Pfizer, Bristol Myers Squibb, Protarga, and
Wyeth Ayerst.

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57
The Economics of the Treatment of Schizophrenia

Susan M. Essock

Linda K. Frisman

Nancy H. Covell

Susan M. Essock: Department of Psychiatry, Mount Sinai School of Medicine, and the Mental Illness Research, Education, and
Clinical Center, VA Medical Center, Bronx, New York.

Linda K. Frisman and Nancy H. Covell: Connecticut Department of Mental Health and Addiction Services (DMHAS), Hartford,
Connecticut, and the Department of Psychology, University of Connecticut, Storrs, Connecticut.

 ECONOMICS OF MENTAL HEALTH AND SCHIZOPHRENIA

 COST-EFFECTIVENESS
 COST OF THE NEWER ANTIPSYCHOTIC MEDICATIONS
 CONCLUSION AND ADDITIONAL RESOURCES
 ACKNOWLEDGMENTS

ECONOMICS OF MENTAL HEALTH AND SCHIZOPHRENIA

Part of "57 - The Economics of the Treatment of Schizophrenia "

Prior to the 1980s, economists paid scant attention to schizophrenia, or indeed to mental health in general (1 ). Early work in the
field included efforts to consider the impact of organization and financing on system efficiency and to address the supply of
personnel in caring for persons with mental illness. For example, McGuire (2 ) reviewed the market for psychotherapy and the
insurability of mental health care, and Frank (3 ) examined the supply of psychiatrists. Researchers at RAND analyzed the impact of
cost-sharing on demand for mental health care (4 ). The use of diagnosis-related groupings to pay for care under prospective
payment was considered by Taube and his colleagues (5 ). Dickey and Goldman (6 ) reviewed the impact of various funding
mechanisms in public mental health.

During the 1990s, work on insurance, regulation, and the organization of mental health services continued. Studies of insurance
mandates for mental health care, such as Frank and colleagues' (7 ) simulation of mandates and related costs, provided valuable
information to state legislators considering such laws. Observers of systems change considered major reorganizational efforts, such
as those implemented through the Robert Wood Johnson Program on Chronic Mental Illness (8 ) and other types of organizational
reforms (9 ,10 ,11 and 12 ). The 1990s also brought analysis of the increasing implementation of managed care with behavioral
health carve-outs (13 ).

These examples of the contributions of mental health economists indicate the range of activities in the field of mental health
economics, including the economics of schizophrenia. This chapter focuses on two aspects of the economics of schizophrenia: cost
studies and cost-effectiveness studies. Studies of the cost of mental illnesses appeared before other types of work in the economics
of mental health, and they have continued throughout the past two decades. Cost studies lay the foundation for cost-effectiveness
and cost-benefit studies because they identify the range of resources that are consumed as a result of an illness. Cost-effectiveness
analyses of mental health programs began to appear in the early 1980s with the hallmark study by Weisbrod and colleagues (14 ) on
the cost-benefit of assertive community treatment teams. Collectively, studies of costs and cost-effectiveness are perhaps the most
important foci of the economics of schizophrenia. First, the sizable cost to society captures the attention of policy makers and
taxpayers, and convinces them of the huge fiscal impact of schizophrenia. Second, decisions by clinicians, managers, and policy
makers that are informed by research on costs and cost-effectiveness lead to better distribution of the resources available for
mental health care.

Costs Of Schizophrenia
Early studies of the costs of mental illness (15 ,16 and 17 ) did not distinguish between the costs of different diagnostic categories
(18 ). More recent studies have estimated specific costs for schizophrenia and other illnesses. Rice has estimated the cost of
schizophrenia in the United States at $32.5 billion in 1990 (19 ) and $44.9 billion in 1994 (20 ). Goeree and colleagues (21 ) have
calculated costs in Canada in 1996 to be approximately $2.35 billion, whereas Knapp (22 ) has estimated the costs of schizophrenia
in the United Kingdom to be 2.6 billion pounds. Some schizophrenia cost studies focus only on service costs, such as Rund and Ruud's
(23 ) study of costs in Norway and Martin and Miller's (24 ) study
 P.810

of Georgia Medicaid recipients. In contrast, Rice's and Knapp's studies include both direct costs (treatment and other service costs)
and indirect costs, such as lost income. But no study of the cost of schizophrenia can claim to capture all costs. As noted by McGuire
(18 ), even comprehensive studies of the cost of schizophrenia often underestimate two types of costs: the costs to families and the
costs of publicly owned capital.

Cost Perspective
Cost-of-illness studies like Rice's and Knapp's typically employ the perspective of society; that is, they consider economic costs.
Economic, or social, costs are the costs of resources consumed because of an illness. Cost-effectiveness and cost-benefit analysis
should always state the perspective from which the study is undertaken. Although a societal perspective presumably provides the
balanced view of the neutral scientist, it is also helpful to examine costs from perspectives of particular stakeholders. For example,
in an analysis of the impact of Assertive Community Treatment in Connecticut, Essock and colleagues (25 ) present costs from the
perspectives of society, the state, and the Department of Mental Health. Comparison of the results from multiple perspectives may
identify areas of cost-shifting that results from certain programs and policies. For example, a treatment that reduces hospital days
may shift costs from state-run inpatient facilities to private nonprofit outpatient settings.

Cost Components

Costs of Treatment and other Services

The examples provided by Rice and Knapp are instructive for those conducting cost-of-illness studies and cost-effectiveness studies
in the area of schizophrenia. As they show, there are many ways in which the illness is associated with greater costs. First are the
costs of treatment, including medication. Treatment may be offered by public, private, or voluntary sector settings, and many
persons with schizophrenia receive care in multiple places. Besides treatment, services like case management, vocational
rehabilitation, and psychosocial clubhouses generate significant costs. Medical and surgical costs also may be relevant in cost-
effectiveness analyses, because utilization of these services may vary depending on the adequacy of mental health care (26 ,27 ).

Lost Productivity and Family Burden

Mental illnesses, like other disorders, cause people to lose workdays (28 ) and sometimes even to forfeit aspirations of having any
career at all. Although lost productivity is usually addressed in comprehensive studies of the costs of schizophrenia, many studies of
interventions for persons with schizophrenia have ignored productivity losses, because of the high rates of disability in this
population. However, new strategies for improving the employment outcomes for persons with serious mental illness, such as
Individual Placement and Support (29 ), have made employment a realistic goal of rehabilitation. These new successes suggest that
loss of productivity should be included when calculating the cost of interventions for persons with schizophrenia. In addition to the
productivity losses of the individual, cost studies should attend to the work losses of family members, and other contributions of
time and in-kind services (30 ).

Capital Costs
Economic cost studies appropriately study the opportunity costs of all resources, that is, the value of those resources in their best
alternative use. In a cost-effectiveness study of a new residential model for persons with serious mental illness, Cannon and her
colleagues (31 ) carefully considered the value of capital costs of a public hospital, which would have been underestimated if valued
through traditional methods of depreciation. Capital costs can be large enough to change the most basic findings of a cost study, as
shown by Rosenheck and colleagues (32 ). Public administrators may not consider the value of buildings and property to be part of a
cost equation because it is not always part of the operating costs, but the value of the property in alternative use may be
considerable.

Other Components
Especially where an intervention is expected to have an impact on co-occurring substance use disorders, it is important to attend to
criminal justice costs (33 ). Another neglected aspect of cost studies is the costs of administering transfer payments (such as social
security). Although disability payments themselves do not represent the use of new resources, the cost of administering these
payments is a cost that should be counted, especially if the intervention could change the rate of receipt of disability payments or
other public benefits (34 ). For example, an intervention that returns people to work will not only increase their productivity (a
benefit), but also decrease disability payments (decrease a cost).

The importance of including any type of cost in a cost-effectiveness study is related to the potential impact of the cost type on the
study findings. The larger the cost per unit, or the more frequently it is used, the more carefully it should be assessed (35 ). But
should cost-effectiveness analyses always be conducted? As indicated by the range of costs and cost perspectives that might be
included, these studies can be expensive to implement. This expense is further increased because, to detect meaningful differences
in a highly variable outcome such as cost, significantly more study participants
 P.811

may be needed than for an effectiveness study alone. The usefulness depends in large part on the likelihood that the treatment or
intervention under study will have an effect on costs—either positive or negative. Cost studies are critical in the analysis of novel
antipsychotic agents because of the relatively high acquisition costs of the drugs, compared to conventional antipsychotic
medications, and because of the potential of these drugs to reduce the number of days that people with schizophrenia are
hospitalized (36 ).

COST-EFFECTIVENESS
Part of "57 - The Economics of the Treatment of Schizophrenia "

The success of interventions in schizophrenia, whether medications or psychosocial rehabilitation programs, is reflected in multiple
domains. An antipsychotic may have an impact on cognition, on hallucinations, on affect, on disruptiveness, on sexual functioning,
on extrapyramidal side effects, on weight, on employment—the list goes on and on. These are all measures of the effectiveness of
the agent, some positive and some negative. Some, such as hallucinations and delusions, may be influenced much more directly by
the medication than more distal outcomes such as housing or employment. Different individuals value changes in these domains
differently (elimination of hearing voices that other people don't hear may be much more important to the person who is troubled
by harassing voices making a steady stream of demeaning comments than to the person whose voices are good company or connect
the patient to an imagined network of internationally renowned researchers). Similarly, some people are very troubled by changes in
weight or sexual functioning, whereas such changes mean little to others. Hence, much as we would like a composite measure
across all effectiveness domains, this reductionistic approach is fraught with untenable compromises. Just as there is variation
among different patients and different providers, patients and payers ascribe different values to the same outcome (e.g., legislators
who make funding allocations to public mental health systems may be more concerned about decreases in violence and patients may
be more concerned with increases in quality of life). How much is a symptom-free day worth? It depends on who is asking and who is
paying.

Cost-effectiveness analyses have evolved to deal with the multiple domains touched by a single treatment. Such analyses report the
change in a given effectiveness measure associated with a particular cost investment in treatment. A medication may be cost-
effective with respect to certain outcomes, cost-neutral for others, and costly for yet others. Lehman (37 ) reminds us that the
current explosion of new knowledge about effective treatments and the advent of evidence-based quality standards for treating
schizophrenia come at a time when cost containment is paramount in the health policy agenda. Policy makers need to know the
impact of dollars invested in treatment—but not just in a single domain like reductions in hospital care. Those who make purchasing
decisions for the public systems of care under which most of the country's treatment for schizophrenia is funded need information
on multiple domains of effectiveness.

An alternative to cost-effectiveness analysis is cost-utility analysis, where a comprehensive outcome indicator is calculated as a
preference-weighted sum of the outcome measures. An example of a cost-utility approach is the use of quality-adjusted life years
(QALYs) (38 ,39 ). As noted above, different stakeholder groups value different outcomes differently; hence, approaches such as
QALYs create an effectiveness metric representative of at best only one stakeholder group, and at worst the resulting metric is
representative of no one. Although elegant in presentation, as with sausages, observing their creation can reduce enthusiasm for
their use. One must find or create weights to apply to the various effectiveness measures and then decide on a combination
scheme—deciding, for example, what weight gain is the equivalent of what change in extrapyramidal side effects (EPSs) and what
change in psychotic symptoms. Typically, one does this either by interviewing individuals representative of the population under
study (e.g., treatment refractory patients with schizophrenia) or by adopting someone else's measures as close enough. Because
“close enough” is a very subjective call, it is important for researchers to disclose the sources of the weighting estimates so that
readers can make their own call. For example, Rosenheck and colleagues' (40 ) report of changes in QALYs among mostly male
veterans with schizophrenia used weights derived as part of a doctoral dissertation by Kleinman (Johns Hopkins University, 1995) of
mainly African-American women, only about half of whom (55%) were diagnosed with schizophrenia (the rest were diagnosed with
major depression, bipolar, and other affective disorders). We were unwilling to take the leap of faith needed to generalize from
groups this disparate when presenting cost-effectiveness results from our own work (41 ). Nevertheless, Rosenheck and colleagues
are to be commended for providing the information necessary to follow back their methods to see what was used. This is not always
the case.

Another type of utility analysis is the measure of symptom-free days (42 ). Under such analyses, interventions are compared with
respect to the number of symptom-free days they produce. Following the methodology of Lave and colleagues (43 ), Simon and
colleagues (42 ) credited a study participant with having one depression-free day if the study participant had 2 days with a
depression score of 0.5. Many people with depression, as well as many researchers, would take issue with saying that someone was
symptom free for half a year if they reported having 50% of full symptoms for each day of that year. Symptom-free days may be a
poor measure within schizophrenia studies simply because, unlike with depression, symptoms and functioning are poorly correlated,
 P.812

and the likelihood of having a completely symptom-free day is rather small.

Disability-adjusted life years (DALYs), where one DALY equals one lost year of healthy life, can also be used to express years lost,
both to premature death and to disabilities associated with living with schizophrenia (44 ). In contrast to QALYs and estimates of
symptom-free days, DALYs are proxies for negative outcomes (45 ) and, as such, the calculation of cost-effectiveness centers around
how many DALYs are saved by using a particular intervention. In a population where individuals may live for a long period of time
with a relatively debilitating illness like schizophrenia, measures of mortality alone do not adequately capture the impact of the
disability. DALYs are calculated by adding together the number of years between mortality and life expectancy (years of life lost,
YLL) and the number of years lived with a disability (YLDs). Calculating YLDs requires making assumptions about the relative impact
of illness onset, duration, and severity on healthy living (for example, making an assumption that a first psychotic episode at age 15
is worse than a first episode at age 25). As with QALYs, these metrics can be derived by surveying individuals with schizophrenia or
their proxies, with the accompanying assumptions that how one weights hypothetical events is the same as the trade-offs one would
make if one could trade fewer days of healthy life for more days of life with particular disabilities. Because such ratings are
inherently untestable by rigorous methods, whether reliable or not, their validity remains suspect. Further, the calculation of DALYs
“presupposes that life years of disabled people are worth less than life years of people without disabilities” (46 ), and may even
rank some individuals' lives as worse than death (47 ). Schizophrenia brings with it an increased risk of suicide (48 ), which is
consistent with DALYs ranking some lives as worse than death. However, assuming that person A and person B, in reality, would
make the same choices as to what fates are worse than death presumes an ecologic validity to DALY ratings that may be
unwarranted.

Cost-utility measures such as QALYs, DALYs, and measures like symptom-free days, have enormous appeal because of their ability to
reduce multiple effectiveness domains to a single bullet measure. By deriving a single measure, one can compare any treatment
approach to any other treatment approach. Where the measure is reduced to dollars (as in QALYs), one may even compare the
values of interventions between different conditions (38 ), for example, if dollars expended on diabetes reap more benefits than
dollars spent on schizophrenia. But the assumptions built into such bullet measures may have limited usefulness for informing
decisions at the level of the individual patient, prescriber, or health care payer. These individuals weigh their particular
circumstances, and may be unwilling to have others' preferences serve as proxies for their own. Instead, these stakeholders are
asking more specific questions. For example, the mental health commissioner asks, “If I put an extra $3 million in the pharmacy
budget for medication X, what can I expect this to buy me in terms of the other domains under my purview, and what is the
downside risk? What will it buy me in terms of reductions in hospital use, improvements in vocational functioning, reductions in
violent episodes, and reductions in side effects?” Similarly, patients and families paying for medications ask, “If I increase/decrease
my spending by changing to medication X, what changes am I likely to see in the voices I hear, in my employability, in my sexual
functioning, and in my body movements?”

An alternative to composite measures are measures that contrast costs invested to a variety of outcome domains, some of which will
be more important to some stakeholders than to others. An analogy is a proposal for a city park to be funded from multiple sources.
Depending on one's perspective (e.g., whether you would use the park, how the park would impact the value of your property, your
safety, your recreational options, what you are called on to invest), the park may or may not be a good idea. And, depending on who
is paying for what, and which outcome domains are most important to you, you may stand to get a lot or a little out of the dollars
going into the park. The challenge is to present the data on costs and effects in such a way that the various payers (the city, private
foundations, neighborhood organizations, individual contributors) can each look from their own perspective, see what the expected
gains and losses are in the outcome domains they care about most (less street noise, more open space, more dogs, more people
drawn to the neighborhood), perform their own idiosyncratic weighting of these factors, and decide if they are in favor of the park
or not.

In contrast to cost-utility analysis, cost-effectiveness analysis does not reduce the impact of an intervention into one measure. Some
outcomes may be clearly preferential or “dominant choices” (e.g., lower costs and higher effectiveness). Other outcomes are not as
clearly dominant, and in these cases it may be useful to show the likely range of cost compared to multiple domains of effectiveness.
One method of examining these ranges is to create sampling distributions for costs and effectiveness measures to show the precision
of estimates as well as their mean. For example, bootstrap techniques use every study participant's data to create an empirical
sampling distribution of the test statistic and plot these estimates as a cost-effectiveness plane. Bootstrapping techniques offer one
means of describing confidence intervals for incremental cost-effectiveness ratios (ICERs) (49 ,50 ). Cost data are often highly
positively skewed, and ICERs provide less biased estimates of confidence intervals in highly skewed cost data (43 ,51 ,52 ).

Figure 57.1 shows such an approach when considering the cost-effectiveness of clozapine compared to conventional agents among
long-stay state hospital patients (41 ). The cluster of points displays the sampling distribution of the ICER. Most of the points fall in
the lower-right quadrant, indicating that clozapine is most likely to be less costly and
 P.813

more effective than conventional antipsychotic agents from the cost perspective (total societal cost) and for the effectiveness
measure in question (reduction in EPS). Such displays of information give the reader/policy maker a sense of the tightness of the
point estimate and the risk of falling in a quadrant other than the one indicating cost-effectiveness. One can use these sampling
distributions to create cost-acceptability curves from the viewpoint of particular payers for particular outcomes (e.g., the likelihood
that the intervention will be cost effective for the payer who is willing to risk $1, $10, or $100 to obtain an 80% likelihood of return
in sexual functioning).

FIGURE 57.1. Ten thousand bootstrap replications plotted in the cost-effectiveness plane (intent-to-treat, N = 136 clozapine
and N = 87 usual care; treatment crossovers excluded, N = 89 clozapine and N = 30 usual care). The x-axis and y-axis,
respectively, show the difference between clozapine and usual-care groups in estimated number of extrapyramidal side effects
(EPS)-free months and total cost during a 2-year period. The quadrant to the lower right of the origin (0,0) contains those
estimates where clozapine was found to be less costly and more effective than the usual care (80% of the estimates for the
intent-to-treat analyses and 81% of the estimates when treatment crossovers are excluded). (From Essock SM, Frisman LK,
Covell NH, et al. Cost-effectiveness of clozapine compared with conventional antipsychotic medication for patients in state
hospitals. Arch Gen Psychiatry 2000;57:992, with permission. Copyright 2000 American Medical Association.)

Saul Feldman (53 ) has held positions as the head of the National Institute of Mental Health (NIMH) Staff College and chief executive
officer of one of the country's largest managed behavioral health care organizations. Thus, he has been in a position to make policy
based on research, and to inform policy makers with research. He has posed the question, Is good research good if it does not inform
policy and practice? It is incumbent on mental health services researchers to report their findings in ways that speak to funders and
service system managers, which means providing estimates of the most likely outcome as well as the likelihood of alternative
outcomes.

COST OF THE NEWER ANTIPSYCHOTIC MEDICATIONS

Part of "57 - The Economics of the Treatment of Schizophrenia "

In general, the acquisition cost of the newer antipsychotic medications is greater than that of conventional ones. These acquisition
costs are reflected in formulary budgets. Once a relatively small component of treatment costs, formulary budgets in psychiatric
settings have risen dramatically in the past decade, and the market share of the newer agents has risen as they have replaced the
less costly conventional agents. Figure 57.2 shows the distribution of antipsychotic prescriptions paid for by Medicaid in 1998 (left)
and the
 P.814

dollars Medicaid paid for these prescriptions (right). These data show that the newer agents account for 58% of all antipsychotic
prescriptions paid for by Medicaid but for $1.15 billion (90%) of the $1.28 billion in Medicaid costs for antipsychotic prescriptions.
These charts dramatically display the disparity in medication costs associated with the newer versus the conventional agents.

FIGURE 57.2. Distribution of (left circle) and total dollars paid (right circle) by Medicaid for antipsychotic medication
prescriptions during 1998. Newer antipsychotic medications represented slightly over half of the total prescriptions, and they
were responsible for 90% of the total cost.

This price difference between the older and the newer antipsychotic medications, which can be a 100-fold difference (e.g., when
contrasting generic oral haloperidol with nongeneric Clozaril), prompted scores of studies asking the cost impact of using the newer
medications when more than simply the cost of the medication was considered. For example, if using new and expensive medication
X results in fewer days hospitalized than some alternative, then, all else being equal, using X will reduce overall costs as long as the
cost savings associated with fewer days in the hospital is greater than the cost difference between medication X and the alternative.

Clozapine Cost Effectiveness Studies As Case Examples

The rub, of course, is that “all else” is rarely equal in effectiveness or cost-effectiveness studies, and the early cost projections
concerning the impact of using clozapine often suffered from faulty assumptions about what was equivalent. Many of these studies
were pre–post comparisons that examined changes in hospital use and lacked a comparison group (54 ,55 ,56 ,57 ,58 ,59 and 60 ).
For example, the study by Meltzer and colleagues (59 ) of patients with schizophrenia who were taking clozapine collected
retrospective cost data for 2 years before and after these 47 individuals began taking clozapine and concluded that clozapine was
associated with a 23% drop in treatment costs. This study generated a series of letters criticizing the study's methodology (61 ,62
and 63 ) [see also the response by Meltzer and Cola (64 )]. Critics focused on the problem of the regression toward the mean that
can be expected whenever study participants are enrolled during a low point in their functioning (such as may have prompted the
initiation of clozapine), and on the other potential temporal and case-mix confounds associated with mirror-image studies of
individuals who were selected to begin a medication through other than a random-assignment process among all eligibles.

The results of the two randomized clinical trials of clozapine's cost-effectiveness each showed much more modest benefits
associated with clozapine, both in the 2-year, open-label trial comparing clozapine to the usual care with a range of conventional
antipsychotics among long-term patients in state hospitals (41 ,65 ,66 ), and in the 1-year masked trial comparing clozapine to
haloperidol among veterans hospitalized for a year or less (67 ). Each trial showed clozapine to be somewhat more effective than
the comparison agents, and this increase in effectiveness comes at no additional cost when costs are viewed from a societal
perspective. Each trial also showed that clozapine is more effective than the usual care in minimizing days hospitalized, enough so
that the reduction in hospital days more than covers the increased cost of the medication plus increased outpatient services. But,
from more narrow perspectives (e.g., the hospital formulary budget, capitated outpatient service providers), clozapine would be
viewed as increasing their costs. For cost-effectiveness studies to influence planning and policy making, the perspectives of these
different payers need to be taken into account because it is these local incentives and disincentives that must be addressed to be
sure that the fiscal incentives are lined up to promote good care. A hospital would have a great incentive to use clozapine for a
heavy user of hospital services if it has a fixed budget (the case with most state hospitals), but a hospital paid a per diem would
have no such incentive.

Lengthy randomized clinical trials in routine practice settings, such as the clozapine study in Connecticut state hospitals and the
clozapine study in Veterans Administration (VA) hospitals, suffer from treatment crossovers. By the end of 6 months in the
Connecticut study, only 11% of the usual care patients had begun a trial on clozapine, but by the end of 24 months in the study, 66%
had. In the VA clozapine study, 72% of the patients assigned to masked haloperidol had ceased taking the masked medication by the
end of the 1-year study period, with 49 of 157 (31%) of them switching to clozapine and the rest to conventional antipsychotics,
including unmasked haloperidol (67 ). Because of the biases introduced by what is likely to be highly nonrandom discontinuation of
the assigned treatment, the importance of intent-to-treat analyses and the unspecified biases of crossovers-excluded analyses are
well documented (68 ). Regardless, when crossovers are common, analyses excluding crossovers offer a proxy for the best-case
scenarios for each treatment condition by comparing only those who do well enough on treatment A to stay on it with only those
who do well enough on treatment B to stay on it. Figure 57.1 illustrates this using data from the Connecticut clozapine study. The
exclusion of treatment crossovers increases the apparent effectiveness of clozapine (the crossoversexcluded oval is shifted to the
right of the intent-to-treat oval in Fig. 57.1 ) and decreased the estimate of the relative costliness of clozapine (the crossovers-
excluded oval is shifted lower by about $5,500) (41 ). Clearly, individuals who leave their assigned treatment are different in terms
of costs and outcomes from those who remain in their assigned treatment condition.

Another difficulty when trying to assess relative costs is the great variability in costs across patients. For example, in the VA study
just cited, health care costs in the 6 months prior to randomization were approximately $27,000 with a standard deviation of about
$17,000 (67 ). For the Connecticut clozapine study, the 95% confidence interval for patients assigned to clozapine was $96,847 to
$114,308 for year 2 versus $103,665 to $121,144 for those assigned to the usual care. With such variability, cost differences are
 P.815

very difficult to detect, even with the relatively large sample sizes of the VA and Connecticut trials (N = 423 and 227, respectively).
Even for individuals who are heavy service users at study entry, mounting a trial powered to detect cost differences requires
hundreds of individuals per treatment arm. If the trial were a study of outpatients who are infrequent users of expensive services
like hospitals, it would require even larger samples to detect cost differences apart from medication.

From a public health perspective, an emphasis on point estimates of costs and effectiveness is misguided when the confidence
intervals are so broad. Economists would call clozapine the dominant alternative in these randomized trials (because most of the
range spanned by the cost confidence intervals includes the values where clozapine costs less than or the same as the usual care and
the effectiveness measures favor clozapine or are neutral). The reduction of data to such a point estimate belies the broad
distribution of possible outcomes that are likely to occur across patients. Planners and policy makers, as well as patients and their
treating clinicians, need a sense of the range of possible outcomes and their relative likelihood to inform their decisions about what
chances they want to take.

Costs Associated with Risperidone, Olanzapine, and Quetiapine

Figure 57.3 shows the frequency of prescribing by type of antipsychotic in three large states in different parts of the country among
individuals whose medications are paid via
 P.816

Medicaid. Because Medicaid formularies allow unrestricted access to any of these medications independent of location in the
country and the same financial incentives apply, one would expect to see similar rates of prescribing these medications. Indeed, the
distributions do appear quite similar to each other and to the national data (Fig. 57.2 ). That these distributions do not reflect what
we know about the relative effectiveness of these agents suggests that other factors are strong influences on medication choice and
that these influences combine to create similar patterns of antipsychotic prescribing under Medicaid nationwide. In addition to
factors such as effectiveness, factors as disparate as patients' past histories of medication use, order of receiving Food and Drug
Administration (FDA) approval, convenience of use, acquisition costs, relative marketing budgets, and side-effect profiles may also
be at play. These figures serve as reminders that medications are started and discontinued for reasons other than effectiveness.
(Data for these pie charts were extracted from the Health Care Financing Administration's (HCFA) Web site
http://medicare.hcfa.gov/medicaid/drug5.htm; potential users take note that, as of March 2000, the Web site reported Medicaid
expenditures in cents rather than dollars and does not label the cost units.)

FIGURE 57.3. Distribution of (left circles) and total dollars paid (right circles) by Medicaid for antipsychotic medication
prescriptions in California, Ohio, and New York during 1998.

Several studies of risperidone and olanzapine also suggest that the acquisition costs of these medications may be offset by reduction
in use of more expensive health care services such as inpatient treatment. Many of these studies have methodologic shortcomings
similar to those of the earlier cost studies of clozapine described above. Another concern is that industry sponsorship of many of
these studies means that they do not meet the criteria for lack of an incentive for bias set forth by the New England Journal of
Medicine (69 ), whose editors noted that the opportunities for introducing bias into economic studies are far greater than in studies
of biological phenomena because of the unusually discretionary nature of model building and data selection in such analyses, and
because drug costs in particular can be quite arbitrary as they are prices (not costs) set by the manufacturer. Hence, additional
work is needed in this area.
In general, the studies by the medications' manufacturers show support for cost reductions favoring that manufacturer's medication
[e.g., for risperidone (70 ) and for olanzapine (71 ,72 )]. Although such studies form good starting points for further investigation,
they need follow-up by independent investigators to assess how the agents' cost-effectiveness plays out in broader settings with
representative patients, lest best-case examples be generalized to settings where they are not applicable and used to set policy
there. An example of an important follow-up study is that of Conley and colleagues (73 ), who found that, among 84 treatment-
refractory patients randomly assigned to a double-blind 8-week fixed-dose trial of either olanzapine or chlorpromazine, olanzapine
appeared to have limited efficacy, showing only a 7% response. Hence, the reduction in treatment costs associated with olanzapine
noted in the reviews of Palmer and colleagues (74 ) and Foster and Goa (75 ) would not be expected among treatment-refractory
patients, even though these patients are heavy users of inpatient services. Under other scenarios, these patients are the very ones
for whom new interventions are associated with cost savings because they have higher initial rates of utilization on which to show an
impact (25 ,40 ). An independent study of risperidone compared to conventional antipsychotics among outpatients with
schizophrenia using a matched comparison group found no difference in total treatment costs or effectiveness measures, although
there was a trend for the risperidone-treated group to have higher costs, attributable to higher medication costs (76 ).

CONCLUSION AND ADDITIONAL RESOURCES

Part of "57 - The Economics of the Treatment of Schizophrenia "
This brief review of the economics of treating schizophrenia has emphasized some of the methodologic complexities that must be
acknowledged and surmounted when addressing treatment costs. The emphasis has been on illustrating the importance of bias
minimization and estimation when constructing studies and reporting results, and on the importance of reporting results from the
perspective of different payers and giving estimates of the variability associated with any findings reported as point estimates. It is
important to tell patients, prescribers, and payers not just the best estimate of costs and effectiveness, but the likelihood that their
costs and outcomes will fall within their acceptable ranges for what they are willing to pay and/or risk to gain a given outcome.
Fortunately, this evolving literature has many active champions who publish widely and lead the way in documenting the ways in
which costs and fiscal (dis)incentives have impact on access to treatment, the quality of care received, and patient outcomes.
Although their work cannot be summarized here, useful source books include those by Drummond and colleagues (38 ), Frank and
Manning (77 ), Gold and colleagues (39 ), and Hargreaves and colleagues (35 ). The journal Health Affairs continues to be a
particularly valuable resource for reports on mental health economics and thoughtful analyses of the economic influences on the
treatment of individuals with schizophrenia.

ACKNOWLEDGMENTS
Part of "57 - The Economics of the Treatment of Schizophrenia "
This research is the product of the collaboration of many individuals, both within and outside the Connecticut Department of Mental
Health and Addiction Services (DMHAS). In particular, we would like to thank Carlos Jackson, Ph.D., of the University of Connecticut
for his assistance with the data extraction and statistical analyses of the Medicaid prescription data. The research was funded
 P.817

in part by U.S. Public Health Service (USPHS) grants R01 MH-48830 and R01 MH-52872 from the National Institute of Mental Health (NIMH) to Susan Essock, Ph.D., principal investigator,
as well as by DMHAS. This publication does not express the views of the Department of Mental Health and Addiction Services or the State of Connecticut. The views and opinions
expressed are those of the authors.

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58
Mechanism of Action of Atypical Antipsychotic Drugs

Herbert Y. Meltzer

Herbert Y. Meltzer: Bixler Professor of Psychiatry and Pharmacology, Vanderbilt University School of Medicine, Nashville,
Tennessee.

 ATYPICAL ANTIPSYCHOTIC DRUGS: WHAT IS TO BE EXPLAINED

 ROLE OF D2 RECEPTORS
 D3 AND D4 RECEPTOR MECHANISMS
 ROLE OF SEROTONIN IN ANTIPSYCHOTIC DRUG ACTION
 ATYPICAL ANTIPSYCHOTICS AND THE 5-HT2A RECEPTOR
 SEROTONIN RECEPTORS AND COGNITIVE FUNCTION
 5-HT2A RECEPTOR BLOCKADE AND EXTRAPYRAMIDAL FUNCTION
 THE ROLE OF THE 5-HT2C RECEPTOR IN ANTIPSYCHOTIC DRUG ACTION: 5-HT2A AND 5-HT2C INTERACTIONS
 THE ROLE OF THE 5-HT1A RECEPTOR IN ANTIPSYCHOTIC DRUG ACTION
 THE ROLE OF SEROTONIN RELEASE IN ANTIPSYCHOTIC DRUG ACTION
 α2- AND α1-ADRENERGIC MECHANISMS AND ATYPICAL ANTIPSYCHOTIC DRUGS
 CONCLUSION
 ACKNOWLEDGMENTS

ATYPICAL ANTIPSYCHOTIC DRUGS: WHAT IS TO BE EXPLAINED

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

The designation of chlorpromazine, and subsequently haloperidol, thioridazine, loxapine, thiothixene, molindone, pimozide, and
related compounds as antipsychotic drugs (1 ) reflects their predominant action in humans, namely the suppression of auditory
hallucinations and delusions, in some, but not all, individuals with diagnoses of schizophrenia as well as other psychoses. These
drugs are also called neuroleptics because they caused catalepsy in rodents and extrapyramidal side effects (EPSs) in humans (2 ).
Their ability to diminish psychotic symptoms was convincingly shown to be initiated by blockade of dopamine (DA) D2 receptors in
mesolimbic nuclei, especially the nucleus accumbens, stria terminalis, and the extended amygdala (2 ,3 ,4 and 5 ). Blockade of D2
receptors in terminal regions, e.g., the striatum, nucleus accumbens, and prefrontal cortex, by these agents initially causes
compensatory increases in the activity of dopaminergic neurons in the substantia nigra and ventral tegmentum, respectively,
followed by a gradual decrease in the activity of DA neurons, and, ultimately, complete inactivation of DA neuron firing in both
regions (6 ). This so-called depolarization block was suggested to be the reason for the slow onset of antipsychotic action, which is
observed in some, but not all, psychotic patients. The development of depolarization block following subchronic haloperidol
treatment has been challenged by Melis et al. (7 ) on the basis of microdialysis studies of DA release in the nigrostriatal system, but
those findings have been suggested by Moore et al. (8 ) to be an artifact.

These first-generation antipsychotic drugs are often referred to as typical antipsychotic drugs because they typically produce EPSs,
e.g., acute dystonic reactions, subacute parkinsonism, and akathisia, and, after chronic use, tardive dyskinesia or dystonia (see
Chapter 56 ) as a direct or indirect result of blockade of D2 receptors in the dorsal striatum, in vulnerable individuals. The
immediate cause of acute and subacute EPSs is considered to be blockade of the dopaminergic inhibition of striatal cholinergic
neurons, leading to increased cholinergic activity in the basal ganglia (2 ). Subsequently, clozapine was found to achieve an
antipsychotic effect without causing EPSs, whereas loxapine, a clozapine congener, was equipotent in producing its antipsychotic
action and EPS in humans and laboratory animals (10 ). This led Hippius and Angst to describe clozapine as an atypical antipsychotic
drug (11 ). Preclinical scientists almost invariably refer to clozapine and other drugs that have antipsychotic properties and low EPSs
as atypical antipsychotics but, as will be discussed, clinical investigators do not universally accept this designation.

The atypical profile of clozapine was initially attributed to its anticholinergic properties, which, along with other unknown features,
caused selective depolarization of the A9 DA neurons in the substantia nigra, which project to the dorsal striatum, sparing those of
the A10 ventral tegmentum, which project to the cortex and mesolimbic systems (12 ). The subsequent evidence that clozapine,
compared to neuroleptic drugs such as haloperidol, had at least six advantages in addition to producing significantly less EPS and
tardive dyskinesia, has attracted enormous interest to clozapine and other subsequently developed atypical antipsychotic drugs.
These six effects of clozapine, not all of which are fully shared with other atypical antipsychotic drugs, are (a) absence of tardive
dyskinesia; (b) lack of serum prolactin elevations in humans; (c) ability to eliminate positive symptoms without exacerbating motor
symptoms in patients with Parkinson's disease who become psychotic due to exogenous dopaminergic agents such as levodopa (L-
DOPA); (d) ability to decrease or totally eliminate psychotic symptoms in approximately 60% of the patients with schizophrenia who
fail to respond to typical neuroleptic drug; (e) ability to improve primary and secondary negative symptoms;
 P.820

and (f) ability to improve some domains of cognition in patients with schizophrenia, especially secondary memory and semantic
memory (verbal fluency) (9 ,11 ,13 ). Some of the atypical antipsychotic drugs have also been shown to be more effective than the
typical neuroleptic drugs in improving depression, stabilize mood, and decrease suicidality (9 ,10 ). These collective advantages of
clozapine led to the search for the mechanism(s) involved in these effects and to find drugs that did not have the panoply of side
effects of clozapine, especially agranulocytosis (9 ).

The other widely available antipsychotic drugs that are classified as atypical, by consensus, are, in order of their introduction,
risperidone, olanzapine, sertindole, quetiapine, and ziprasidone. Melperone, a butyrophenone, introduced at about the same time
as clozapine, has also been suggested to be an atypical antipsychotic drug because of its many clinical similarities with clozapine
(14 ). Other agents with the essential clinical characteristics of an atypical antipsychotic drug that are currently at an advanced
stage of clinical testing are iloperidone, ORG-5222, and aripiprazole (9 ,10 ). Zotepine and amisulpride, both of which are widely
used antipsychotic drugs in Europe, are also sometimes grouped with the atypical antipsychotic drugs (9 ,10 ). With the exception of
aripiprazole, a partial DA agonist (15 ), and amisulpride, a selective D2/D3 antagonist (16 ), all of the drugs listed above cause
potent serotonin (5-hydroxytryptamine, 5-HT) receptor subtype 5-HT2A relative to DA D2 receptor blockade (17 ,18 and 19 ).

There are a very large number of drugs in development as antipsychotics that have the property of being active in various animal
models that predict antipsychotic action, e.g., blockade of amphetamine-induced locomotor activity or of the conditioned
avoidance response, at doses 5- to 20-fold lower than that which produce catalepsy, a predictor of EPSs. All of these drugs are
routinely referred to as putative atypical antipsychotic drugs, at least by preclinical scientists, because of their ability to produce an
antipsychotic action at doses that do not cause significant EPSs in humans and a comparable dissociation in animal models of
psychosis and EPSs, e.g., blockade of conditioned avoidance response and blockade of DA-induced locomotor activity, and the
induction of catalepsy, respectively. These drugs differ greatly in chemical structure and, to some extent, pharmacologic profile,
and thus cannot be referred to as a group by either chemical class or pharmacologic profile. However, some clinical investigators
find the term atypical antipsychotic drug misleading because there are important clinical differences among the compounds with
regard to the six clinical features of clozapine noted above, and they prefer the term novel or new generation over atypical to
describe these agents. However, this temporal-based nomenclature is not routed in any meaningful or enduring characteristic of
these agents. Others prefer to call them multireceptor antipsychotics, which is clearly preferable to 5-HT/DA antagonists, another
commonly used term. It is our view that these other designations have no specific advantages and some disadvantages compared to
the classic term atypical. Thus, this chapter continues to use the term atypical to designate antipsychotic drugs that have a major
advantage with regard to EPSs in patients with schizophrenia or Parkinson's disease, or both, to contrast with the typical
antipsychotic drugs, and to update some of the key hypotheses for explaining some of the other highly valued advantages of these
agents, as well as their unique side effects.

As can be expected, there has been an intensive effort to determine the basis for the differences between the typical and atypical
antipsychotic drugs. This chapter reviews the major hypotheses, which are based on the pharmacologic profiles of the numerous
classes of agents with atypical properties as well as current theories of the action of drugs effective in animal models of psychosis,
but not yet adequately tested in humans, e.g., AMPA antagonists and metabotropic glutamate receptor antagonists (20 ,21 ).

The affinities of clozapine and some of its congeners for monoamine receptors are given elsewhere in this volume (see Chapter 56 )
(22 ). The affinities reported therein are for the D1, D2, D3, D4, 5-HT1A, 5-HT1D, 5-HT2A, 5-HT2C, 5-HT6, 5-HT7, α1, α2, H1, and
muscarinic M1 receptors. Of these, the greatest interest is in the role of D2, D4, 5-HT1A, 5-HT2A, 5-HT2C, α1, and α2 receptors.
Clozapine does have effects on glutamate and GABA neurons and interneurons, respectively, but space considerations preclude their
discussion here.

ROLE OF D2 RECEPTORS
Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

Most effective antipsychotics, typical as well as atypical, have affinities for the DA D2 receptor high enough to suggest that they
produce effective blockade of these receptors in vivo (23 ,24 ). The model for atypical antipsychotic drug action proposed by
Meltzer et al. (17 ) postulated that atypical antipsychotic drugs had to have some D2 receptor blockade in vivo, although weaker
than 5-HT2A receptor blockade, to achieve a low EPS profile and, possibly, some of the other advantages of clozapine. An exception
to this may be amperozide, with is a potent 5-HT2A antagonist and DA reuptake inhibitor with very low affinity for the D2 receptor
(25 ). Recently, NRA0045, which has potent 5-HT2A, D4, and α1 but no D2 or D3 receptor blockade has been found to have atypical
antipsychotic properties (26 ). Partial DA agonists, which may act as agonists at presynaptic DA receptors, and antagonists at
postsynaptic DA receptors are a new class of antipsychotic drugs that has promise (15 ,27 ,28 ). However, clinical testing of these
agents is just beginning, and current data support only the view that they are atypical in the classic sense, i.e., they are
antipsychotic in preclinical or clinical testing at doses that produce weak or absent EPSs. Other evidence of the importance of α1
antagonism for atypical antipsychotic drug activity will be discussed subsequently. The in vitro affinity of a drug at the DA D2
receptor
 P.821

is a useful predictor of the dose that produces EPSs and control of positive symptoms for typical neuroleptic drugs (2 ,29 ), although
it does not do so for some atypical antipsychotic drugs, e.g., ziprasidone. Furthermore, there is no agreement on how to determine
the doses used in such correlations because of the differences in dosage requirements as a function of stage of illness, body mass
index, and age. There is little agreement even on the best dose for haloperidol, the most widely used antipsychotic drug. A wide
range of 2 to 15 mg/day has been suggested, far removed from the 20 to 40 mg/day thought to be most effective in 1966 (9 ,10 ). To
date, no clinically proven antipsychotic with the possible exception of amperozide lacks significant D2 receptor antagonist
properties. As will be discussed, the combination of D2 and 5-HT2A receptor blockade, in the right ratio, produces some of the
effects of clozapine and other atypical antipsychotic drugs in rodents, e.g., increases DA efflux in the cortex and striatum of rats
(30 ) and blockade of the conditioned avoidance response, an indication of antipsychotic activity (31 ). There have been only limited
tests of this hypothesis in humans, mainly using ritanserin, which is a mixed 5-HT2A/2B/2C antagonist (32 ). Nevertheless, various
comprehensive reviews of the action of the atypical antipsychotic drugs have concluded that the combination of 5-HT2A, D2, and α1
receptor blockade is the probable basis of their antipsychotic action (10 ,18 ,19 ,22 ,33 ,34 ,35 ,36 and 37 ). The evidence for this
hypothesis will be discussed subsequently.

Counter to the hypothesis of the importance of 5-HT2A receptor antagonism to the action of clozapine and other atypical
antipsychotic drugs is the proposal of Seeman and Tallerico (24 ) and Kapur and Seeman (38 ) that the basis of atypical
antipsychotics may lie in their rapid dissociation from the DA D2 receptor and their relatively easily displacement by surges of
endogenous DA. It has also been proposed that rapid and extensive displacement of clozapine and quetiapine from binding sites
accounts for the reported low occupancy of striatal D2 receptors by these drugs (24 ). The authors also suggested that this might
account for more rapid relapse following clozapine and quetiapine withdrawal (24 ). Although the evidence cited for clozapine-
induced relatively rapid relapse is robust (39 ), the evidence with regard to quetiapine and rapid relapse has never been published
and does not accord with general clinical experience. Seeman and Tallerico found that the affinity for and rate of dissociation of
antipsychotics from the D2 receptor are highly correlated. Drugs with low affinity for the D2 receptor, e.g., clozapine and
quetiapine, were found to have a higher dissociation rate constant than drugs with higher affinity, e.g., haloperidol. Rapid
dissociation from the D2 receptor was reported to also permit easier displacement of clozapine and quetiapine by endogenous DA,
thereby avoiding side effects related to DA receptor blockade such as EPSs and hyperprolactinemia (38 ). It was also reported that
olanzapine, risperidone, and sertindole, all of which are well established as atypical antipsychotic drugs, are comparable to
haloperidol in their rate of dissociation from the D2 receptor and are not displaced by raclopride or iodobenzamide, as are clozapine
and quetiapine (24 ). Thus, this hypothesis could not explain the basis for their low EPSs. Moreover, for these agents to achieve their
antipsychotic action, they would have to be less easily displaced from limbic and possibly cortical D2 receptors. There are no data to
support this selectivity with regard to displacement as yet. Although there is evidence for higher occupancy of extrastriatal D2
receptors by clozapine and quetiapine in patients with schizophrenia (40 ,41 ), and for atypical antipsychotic drugs that show more
potent 5-HT2A receptor blockade in rodents (42 ), the same appears to be true for olanzapine, which does not show the higher off-
rates of clozapine and quetiapine (R. Kessler and H. Meltzer, in preparation). Because clozapine produces a greater increase in DA
release in the cortex than in the accumbens or striatum, at least in rodents and monkeys (43 ), clozapine might be expected to
produce greater occupancy of D2 receptors in these regions than the cortex, but, as noted above, this is not the case. It is also not
clear how this model could explain any of the advantages of clozapine with regard to efficacy in neuroleptic-resistant patients or for
cognition.

Studies on the regulation of prefrontal cortical or limbic DA release also provide no evidence that blockade of D2 receptors alone,
regardless of degree of occupancy, can mimic the effects of the multireceptor antagonists such as clozapine (31 ,44 ,45 ).
Pharmacologic analysis of this important model for the action of atypical antipsychotic drugs on cognition and negative symptoms
strongly supports the importance of combined blockade of 5-HT2A, D2, and possibly α1 receptors (36 ,37 ,44 ,45 ).

D3 AND D4 RECEPTOR MECHANISMS

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

Ever since the cloning and characterization of the distribution of the D3 and D4 receptors, which revealed a limbic and cortical
distribution, there has been considerable speculation about the role of these receptors in schizophrenia and the mechanism of
action of antipsychotic drugs (19 ,46 ,47 and 48 ). As specific antagonists have become available, it has been possible to test their
efficacy in animal models of psychosis, cognition, and motor function, as well as to carry out some clinical trials in schizophrenia.
Recently, Reavill et al. (49 ) reported that SB-277011-A, which has high affinity and selectivity for the D3 receptor and good brain
bioavailability, has an atypical antipsychotic drug profile. This compound was active in preventing isolation-induced deficits in
prepulse inhibition but was not effective in blocking either amphetamine- or phencyclidine (PCP)-induced locomotor activity or, by
using microdialysis, to increase prefrontal cortical DA release in rats (49 ). However, subchronic administration of SB-277011-A
selectively decreased the firing rate of A10, but not A9, DA neurons in the rat, indicating a clozapine-like profile (50 ). These are the
most promising
 P.822

data yet that a selective antagonist of D3 receptors might be useful in the treatment of psychosis.

On the basis of the finding that the affinity of clozapine for the cloned DA D4 receptor was two to three times greater than that for
the D2 receptor, Van Tol et al. (51 ) suggested that blockade of the D4 receptor was the basis for the superiority of clozapine over
the typical neuroleptic drugs in the treatment of schizophrenia. Other investigators have found less of a difference between the
affinity of clozapine for D2 and D4 receptors (48 ). Several typical antipsychotic drugs, including haloperidol, have nearly equivalent
affinity for D2 and D4 receptors, suggesting that D4 affinity per se does not convey any special advantages for an antipsychotic drug
(48 ). The preclinical behavioral and electrophysiologic profile of highly selective D4 antagonists provides mixed evidence with
regard to antipsychotic action associated with this receptor (52 ,53 and 54 ). A D4/α1 antagonist, NRA0025, appears to have promise
as an antipsychotic agent based on its preclinical profile (26 ). A clinical trial of a selective D4 antagonist showed no sign of activity
(55 ). A compound with potent 5-HT2A and D4 antagonist properties, finanserin, was also ineffective (56 ). Further clinical trials of
compounds that have D4 or D3 antagonism, or both, together with high affinities for 5-HT1A or α1 receptors, and without D2 affinity
seems indicated.

ROLE OF SEROTONIN IN ANTIPSYCHOTIC DRUG ACTION

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

Determining the biological basis for the advantages of clozapine and other atypical antipsychotic drugs cited above and described in
detail by Miyamoto et al. in Chapter 56 has led to considerable interest in the role of 5-HT in antipsychotic drugs action. We now
consider some of this evidence. Other reviews of this topic should also be consulted (18 ,19 ,22 ,34 ,35 ,36 and 37 ,57 ).

Serotonin Receptors Involved in Antipsychotic Drug Action

The hypothesis that a relatively high affinity for the 5-HT2A receptor compared to an affinity for the D2 receptor was the basis for
the difference between atypical and typical antipsychotic agents contributed to the development of the newer antipsychotic agents
listed above, all of which support the previously mentioned hypothesis of high affinity for 5-HT2A and low affinity for D2 receptors
(17 ,58 ,59 ). However, other 5-HT receptors may be important to the action of clozapine and other recently introduced
antipsychotic agents, or of potential value for developing more effective or better tolerated antipsychotic agents. These include the
5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT6, and 5-HT7 receptors (19 ,22 ,33 ). Although some of the atypical antipsychotic drugs developed
on the basis of the 5-HT2A/D2 hypothesis also have affinities for 5-HT2C, 5-HT3, 5-HT6, or 5-HT7 receptors that are in the same range as
that for the 5-HT2A receptor, this is not characteristic of all of these agents, and thus it is not likely that affinities for these
receptors are primary factors contributing to the low EPS profile of the entire class of agents (19 ,22 ,60 ,61 ). However, this does
not rule out that actions at various 5-HT receptors contribute to low EPSs of specific drugs, or other actions, e.g., cognitive
improvement or improvement in negative symptoms. For example, 5-HT1A receptor agonism has also been suggested to be able to
contribute to an atypical antipsychotic drug profile (62 ), and some of the atypical antipsychotics are 5-HT1A partial agonists as well
as 5-HT2A/5-HT2C antagonists, e.g., clozapine, quetiapine, ziprasidone, and S16924 (63 ,64 ). The role of the 5-HT4 receptor in
cognition will be discussed subsequently. Furthermore, there is some evidence of interactions among the 5-HT1A, 5-HT2A, and 5-HT2C
receptors (19 ,35 ). Because of space limitations, this chapter focuses on these three 5-HT receptors and briefly considers the others.

ATYPICAL ANTIPSYCHOTICS AND THE 5-HT RECEPTOR 2A

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

5-HT2A receptors have been implicated in the genesis of, as well as the treatment of, psychosis, negative symptoms, mood
disturbance, and EPSs (17 ,19 ,33 ,34 ,35 and 36 ,45 ). The hallucinogenic effect of indole hallucinogens has been related to
stimulation of 5-HT2A rather than 5-HT2C receptors (65 ). Numerous studies have examined the density of 5-HT2A receptors in various
cortical regions of patients with schizophrenia with decreased (66 ,67 ), increased (68 ), or normal levels reported. It is well
established that some typical and atypical antipsychotic drugs can decrease the density of 5-HT2A receptors (69 ,70 ), so the
postmortem results noted above may be related to drug treatment. Positron emission tomography (PET) studies have not found
decreased 5-HT2A receptors in the cortex of never-medicated or unmedicated patients with schizophrenia (71 ). As mentioned above,
the antipsychotic effect of clozapine has been attributed, in part, to its ability to block excessive 5-HT2A receptor stimulation
without excessive blockade of D2 receptors (17 ). This conclusion is consistent with the high occupancy of 5-HT2A receptors produced
by clozapine at clinically effective doses and its low occupancy of D2 receptors (in the 30% to 50% range as measured with
[3H]raclopride), the latter being significantly below the 80% to 100% occupancy usually produced by typical neuroleptic drugs
(35 ,72 ,73 ,74 and 75 ). The occupancy of 5-HT2A and D2 receptors has been studied with other novel antipsychotic drugs such as
risperidone, olanzapine, sertindole, and quetiapine with results similar to those of clozapine; all are more potent 5-HT2A and D2
antagonists at appropriate doses, but less so than clozapine (72 ,73 ,74 and 75 ).
 P.823

Some of these agents (e.g., risperidone and olanzapine) produce high D2 occupancy at high doses (76 ,77 ).

The bell-shaped dose–response curve of risperidone, with higher doses being less effective than lower doses (78 ), is consistent with
the hypothesis that excessive D2 receptor antagonism may diminish some of the beneficial effects of 5-HT2A receptor blockade
(17 ,19 ,33 ,35 ,36 ). The highly selective 5-HT2Aagonist M100907, formerly MDL 100907, has been found in a controlled study to have
some efficacy for treating positive and negative symptoms in hospitalized schizophrenic patients (79 ). However, because it was less
effective than haloperidol, no further testing in schizophrenia has been scheduled at present. Nevertheless, the concept that 5-HT2A
antagonism may be useful to treat some forms of psychosis, especially when combined with weak D2 receptor blockade, warrants
further study. Other 5-HT2A selective agents such as SR 46349B (80 ) are currently being tested. Additional clinical evidence
supporting the role of 5-HT2A receptor blockade in the action of clozapine and possibly other drugs with potent 5-HT2A affinities is
available from the several reports that the His452Tyr allele of the 5-HT2A receptor, which is present in 10% to 12% of the population,
is associated with a higher frequency of poor response to clozapine (81 ,82 ). Taken together, the evidence from clinical trial data
suggests that 5-HT2A receptor blockade may contribute to antipsychotic drug action.

There is additional basic research that is also consistent with the relevance of 5-HT2A receptor blockade for antipsychotic drug action.
Thus, M100907 or other selective 5-HT2A receptor antagonists, either alone or in combination with selective antagonists of other
receptors, have been found to be effective in various animal models of psychosis. These include (a) blockade of amphetamine-
induced locomotor activity and the slowing of ventral tegmental area (VTA) (A10) dopaminergic neurons (34 ); (b) blockade of PCP-
and dizocilpin (MK-801)–induced locomotor activity (83 ,84 ); (c) blockade of MK-801–induced prepulse inhibition (85 ); and (d)
antipsychotic-like activity in the paw test (86 ) among others. Of particular interest is the report of Wadenberg et al. (31 ) that the
combination of a median effective dose (ED50) of raclopride, a D2 receptor antagonist, and M100907, but not M100907 alone, was
effective in blocking the conditioned avoidance response. The authors concluded that 5-HT2A antagonism alone could not achieve an
antipsychotic action, but that minimal blockade of D2 receptors was required to achieve such an effect. This corresponds much
more closely to the apparent clinical situation than do the models where M100907 alone was effective, e.g., blockade of PCP- or MK-
801–induced locomotor activity (34 ,83 ,84 ,87 ,88 and 89 ). As mentioned previously, administration of even a single dose of
atypical antipsychotic drugs, which are relatively more potent 5-HT2A than D2 blockers, has been shown to down-regulate 5-HT2A
receptors in rat brain (69 ,70 ). This has now been shown to be due to internalization of the receptors (70 ). Recovery from this
process requires the synthesis of new receptors.

An important effect of 5-HT2A (and 5-HT2C) receptors that may be relevant to their contribution to psychosis is their ability to
influence dopaminergic activity in the mesolimbic and mesostriatal systems (19 ,33 ,90 ,91 ,92 ,93 and 94 ). Increased dopaminergic
activity in the nucleus accumbens and other mesolimbic and possibly cortical regions may contribute to positive symptoms, including
formal thought disorder (2 ,5 ). Increased dopaminergic activity in the striatum would be expected to diminish EPSs (2 ,5 ). The 5-
HT2A/2C agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane], which itself had no effect on basal DA release, potentiated
amphetamine-induced DA release and attenuated the ability of apomorphine, a direct acting D1/D2/D3 agonist, to decrease DA
release in the striatum (93 ). Increasing serotoninergic activity, e.g., by administration of selective serotonin reuptake inhibitors
(SSRIs) alone or in combination with the 5-HT1A receptor antagonist WAY 100635, has no effect on basal DA output in the striatum.
However, the SSRIs can significantly enhance the increase in DA outflow induced by haloperidol. These findings indicate that in the
striatum, endogenous 5-HT positively modulates DA outflow when nigrostriatal DA transmission is activated (94 ). There is now
considerable evidence from both behavioral and neurochemical studies involving N-methyl-D-aspartate (NMDA) antagonists such as
PCP and MK-801 that 5-HT2A receptors modulate activated but not basal mesolimbic DA function (84 ,95 ). Thus, stimulated DA
release, e.g., with stress, may be increased in the forebrain terminal regions secondary to enhanced stimulation of 5-HT2A receptors.
Agents that block the effect of excessive, but not basal, 5-HT2A receptor stimulation may be the most useful clinically. M100907 has
been found to diminish the increase in DA efflux in the nucleus accumbens produced by haloperidol (30 ) or S-sulpiride (92 ). Taken
together, these data suggest that 5-HT2A antagonism by itself may have antipsychotic action when dopaminergic activity is slightly to
moderately increased. More studies are needed to define the ability of 5-HT2A receptor antagonists to potentiate the action of low
doses of D2 receptor blockers in animal models as well as in humans.

Jakab and Goldman-Rakic (96 ) have proposed that the 5-HT2A receptors on cortical pyramidal neurons may play a crucial role in
psychosis by virtue of their ability to modulate intracortical and cortical-subcortical glutamatergic neurotransmission. This could
contribute to the ability of 5-HT2A antagonists to attenuate some of the behavioral effects of PCP and ketamine. Aghajanian and
Marek (97 ) have proposed a link between the glutamate hypothesis of schizophrenia and the hallucinogen hypothesis. Briefly,
stimulation of 5-HT2A receptors on layer V pyramidal cells increases the frequency of postsynaptic potentials (PSPs). These are mostly
blocked by the AMPA/kainate glutamatergic receptor antagonist LY293558, indicating that
 P.824

they are mainly excitatory PSPs (EPSPs), in contrast with the piriform cortex, where 5-HT produces inhibitory PSPs (IPSPs). The
selective group II metabotropic agonist LY354740, which inhibits glutamate release by stimulating inhibitory presynaptic
autoreceptors on glutamatergic nerve terminals, suppresses the 5-HT–induced increase in the frequency of EPSPs. However,
Aghajanian and Marek suggest that the main way in which 5-HT2A receptor agonists increase glutamate release is a retrograde action
from stimulation of postsynaptic 5-HT2A receptors. The type of glutamate release induced by 5-HT2A receptor stimulation differs from
ordinary depolarization-induced neurotransmitter release, which is called synchronous release. The type of release induced by 5-HT
is delayed in onset, slow, and produces small excitatory postsynaptic currents (EPSCs) and is called asynchronous release.
Aghajanian and Marek propose that hallucinogens such as LSD enhance asynchronous EPSCs. They suggest that stimulation of other
types of 5-HT receptors may oppose this action and that the effect of 5-HT2A receptor antagonists unmasks the effect of these other
types of 5-HT receptors (97 ). A similar proposal has been made by Martin et al. (95 ). Although Aghajanian and Marek do not
mention the 5-HT1A receptor specifically, it would be a good candidate to counter the effect of 5-HT2A receptor stimulation, as will
be discussed. They propose that the thalamic filter hypothesis of Carlsson (98 ) might be related to the effect of 5-HT2A receptor
stimulation on thalamocortical afferents to affect cortical function or on corticostriatal or corticothalamic efferents to affect the
thalamic filter.

SEROTONIN RECEPTORS AND COGNITIVE FUNCTION

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

Clozapine, risperidone, ziprasidone, quetiapine, and olanzapine have been shown to improve selected areas of cognitive function in
patients with schizophrenia, with the available data suggesting differential effects on specific functions (13 ). The available data
suggest that each of the atypical drugs has a different pattern of effects on cognitive dysfunction in schizophrenia, but more head-
to-head studies are needed to confirm this impression. Whether relatively more potent 5-HT2A receptor compared to D2 antagonism
has a major or, indeed, any role in the cognitive effects of these agents is not known. However, this is the major characteristic that
these drugs share in common. It may be that the effect of these agents on cognition is mainly dependent on their ability to increase
the release of DA (99 ,100 ) and acetylcholine in prefrontal cortex (101 ), which may depend, in part, on their serotoninergic actions.
The effect of the atypical agents to increase DA efflux in the medial prefrontal cortex (mPFC) of rats appears to be due mainly to
actions at the terminal regions rather than cell bodies and not to be related to D2 receptor blockade because local administration of
haloperidol is without effect whereas clozapine and olanzapine produce huge increases in DA efflux (102 ). Studies have suggested
that the clozapine, olanzapine, risperidone, and ziprasidone, but not haloperidol, may enhance acetylcholine release in the rat
prefrontal cortex (101 ,103 ). Interactions between the 5-HT and cholinergic systems have been previously reported (104 ). 5-HT1A, 5-
HT2C, 5-HT3, and 5-HT4 receptors have also been reported to have significant effects on acetylcholine release in the rat prefrontal
cortex (105 ,106 ,107 ,108 and 109 ).

Because cognitive enhancement is critical for functional improvement in schizophrenia, establishing the mechanism for the ability of
the atypical antipsychotic drugs to increase DA efflux is of the greatest importance. The evidence concerning 5-HT receptors and
cognition has been reviewed in detail elsewhere (110 ,111 ). 5-HT2A/2C antagonists have little adverse effect and no apparent
beneficial effects on learning and memory (112 ). There is some evidence that 5-HT4 agonists, e.g., RS 67333, can improve learning
and memory in rodents (113 ). Impairment of working memory in humans following administration of the 5-HT1Aagonist flesinoxan has
been reported (114 ). However, Sumiyoshi et al. (115 ) have found that tandospirone, a 5-HT1A partial agonist, can improve other
domains of cognition in patients with schizophrenia treated with typical neuroleptic drugs. Neurochemical differences in the patient
populations, including possible abnormalities in the density of 5-HT1A receptors in schizophrenia, concomitant administration of a
neuroleptic to the patients with schizophrenia, and differences in the type of cognitive domain studied, may account for this
discrepancy. Further study in patients with schizophrenia is clearly indicated.

5-HT RECEPTOR BLOCKADE AND EXTRAPYRAMIDAL FUNCTION

2A

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

There have been numerous suggestions to explain the low EPSs of clozapine, namely its anticholinergic properties, lack of ability to
increase acetylcholine in the striatum, D1 or D4 receptor blockade, and its effects as an α1- or α2-adrenoceptor antagonist
(2 ,18 ,33 ,116 ). In addition, several lines of evidence suggest that potent 5-HT2A receptor blockade is relevant to the low EPS
profile of clozapine, but that 5-HT2A receptor blockade by itself cannot explain the low EPS liability of these agents (17 ,58 ,59 ).
Meltzer et al. (17 ) studied a group of compounds that had antipsychotic activity in humans or in animal models that are thought to
be predictive of antipsychotic activity, e.g., conditioned avoidance response or blockade of amphetamine-induced locomotor
activity, and which produced weak EPSs in humans or weak catalepsy in animals relative to their antipsychotic efficacy. These
compounds shared in common relatively weaker D2 compared to 5-HT2A receptor affinities, whereas D1 receptor affinities did not
contribute to this effect.
 P.825

Among the drugs studied were melperone, a butyrophenone long used in Europe and Scandinavia as an antipsychotic and reported to
produce low EPSs (14 ). Indeed, it has been found to be tolerable to patients with Parkinson's disease (117 ), even more so than
risperidone and olanzapine. The ∩-shaped dose–response curve of risperidone as well as the increasing incidence of EPSs as the dose
increases for olanzapine and ziprasidone, together with PET studies of DA receptor occupancy previously discussed, strongly suggests
that lower D2 receptor occupancy, possibly in relation to high 5-HT2A receptor is necessary to avoid EPSs with these compounds.

As previously discussed, numerous compounds of diverse chemical structure that share this pharmacologic profile have been
identified or deliberately synthesized and tested for antipsychotic action and EPS liability. These include risperidone, olanzapine,
sertindole, quetiapine, ziprasidone, and iloperidone. All of these compounds can produce fewer EPSs than haloperidol at comparable
doses. Clozapine and quetiapine have been shown to produce the least EPSs in studies of patients with Parkinson's disease.
Consistent with this concept, the 5-HT2A antagonist mianserin has been reported to be effective in neuroleptic-induced akathisia
(118 ). There are also a variety of preclinical data to support the importance of relatively high 5-HT2A compared to D2 receptor
affinity to preserve striatal function. For example, Ishikane et al. (119 ) reported that M100907 is able to block haloperidol-induced
catalepsy only at low doses of haloperidol. Consistent with this, Spampinato et al. (120 ) reported that specific 5-HT2A and 5-HT2C
antagonists were able to modulate the ability of haloperidol at 0.01 mg/kg but not at a higher dose (1.0 mg/kg) to increase striatal
DA release in freely moving rats.

THE ROLE OF THE 5-HT RECEPTOR IN ANTIPSYCHOTIC DRUG ACTION:

2C

5-HT AND 5-HT INTERACTIONS

2A 2C

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

There has been some consideration given to the role of 5-HT2C receptors in the action of atypical antipsychotic drugs. The 5-HT2C
receptor is found throughout the central nervous system (CNS), including the ventral tegmentum and the nucleus accumbens (121 ).
With the availability of specific 5-HT2C agonists and antagonists, evidence for a tonic inhibitory action of 5-HT2C receptors on the
burst firing of mesolimbic and mesocortical dopaminergic neurons has been obtained. Thus, the firing rate of VTA DA neurons is
inhibited or increased by 5-HT2C agonists or antagonists, respectively. This is consistent with microdialysis studies that show that 5-
HT2C antagonists increase extracellular concentrations of DA in the nucleus accumbens, striatum, and medial prefrontal cortex
(91 ,122 ). Early studies found no significant differences between groups of novel antipsychotic drugs and typical neuroleptics with
regard to the affinity for 5-HT2C receptor or the difference between 5-HT2C and D2 affinities (22 ,60 ). Of the approved novel
antipsychotic drugs, some have equivalent affinities for the 5-HT2A and 5-HT2C receptors (clozapine, olanzapine, sertindole), whereas
others are more selective for the 5-HT2A receptor (risperidone, quetiapine, ziprasidone). This difference roughly corresponds with
the potential to produce weight gain, in that clozapine and olanzapine cause the greatest weight gain and risperidone and
ziprasidone the least (see Chapter 56 ). There is little available data for sertindole and quetiapine, but they appear to be
intermediate. There is no apparent relationship between 5-HT2C affinity relative to 5-HT2A affinity with regard to EPSs because
quetiapine and ziprasidone are comparable to olanzapine and sertindole in this regard. Similarly, there is no apparent relationship
to efficacy in treatment-resistant schizophrenia. There could be a relationship to differences among the atypical antipsychotic drugs
with regard to improvement in specific types of cognitive function in schizophrenia (13 ).

An interesting aspect of the 5-HT2C receptor with regard to antipsychotic action is that 5-HT2C antagonism may be functionally
opposed to 5-HT2A antagonism. Meltzer et al. (123 ) reported that atypical antipsychotic drugs were more likely to be weak 5-HT2C
and potent 5-HT2A antagonists compared to typical neuroleptic drugs. Subsequently, neurochemical (120 ) and behavioral (96 ,124 )
data have been reported that support the notion of a functional antagonism of these two receptors that may coexist on the same
neurons. Thus, Martin et al. (95 ) found that ritanserin, a mixed 5-HT2A/2C antagonist, blocked the ability of M100907 to antagonize
the effect of MK-801 to increase locomotor activity in mice.

THE ROLE OF THE 5-HT RECEPTOR IN ANTIPSYCHOTIC DRUG ACTION

1A

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

The 5-HT1A receptor is located pre- and postsynaptically. The presynaptic 5-HT1A receptor is an autoreceptor located on cell bodies of
raphe neurons; stimulation leads to inhibition of firing of 5-HT neurons. Stimulation of postsynaptic 5-HT1A receptors generally leads
to hyperpolarization of neurons, which is opposite of the effect of stimulation of 5-HT2A receptors. There is extensive evidence that
cannot be reviewed in detail here that indicates that 5-HT1A receptor agonists and 5-HT2A receptor antagonists produce similar
neurochemical and behavioral effects on a variety of measures (125 ,126 ). For example, DOI injected bilaterally into the rat medial
prefrontal cortex elicits a dose-dependent head twitch response. This effect is inhibited by M100907 and ketanserin, relatively
selective for 5-HT2A receptors at appropriate doses, but not the selective 5-HT2C antagonist SDZ SER082. Pretreatment with the 5-HT1A
agonist 8-OH-DPAT also inhibited the head twitch response to DOI (127 ). Ahlenius (128 ) first suggested that stimulation
 P.826

of 5-HT1A receptors might produce an antipsychotic like action on the basis of behavioral studies in animals using the direct 5-HT1A
agonist 8-OH-DPAT. Subsequent studies demonstrated that 8-OH-DPAT enhanced the antipsychotic-like effect of the D2/D3
antagonist raclopride (129 ) and of haloperidol (130 ), and antagonized the catalepsy induced by the D1 agonist SCH23390 in rats
(131 ). The ability of clozapine to reverse olanzapine-induced catalepsy is blocked by the selective 5-HT1A antagonist WAY 100635,
suggesting the effect of clozapine was mediated by stimulation of 5-HT1A receptors. The beneficial effect of 5-HT1A agonists appears
to be mediated by inhibition of median raphe serotoninergic neurons (132 ).

5-HT1A agonists have different regional effects on DA release in the rat brain. 5-HT1A receptor stimulation appears to inhibit DA
release in subcortical regions. Thus, Ichikawa et al. (133 ) demonstrated that the 5-HT1A agonist 8-OH-DPAT inhibited the ability of
amphetamine to increase extracellular DA levels in the nucleus accumbens and the striatum of conscious rats. The effect of 5-HT1A
receptor stimulation in the nucleus accumbens would be expected to enhance the antipsychotic effect of these agents by reducing
dopaminergic activity. Several atypical antipsychotic drugs, including clozapine, ziprasidone, quetiapine, and tiospirone, are partial
agonist at the 5-HT1A receptor. Their affinities for the 5-HT1A receptor are similar to their affinities for the human D2 receptor (22 ).
Rollema et al. (134 ) demonstrated that the ability of clozapine to increase DA release in the rat prefrontal cortex was due, in part,
to its 5-HT1A agonist properties, as it could be blocked by WAY-100635, a 5-HT1A antagonist. Ichikawa et al. (92 ) have extended these
findings in a variety of ways; e.g., the ability of risperidone and the combination of M100907 and sulpiride to increase prefrontal
cortical PFC DA efflux were both blocked by WAY100635. These findings suggest that the combination of D2 antagonism and 5-HT1A
agonism provides some of the key features of atypical antipsychotic agents. S16924, a 5-HT1A partial agonist D2 antagonist, is an
example of a putative atypical antipsychotic drug based on this model. It has atypical antipsychotic properties very similar to those
of clozapine in a variety of relevant animal models (64 ). Whether this or similar compounds will have the same spectrum of efficacy
and side effect advantages as the multireceptor antagonists that are relatively more potent 5-HT2A than D2 antagonists remains to be
determined. Significant differences should be expected. It is noteworthy that clozapine has both relatively more potent 5-HT2A
antagonism than D2 antagonism as well as 5-HT1A partial agonism. This may be part of the mixture that accounts for its particular
advantages over other atypical antipsychotic drugs. Wedzony et al. (135 ) found that WAY100135, a 5-HT1A antagonist, attenuated
the effect of MK-801, an NMDA antagonist on locomotor activity, prepulse inhibition, and the detrimental effect of MK-801, a
noncompetitive NMDA antagonist on working memory and selective attention in rats. They cite other evidence that 5-HT1A
antagonists may improve learning and memory in animal models and suggest this may be due to blocking the inhibitory effects of 5-
HT1A receptor stimulation on the firing of hippocampal neurons. This suggests that a partial agonist, acting as an antagonist, may
sometimes be of benefit with regard to effects relevant to schizophrenia.

THE ROLE OF SEROTONIN RELEASE IN ANTIPSYCHOTIC DRUG ACTION

Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

The antagonism of multiple 5-HT receptors by clozapine would be expected to enhance the release of 5-HT by feedback mechanisms.
Thus, it is surprising that Ferré and Artigas (136 ) reported that clozapine decreased 5-HT release in the nucleus accumbens.
However, Ichikawa et al. (137 ) reported that clozapine (20 mg/kg) and risperidone (1 mg/kg) significantly increased extracellular 5-
HT levels in the nucleus accumbens and medial prefrontal cortex, respectively, whereas amperozide (1 and 10 mg/kg) increased
extracellular 5-HT levels in both regions. Hertel et al. (138 ) reported similar results with risperidone and suggested that this might
be relevant to its ability to improve negative symptoms. If so, this is not the explanation for the effects of clozapine or olanzapine
on negative symptoms because olanzapine, sulpiride, haloperidol, and M100907 had no effect on extracellular 5-HT levels in either
region. The latter consideration also indicates that blockade of 5-HT2A receptors is not the basis for the ability of clozapine,
risperidone, or amperozide to increase 5-HT levels. The enhancement of 5-HT efflux in the prefrontal cortex may contribute to the
ability of these agents to improve mood disorders and cognition.

α - AND α -ADRENERGIC MECHANISMS AND ATYPICAL ANTIPSYCHOTIC

2 1

DRUGS
Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

Most of the atypical antipsychotic drugs are potent antagonists of the α1 or α2 adrenoceptors, or both. Thus, risperidone 9-
hydroxyrisperidone, clozapine, olanzapine, zotepine, quetiapine, ORG-5222, sertindole and ziprasidone are potent α1 antagonists
(22 ). Prazosin, an α1 adrenoceptor antagonist, has, like clozapine and other atypical antipsychotic drugs, been shown to increase DA
efflux in the shell but not the core of the nucleus accumben, signifying a limbic rather than a striatal effect of α1 antagonism (91 ).
These authors also suggested that α1 antagonism may explain the atypical properties of sertindole, which has been reported to
achieve as high an occupancy of D2 receptors as typical antipsychotic drugs (36 ). All of the atypical agents mentioned above are
also potent α2 antagonists, with the exception of zotepine and sertindole (22 ). Kalkman et al. (116 ) raised the possibility that the
α2C subtype may be particularly relevant to the anticataleptic as well as other actions of clozapine and iloperidone. However,
McAllister and Rey (139 ) were unable to reverse the effects of loxapine or haloperidol
 P.827

on catalepsy with α2 antagonists and showed that the effect of clozapine to reverse loxapine-induced increase in catalepsy was due
to its anticholinergic rather than its adrenoceptor blocking properties. Clozapine produces massive increases in plasma
norepinephrine, which may indicate that it can cause effective stimulation of α-adrenoceptors receptors in brain (140 ). The
addition of idazozan, an α2 antagonist, to fluphenazine, a typical neuroleptic, was reported by Littman et al. (141 ) to have efficacy
comparable to clozapine in a small group of neuroleptic-resistant patients with schizophrenia. These results need to be replicated.
Idazoxan has also been shown to improve attentional and executive dysfunction in patients with dementia of the frontal type (142 ),
suggesting that some of the cognitive enhancing effects of the atypical antipsychotic drugs might be related to their α2 blocking
properties. Another α2 antagonist, atipamezole, has been reported to improve cognitive performance in aged rats (143 ).
Polymorphisms of the α1 and α2 receptors have been reported not to predict response to clozapine (144 ).

In this regard, it is of interest that idazoxan has been shown to preferentially increase DA efflux in the rat mPFC by an action at the
terminal area (145 ). This effect appears to be independent of dopaminergic activity (146 ). Westerink et al. (147 ) demonstrated
that systemic administration of clozapine, risperidone, ziprasidone, and olanzapine, as well as haloperidol, produced a dose-
dependent increase in noradrenaline in the mPFC of rats. This effect was closely coupled to the increase in DA efflux. Increased
levels of norepinephrine might also be related to the cognitive and antidepressant effects of the atypical antipsychotic drugs
(148 ,149 ).

CONCLUSION
Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

Typical neuroleptic drugs such as haloperidol have been reliably shown to produce their antipsychotic action by blockade of D2
receptors in the mesolimbic system, suggesting that increased dopaminergic activity in these terminal areas of the ventral
tegmental DA neurons are of importance to the etiology of schizophrenia. Striatal D2 receptor antagonism is the critical element in
the EPSs produced by these drugs. Atypical antipsychotic drugs are those antipsychotics that achieve an antipsychotic action with
quantitatively less EPSs in humans or a clear distinction between doses that affect mesolimbic and striatal dopaminergic function in
rodents. Clozapine was the first atypical antipsychotic drug by the definition noted above, but more importantly it showed that
antipsychotic drugs might also be effective in some patients with schizophrenia whose positive symptoms do not respond to
neuroleptic-type agents and to improve negative symptoms, cognitive impairment, depression, and possibly suicidality of
schizophrenia and other psychotic disorders as well (150 ,151 and 152 ). There is strong evidence of the role of 5-HT2A receptors and
suggestive evidence of the roles of the 5-HT1A, 5-HT2C, and α1 receptors in various actions of clozapine, risperidone, olanzapine,
quetiapine, ziprasidone, iloperidone, sertindole, and related atypical antipsychotic drugs. Atypical antipsychotic drugs that are
potent 5-HT2A antagonists relative to their D2 receptor blocking property appear to potentiate 5-HT1A-mediated effects on
dopaminergic neurons in the mesocortical, mesolimbic, and mesostriatal regions. The effects in the mesocortical regions appear to
be mediated by modulation of glutamate release from pyramidal neurons. These agents have been found to preferentially increase
DA efflux in the mPFC compared to limbic and striatal regions. They also increase acetylcholine release in the PFC. Effects on 5-HT2C,
5-HT3, 5-HT4, 5-HT6, and 5-HT7 receptors may also be relevant to some of their actions, e.g., improvement of cognition, weight gain,
etc. Other models of atypicality appear to be effective, including partial DA agonists such as aripiprazole. Selective D2/D3
antagonists such as amisulpride may also have atypical properties. At this time, multireceptor agents appear to be more promising
as antipsychotic agents for the majority of psychiatric patients because of important interactions between neural circuits that
employ multiple neurotransmitters.

ACKNOWLEDGMENTS
Part of "58 - Mechanism of Action of Atypical Antipsychotic Drugs "

This work is supported in part by grants from Mr. Donald Test, Mrs. Test, and the Warren Foundation. The assistance of Ms. Karen
Espenant and Ms. Alice Hammond in the preparation of this manuscript is greatly appreciated.

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59
Neurochemical and Neuropharmacological Imaging in Schizophrenia

Daniel R. Weinberger

Marc Laruelle

Daniel R. Weinberger: Clinical Brain Disorders Branch, Intramural Research Program, National Institute of Mental Health, Bethesda,
Maryland.

Marc Laruelle: Division of Functional Brain Mapping, New York State Psychiatric Institute; Departments of Psychiatry and Radiology,
Columbia College of Physicians and Surgeons, New York, New York.

Over the last 15 years, the ability to measure specific molecules and proteins in the living human brain underwent enormous
developments, opening direct windows into neurotransmitter functions and cellular processes associated with health and disease.
These techniques are based either on the injection of radioactive moieties whose distribution is recorded with positron emission
tomography (PET) or single photon emission computed tomography (SPECT), or on the direct detection of molecules based on their
intrinsic magnetic properties with magnetic resonance spectroscopy (MRS).

Although lacking the level of resolution of postmortem studies and limited by the relatively small number of targets that can be
currently studied in vivo, these imaging techniques provide unique opportunities for elucidation of pathophysiology associated with
neuropsychiatric conditions. In vivo imaging techniques enable studying patients with well-documented psychopathology, relating
biochemical observations to psychiatric symptoms and cognitive processes, and clarifying abnormalities associated with the disease
as opposed to its treatment. Neurochemical imaging allows longitudinal studies to investigate mechanisms of actions and
consequences of treatments, as well as to characterize neurochemical abnormalities in relation to treatment response and illness
outcome. Neurochemical imaging further provides insight as to the pathophysiologic bases of alterations measured with flow and
metabolism imaging studies, and can provide a direct link with animal models of the illness. Moreover, these techniques enable the
study of patient's relatives, in order to clarify the endophenotypes associated with illness vulnerability. These potentialities are
discussed in this chapter.

This chapter also critically summarizes results obtained using neurochemical imaging techniques in schizophrenia research, and the
various insights on the pathophysiology and treatment of schizophrenia gained by these results. We first consider PET and SPECT
investigations, and then MRS studies.

 PET AND SPECT NEUROCHEMICAL IMAGING

 MAGNETIC RESONANCE SPECTROSCOPY
 ACKNOWLEDGMENTS

PET AND SPECT NEUROCHEMICAL IMAGING

Part of "59 - Neurochemical and Neuropharmacological Imaging in Schizophrenia "

The principles of PET and SPECT neurochemical imaging are reviewed elsewhere in this volume. Numerous PET and SPECT
radiotracers are currently available to study key proteins in the living brain, such as receptors, transporters, and enzymes.
Regarding schizophrenia, the majority of clinical investigations studied various aspects of dopaminergic transmission. Dopamine (DA)
D2 receptors were the first neuroreceptors visualized in the living human brain (1 ). Since then, several DA-related radiotracers have
been developed, allowing the study of many aspects of dopaminergic transmission (DA synthesis, DA release, D1 and D2 receptors,
DA transporters). Given the availability of these tools and the important role that DA transmission is believed to play in
schizophrenia, it is not surprising that most of the research effort focused on this system. Despite marked limitations, these studies
provide a relatively consistent picture suggesting that schizophrenia, at least during periods of clinical exacerbation, is associated
with dysregulation of DA transmission.

Imaging DA Transmission Parameters In Schizophrenia

The classic DA hypothesis of schizophrenia, formulated over 30 years ago, proposed that a hyperactivity of the dopaminergic
transmission is associated with this illness (2 ,3 ).
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This hypothesis was essentially based on the observation that all antipsychotic drugs provided at least some degree of D2 receptors
blockade, a proposition that is still true today (4 ,5 ). As D2 receptor blockade is most effective against positive symptoms, the DA
hyperactivity model appeared to be most relevant to the pathophysiology of positive symptoms. The fact that sustained exposure to
DA agonists such as amphetamine can induce a psychotic state characterized by some salient features of positive symptoms of
schizophrenia (emergence of paranoid delusions and hallucinations in the context of a clear sensorium) also contributed to the idea
that positive symptoms might be due to sustained excess dopaminergic activity (6 ,7 ).

These pharmacologic effects indeed suggest, but do not establish, a dysregulation of DA systems in schizophrenia. For example,
these observations are also compatible with the hypotheses that DA activity per se would be normal and increased only relatively to
other systems that may be deficient, such as the glutamatergic or serotoninergic system (8 ,9 ). Under these conditions, D2 receptor
blockade would reestablish a compromised balance between dopaminergic and glutamatergic or serotoninergic tone. Thus, these
pharmacologic observations do not necessarily imply a specific disturbance of DA activity per se in the brain of patients with
schizophrenia. Indeed, documentation of abnormalities of DA function in postmortem studies in schizophrenia has remained elusive
(10 ,11 and 12 ). Because positive symptoms are mostly prominent in young patients and their intensity decreases with age, the
ability to detect their biochemical correlates in postmortem studies (generally performed in older subjects) may be limited.

On the other hand, negative and cognitive symptoms are generally resistant to treatment by antipsychotic drugs. Functional brain
imaging studies suggested that these symptoms are associated with prefrontal cortex (PFC) dysfunction (13 ). Studies in nonhuman
primates demonstrated that deficits in DA transmission in PFC induce cognitive impairments reminiscent of those observed in
patients with schizophrenia (14 ), suggesting that a deficit in DA transmission in the PFC might be implicated in cognitive
impairments presented by these patients (15 ,16 ). In addition, a recent postmortem study described abnormalities of DA terminals
in the PFC associated with schizophrenia (17 ). Thus, a current view on DA and schizophrenia is that subcortical mesolimbic DA
projections might be hyperactive (resulting in positive symptoms) and that the mesocortical DA projections to the PFC are
hypoactive (resulting in negative symptoms and cognitive impairment). Furthermore, these two abnormalities might be related, as
the cortical DA system generally exerts an inhibitory action on subcortical DA systems (18 ,19 ).

The advent in the early 1980s of techniques based on PET and SPECT to measure indices of DA activity in the living human brain held
considerable promise for investigating these questions.

Striatal D2 Receptor Density

Striatal D2 receptor density in schizophrenia has been extensively studied with PET and SPECT imaging. Studies comparing
parameters of D2 receptor binding in patients with schizophrenia and healthy controls (n = 16 studies) are listed in Table 59.1 , and
included a total of 228 patients (102 were neuroleptic-naive, and 126 were neuroleptic-free for variable periods of time). These
patients were compared to 213 controls, matched for age and sex. Ten studies used PET and six studies used SPECT. Radiotracers
included butyrophenones ([11C]N-methyl-spiperone, [11C]NMSP, n = 3, and [76Br]bromospiperone, n = 3), benzamides ([11C]raclopride, n
= 3, and [123I]IBZM, n = 5) or the ergot derivative [76Br]lisuride, n = 2). A variety of methods and outcome measures were used to
estimate D2 receptors density. Six studies used empirical ratio methods, i.e., the ratio of striatal to cerebellar activities at a given
time after single bolus injection of the radiotracer. Five studies used model-based methods to measure the binding potential (BP),
which is equal to the product of receptor density (Bmax) and affinity (1/Kd). Five studies reported both Bmax and Kd.

TABLE 59.1. IMAGING STUDIES OF D2 RECEPTOR PARAMETERS IN DRUG NAIVE AND

DRUG FREE PATIENTS WITH SCHIZOPHRENIA

Only two out of 13 studies detected a significant elevation of D2 receptor density parameters at a level p <.05. However,
metaanalysis of the 16 studies reveals a small (on the order of 13%) but significant elevation of D2 receptors in patients with
schizophrenia. If D2 receptor density did not differ between patients and controls (null hypothesis), one would expect approximately
50% of the studies to report lower D2 receptor levels in schizophrenics compared to controls. Instead, 12 out of 16 studies reported
an increase (although not significant in 10 out of 12 cases), two reported no change, and only two studies reported a decrease in
patients compared to controls. This distribution is unlikely (p <.05, sign test) under the null hypothesis. Moreover, under the null
hypothesis, the effect sizes [mean value in schizophrenic group - mean value in control group/standard deviation (SD) in control
group] should be distributed around 0. The average effect size of the 16 studies was 0.57 ± 0.78 (SD), and the probability to yield
such effect size under the null hypothesis is again lower than .05. The aggregate magnitude of this elevation is thus 57% of the SD of
controls. Given an average control SD of 23%, the effect is about 13%. To detect an effect size of 0.50 at the .05 significance level
with a power of 80%, a sample of 64 patients and 64 controls would be needed. Clearly, none of the studies included enough
patients to detect this small effect with appropriate power. Another observation is that the variability in the patient sample was
larger than in the control sample in 13 out of 15 studies, which was also significant (p <.05, sign test). The average variance ratio
(SD schizophrenics/SD controls) was 1.47 ± 0.58. The larger variance in patients compared to controls further increases the sample
size needed to detect this small group difference with reasonable power.
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No clinical correlates of increased D2 receptor binding parameters has been reliably identified. Thus, the simplest conclusions from
these studies are that patients with schizophrenia show a modest elevation in D2 receptor density parameters of undetermined
clinical significance, that all studies were underpowered, and that positive results occasionally reported (20 ,21 ) are due to a
sampling effect. These conclusions are reached under the assumptions that all studies measured parameters from the “same” D2
receptors population.
However, there is another way to look at these data. Studies performed with butyrophenones (n = 6) have an effect size of 1.08 ±
1.06 (n = 6), whereas studies performed with other ligands (benzamides and lisuride, n = 10) have an effect size of 0.20 ± 0.26, a
difference that is significant (p = .022). This observation suggests that schizophrenia might be associated with an increase in
butyrophenone binding and no change in benzamide or lisuride binding.
Unfortunately, no studies have been reported in which the same subjects were scanned with both ligands. Such a study is warranted
to directly test this view. Nevertheless, several hypotheses have been advanced to account for the existence of a differential
increase in [11C]NMSP binding in vivo in patient with schizophrenia in the face of normal benzamide binding. Because [11C]raclopride
and [123I]IBZM binds to D2 and D3 receptors whereas [11C]NMSP binds to D2, D3, and D4 receptors, this difference could reflect a
selective elevation of D4 receptors in schizophrenia (22 ). This hypothesis has not been substantiated. The density of D4 receptors is
negligible in the striatum, and, when measured with a specific ligand, not different in postmortem striatal samples from patients
with schizophrenia and controls (23 ). Another hypothesis derives from the observation that D2 receptors, like several G-protein–
coupled receptors, exist in monomers, dimers, and other oligomeric forms (24 ,25 ,26 and 27 ). Photoaffinity labeling experiments
suggested that butyrophenones detect only monomers, whereas benzamides detect both monomers and dimers. Thus, increased
butyrophenone binding and normal benzamide binding might reflect a higher monomer/dimer ratio in schizophrenia. This interesting
hypothesis warrants further exploration. A third proposition evolved around the idea that the binding of these ligands would display
different vulnerability to competition by endogenous DA (28 ,29 ). This proposition was based on two assumptions: (a) the
concentration of DA in the proximity of D2 receptors might be higher in patients compared to controls, and (b) [11C]NMSP might be
less affected than [11C]raclopride or [123I]IBZM binding by endogenous DA competition. It follows that D2 receptor density measured in
vivo with [11C]raclopride and [123I]IBZM would be “underestimated” to a greater extent in patients with schizophrenia than in control
subjects. This hypothesis played an important role in bringing the endogenous competition concept to the attention of the imaging
field.

Striatal DA Presynaptic Function

Imaging studies of presynaptic DA function in schizophrenia included measurements of dihydroxyphenylalanine (DOPA) decarboxylase
activity, DA release at baseline and following pharmacologic challenges with amphetamine, and DA transporters (DAT). These
studies are summarized in Table 59.2 .

TABLE 59.2. IMAGING STUDIES OF PRESYNAPTIC DA PARAMETERS IN DRUG NAIVE

AND DRUG FREE PATIENTS WITH SCHIZOPHRENIA

DOPA Decarboxylase Activity

Five studies reported rates of DOPA decarboxylase in patients with schizophrenia, using [18F]DOPA (30 ,31 ,32 and 33 ) or [11C]DOPA
(34 ) (Table 59.2 ). Four out of five studies reported increased accumulation of DOPA in the striatum of patients with schizophrenia,
and the combined analysis yielded an effect size of 0.92 ± 0.45, which is significantly different from zero (p = .01). The variability of
the DOPA accumulation was larger in the schizophrenic group compared to the control group. Several of these studies reported the
observation of high DOPA accumulation in psychotic paranoid patients, and low accumulation in patients with negative or depressive
symptoms and catatonia. Although the relationship between DOPA decarboxylase and DA synthesis rate is unclear (DOPA
decarboxylase is not the rate-limiting step of DA synthesis), these observations are compatible with higher DA synthesis activity of
DA neurons in schizophrenia, at least in subjects experiencing psychotic symptoms.

Amphetamine-Induced DA Release
As discussed above, endogenous DA competition is a source of errors for in vivo measurement of D2 receptors. On the other hand,
the recognition of this phenomenon implies that D2 receptor imaging, combined with pharmacologic manipulation of DA release,
could provide a functional evaluation of DA presynaptic activity. Indeed, over the last decade, numerous groups demonstrated that
acute increase in synaptic DA concentration is associated with decreased in vivo binding of [11C]raclopride and [123I]IBZM. These
interactions have been demonstrated in rodents, nonhuman primates, and humans, using a variety of methods to increase synaptic
DA [amphetamine, DAT blockers, levodopa (L-DOPA), nicotine agonists, serotonin receptor subtype 2A (5-HT2A) antagonists, direct
electrical stimulation of DA neurons] (see ref. 35 for review of this abundant literature). It has also been consistently observed that
the in vivo binding of spiperone and other butyrophenones is not as affected as the binding of benzamides by acute fluctuations in
endogenous DA levels (35 ).
The decrease in [11C]raclopride and [123I]IBZM in vivo
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binding following acute amphetamine challenge has been well validated as a measure of the change in D2 receptor stimulation by
DA due to amphetamine-induced DA release. Manipulations that are known to inhibit amphetamine-induced DA release, such as
pretreatment with the DA synthesis inhibitor α-methyl-para-tyrosine (α-MPT) or with the DAT blocker GR12909 also inhibit the
amphetamine-induced decrease in [123I]IBZM or [11C]raclopride binding (36 ,37 ). These experiments support the assumption that the
amphetamine effect on [11C]raclopride and [123I]IBZM binding is mediated by DA release. Combined microdialysis and imaging
experiments in primates demonstrated that the magnitude of the decrease in ligand binding was correlated with the magnitude of
the increase in extracellular DA induced by the challenge (37 ,38 ), suggesting that this noninvasive technique provides an
appropriate measure of the changes in synaptic DA levels.

Three out of three studies demonstrated that amphetamine-induced decrease in [11C]raclopride or [123I]IBZM binding was elevated in
untreated patients with schizophrenia compared to well-matched controls (38 ,39 and 40 ). A significant relationship was observed
between magnitude of DA release and transient induction or worsening of positive symptoms. The increased amphetamine-induced
DA release was observed in both male and female patients, and in both first-episode/drug-naive patients and patients previously
treated by antipsychotic drugs (41 ). Combined analysis of the results of two studies revealed that patients who were experiencing
an episode of illness exacerbation (or a first episode of illness) at the time of the scan showed elevated amphetamine-induced DA
release, whereas patients in remission showed DA release values not different from those of controls (41 ). This exaggerated
response of the DA system to amphetamine exposure did no appear to be a nonspecific effect of stress, as higher self-reports of
anxiety before the experiments were not associated with larger effect of amphetamine. Furthermore, nonpsychotic subjects with
unipolar depression, who reported levels of anxiety similar to, if not higher than, the schizophrenic patients at the time of the scan,
showed normal amphetamine-induced displacement of [123I]IBZM (42 ).

These findings were generally interpreted as reflecting a larger DA release following amphetamine in the schizophrenic group.
Another interpretation of these observations would be that schizophrenia is associated with increased affinity of D2 receptors for DA.
Development of D2 receptors imaging with radiolabeled agonists is needed to settle this issue (42 ). Another limitation of this
paradigm is that it measures changes in synaptic DA transmission following a nonphysiologic challenge (i.e., amphetamine) and do
not provide any information about synaptic DA levels at baseline, i.e., in the unchallenged state.

Baseline DA Release
Several laboratories reported that, in rodents, acute depletion of synaptic DA is associated with an acute increase in the in vivo
binding of [11C]raclopride or [123I]IBZM to D2 receptors (see ref. 35 for review). The increased binding was observed in vivo but not in
vitro, indicating that it was not due to receptor up-regulation (43 ), but to removal of endogenous DA and unmasking of D2 receptors
previously occupied by DA. The acute DA depletion technique was developed in humans using α-MPT to assess the degree of
occupancy of D2 receptors by DA (43 ,44 ). Using this technique, higher occupancy of D2 receptor by DA was recently reported in
patients with schizophrenia experiencing an episode of illness exacerbation, compared to healthy controls (45 ). Again assuming
normal affinity of D2 receptors for DA, the data are consistent with higher DA synaptic levels in patients with schizophrenia. This
observation was present in both first-episode/drug-naive and previously treated patients.

Following DA depletion, higher D2 receptor availability was observed in patients with schizophrenia compared to controls. This
observation supported the proposition that, in schizophrenia, elevated D2 receptor density might be masked by DA occupancy when
imaging studies are performed with ligands vulnerable to endogenous competition (28 ). However, the increase in D2 receptors
measured with [123I]IBZM in DA-depleted patients was moderate (12%), suggesting that other factors than vulnerability to endogenous
DA competition are involved in the butyrophenone-benzamides binding differences discussed above.

Interestingly, higher occupancy of D2 receptors by DA in patients with schizophrenia was not associated with the intensity of positive
symptoms, but was predictive of good therapeutic response of these symptoms following 6 weeks of treatment with atypical
antipsychotic medications. The fact that high levels of synaptic DA at baseline predicted better or faster response to atypical
antipsychotic drugs suggested that the D2 receptor blockade induced by these drugs remains a key component of their initial mode
of action.

DA Transporters
The data reviewed above are consistent with higher DA output in the striatum of patients with schizophrenia, which could be
explained by increased density of DA terminals. Because striatal DATs are exclusively localized on DA terminals, this question was
investigated by measuring binding of [123I]-CIT (46 ) or [18F]CFT (48 ) in patients with schizophrenia. Both studies reported no
differences in DAT binding between patients and controls. In addition, Laruelle et al. (46 ) reported no association between
amphetamine-induced DA release and DAT density. Thus, the increased presynaptic output suggested by the studies reviewed above
 P.839

does not appear to be due to higher terminal density, an observation consistent with postmortem studies that failed to identify
alteration in striatal DAT binding in schizophrenia (47 ,48 ,49 ,50 ,51 and 52 ).

Subcortical DA Dysregulation as a Failure of Inhibitory Pathways

Although the studies reviewed above generally confirmed the classic DA hypothesis of schizophrenia, it is important to examine
these results in light of the more recent views of schizophrenia as a neurodevelopmental illness, involving dysconnectivity of
multiple cortico-subcortical and intracortical networks. Although it cannot be definitively ruled out that the DA dysregulation
revealed by these studies would stem from a primary abnormality of DA neurons, it seems more likely that these abnormalities are a
consequence of cortico-subcortical dysconnectivity. Moreover, given the weight of evidence implicating PFC connectivity as a
central deficient node in the schizophrenic brain, it is tempting to speculate that dysregulation of DA neurons' firing activity might
stem from a failure of the PFC to regulate this process. In fact, it has long been hypothesized that dysregulation of subcortical DA
function in schizophrenia may be secondary to a failure of the PFC to adequately control subcortical dopaminergic function (53 ,54 ).

Activity of midbrain DA neurons is under dual influence of PFC via an activating pathway (the “accelerator”) and an inhibitory
pathway (“the brake”), allowing fine-tuning of dopaminergic activity by the PFC (55 ). The activating pathway is provided by direct
glutamatergic projections onto the dopaminergic cells. The inhibitory pathway is provided by glutamatergic projections to midbrain
γ-aminobutyric acid (GABA)ergic interneurons or striatomesencephalic GABA neurons. The inhibition of dopaminergic cell firing
following amphetamine is an important feedback mechanism by which the brain reduces the effect of amphetamine on DA release.
The inhibition of dopaminergic cell firing induced by amphetamine is mediated both by stimulation of presynaptic D2 autoreceptors
and by stimulation of this inhibitory pathway (56 ). Following administration of amphetamine (i.e., under conditions in which the
inhibitory pathway should be activated), N-methyl-D-aspartate (NMDA) receptor blockade results in a failure of activation of the
inhibitory pathway, resulting in exaggerated amphetamine-induced DA release (57 ). Kegeles et al. (58 ) recently confirmed this
mechanism in humans: ketamine pretreatment significantly enhanced amphetamine-induced decrease in [123I]IBZM BP, from -5.5 ±
3.5% under control conditions to -12.8 ± 8.8% under ketamine pretreatment (p = .023). The increase in amphetamine-induced DA
release induced by ketamine (greater than twofold) was comparable in magnitude to the exaggerated response seen in patients with
schizophrenia. These data are consistent with the hypothesis that the alteration of DA release revealed by the amphetamine
challenge in schizophrenia results from a disruption of glutamatergic neuronal systems regulating dopaminergic cell activity and are
consistent with the hypothesis that schizophrenia might be associated with NMDA receptor hypofunction (59 ,60 and 61 ).

The failure of glutamatergic control of DA release might stem from mechanisms other than NMDA hypofunction. For example,
glutamatergic projections from the PFC to the ventral tegmental area (VTA) are under tonic inhibition by prefrontal GABA and DA
activity (see ref. 62 and references therein). It follows that deficits in GABAergic or dopaminergic function in the PFC (both deficits
also implicated in schizophrenia) are expected to have similar consequences to an NMDA deficiency on the subcortical DA response
to amphetamine. Thus, in patients with schizophrenia, various or multiple mechanisms (NMDA receptor hypofunction, GABAergic or
dopaminergic deficits in the PFC) may lead to the dysregulation of subcortical DA revealed by the amphetamine challenge.

As reviewed below, direct evidence has been provided that disinhibition of subcortical DA activity is associated with prefrontal
pathology in schizophrenia. In patients with schizophrenia, low N-acetylaspartate (NAA) concentration in the dorsolateral prefrontal
cortex (DLPFC), a marker of DLPFC pathology, is associated with increased amphetamine-induced DA release (63 ). Studies in
primates have documented the consequences of neurodevelopmental alteration in PFC connectivity on subcortical DA release
(64 ,65 ). Adult rhesus monkeys with neonatal ablation of the amygdala-hippocampal formation within 3 weeks of birth exhibit lower
NAA concentration in the PFC and abnormal relationships between prefrontal and subcortical DA functions; whereas local perfusion
of amphetamine into the PFC induced a decrease in striatal DA in control monkeys and in monkeys with adult lesions, PFC
amphetamine perfusion increased striatal DA release in monkeys with neonatal lesions. This study documents that dysregulation of
subcortical DA function might be a delayed and enduring consequence of neurodevelopmental abnormalities of PFC connectivity.

Positive Symptoms and Neuroplasticity

The fluctuating nature of the DA abnormalities documented by the imaging studies reviewed above implies that some level of
neuroplasticity is involved in the emergence of this increased subcortical DA activity during episodes of illness exacerbation (66 ).
Neurochemical sensitization of mesolimbic DA systems has been proposed by several authors as one mechanism that might underlie
the progression of a “silent” vulnerability into an overt symptomatology (67 ,68 ,69 ,70 and 71 ). Sensitization of DA systems is a
positive feedback loop, in which increased DA activity leads to more DA
 P.840

activity (70 ,72 ). During late adolescence, the failure of cortical development associated with schizophrenia liability might limit the
brain capacity to modulate stress-related increased activity of mesolimbic DA neurons. This failure of normal homeostatic and
buffering mechanisms would result in an increased vulnerability of DA neurons to develop a process of endogenous sensitization, a
response not observed in humans under normal circumstances. The endogenous sensitization process drives the prodromal and initial
phases of the illness, characterized by increased DA activity and culminating in the expression of positive symptoms. Sustained D2
receptor blockade interrupts this positive feedback loop. Upon neuroleptic discontinuation, the brain becomes again vulnerable to
the stress-induced reemergence of this endogenous sensitization process and clinical relapse.

It should be emphasized that the relationship between stimulation of D2 receptors and psychotic symptoms is complex and
presumably also involves neuroplasticity. NMDA antagonists such as ketamine or 5-HT2A agonists such as lysergic acid diethylamide
(LSD) induce psychotic symptoms in healthy subjects immediately upon drug exposure. In contrast, sustained administration of DA
agonists is required to induce psychotic symptoms in healthy subjects (7 ,73 ). This observation suggests that sustained
overstimulation of D2 receptors leads to remodeling of prefrontal-ventrostriatal-thalamic-prefrontal loops and their modulation by
hippocampal afferents projections, neuronal ensembles that are believed to underlie the psychotic experience (74 ,75 ). In the
amphetamine studies, DA-mediated stimulation of D2 receptors explained only about 30% of the variance in the positive symptom
change in untreated patients with schizophrenia (41 ), indicating that factors downstream from the DA synapse play a role in the
exacerbation of these symptoms following amphetamine. In the α-MPT study, global severity of positive symptoms did not correlate
with occupancy of striatal D2 receptors by DA (45 ), suggesting that, in some patients, the experience of positive symptoms is no
longer (or has never been) dependent on DA overstimulation. Patients with psychotic symptoms in the presence of apparently
normal DA function failed to show significant improvement in these symptoms following 6 weeks of D2 receptor blockade (45 ). Thus,
although these imaging studies have generally confirmed the time-honored dopamine hypothesis of schizophrenia, they also
contributed to pointing out the limitations of an oversimplified model linking psychosis and excess DA activity.

Prefrontal DA D1 Receptor Density

As discussed above, several lines of evidence from preclinical, clinical, and postmortem studies converge to suggest that a
deficiency in DA transmission in the prefrontal cortex is involved in the pathophysiology of negative symptoms and cognitive
impairment in schizophrenia (14 ,16 ). Furthermore, a deficient prefrontal DA function is a potential mechanism to account for the
subcortical DA disinhibition discussed above, as cortical DA function has an inhibitory impact on subcortical DA function (18 ,19 ). In
vivo measurement of prefrontal DA function would provide the tools to directly test these hypothesis.

The majority of DA receptors in the PFC are of the D1 subtype (76 ,77 ). At the ultrastructural level, they are mostly located on
pyramidal spines, and are mostly abundant on the distal dendrites (78 ,79 and 80 ). In postmortem studies, no evidence was found
of an alteration in D1 receptors in the DLPFC of patients with schizophrenia (81 ,82 ), and the expression of the D1 receptor gene is
unaltered (83 ). In contrast, a PET study with [11C]SCH 23390 reported decreased density of D1 receptors in younger patients with
schizophrenia (84 ). No significant differences were found in the other regions examined (anterior cingulate, temporal, occipital,
and striatum). In addition, low-PFC D1 density was associated with the severity of negative symptoms and poor performance on the
Wisconsin Card Sort Test (WCST). This finding is important, because it represents the first direct evidence of an association between
negative symptoms, working memory deficits, and selective alteration in prefrontal DA function. However, the camera used in this
study had a limited resolution, and the low specific to nonspecific ratio of [11C]SCH3390 makes the measurement of D1 receptor in
PFC with this ligand quite vulnerable to noise (85 ). Several groups are currently attempting to replicate this finding, using better
cameras and a superior D1 receptor radiotracer, [11C]NNC 112 (86 ).

In addition, measurement of receptor availability reveals only one aspect of neurotransmission. As D1 receptors are the most
abundant DA receptors in the PFC, the availability of a D1 receptor radiotracer vulnerable to competition by endogenous DA (i.e., a
D1 receptor “[11C]raclopride”) would be invaluable to assess presynaptic DA function in the PFC. Unfortunately, such a ligand is
currently lacking (87 ,88 ).

Studies of Nondopaminergic Receptors in Schizophrenia

Receptors related to the GABA and 5-HT systems have been studied in vivo in schizophrenia. Postmortem studies reported
abnormalities of both systems in schizophrenia. A robust body of findings suggests deficiency of GABAergic function in the PFC in
schizophrenia (see refs. 89 and 90 for reviews). In vivo evaluation of GABAergic systems in schizophrenia has so far been limited to
evaluation of benzodiazepine receptor densities with SPECT and [123I]iomazenil, and three out of three studies comparing patients
with schizophrenia and controls reported no significant regional differences (91 ,92 and 93 ). Although some significant correlations
with symptoms clusters and regional benzodiazepine densities have been observed (91 ,92 ,94 ,95 ), these relationships
 P.841

have not been replicated by other studies. Thus, together, these studies are consistent with an absence of marked abnormalities of
benzodiazepine receptor concentration in the cortex and patients with schizophrenia. Alterations of GABAergic systems in
schizophrenia might not involve benzodiazepine receptors (96 ), or be restricted to certain cortical layers or classes of GABAergic
cells that are beyond the resolution of current radionuclides based imaging techniques. Recent developments in GABA imaging with
MRS (described below) are a promising new avenue to study in vivo GABAergic function in schizophrenia.

Abnormalities of 5-HT transporters (SERT), 5-HT2A receptors and, more consistently, 5-HT1A receptors have been described in
postmortem studies in schizophrenia (see references in ref. 97 ). Given the relatively recent development of radiotracers to study
5-HT receptors, only a limited number of imaging studies have been published. The concentration of SERT in the midbrain measured
by [123I]β-CIT is unaltered in patients with schizophrenia (46 ). Studies with more specific ligands are warranted to assess the
distribution of SERT in other brain areas, such as the PFC, where their density has been reported to be reduced in three out of four
postmortem studies (97 ). Decrease in 5-HT2A receptors has been reported in the PFC in four out of eight postmortem studies
(97 ,98 ). Three PET studies in drug-naive or drug-free patients with schizophrenia reported normal cortical 5-HT2A receptor binding
(98 ,99 and 100 ), whereas one study reported a significant decrease in PFC 5-HT2A binding in a small group (n = 6) of drug-naive
schizophrenic patients (101 ). The most consistent abnormality of 5-HT parameters reported in postmortem studies in schizophrenia
is an increase in the density of 5-HT1A receptors in the PFC, reported in seven out of eight studies (97 ). Several groups are currently
evaluating the binding of this receptor in vivo with PET and [11C]WAY100907.

Receptor Occupancy By Antipsychotic Drugs

Maybe the most widespread use of neuroreceptor imaging in schizophrenia over the last decade has been the assessment of
neuroreceptor occupancy achieved by typical and atypical antipsychotic drugs, a topic that has been the subject of recent reviews
(102 ,103 ). Neuroreceptor studied included essentially D2 receptors, but also 5-HT2A and D1 receptors. The main conclusions from
this line of research are as follows: (a) Studies repeatedly confirmed the existence of a threshold of occupancy of striatal D2
receptors (about 80%) above which extrapyramidal side effects are likely to occur (104 ). (b) In general, studies failed to observe a
relationship between degree of D2 receptor occupancy and clinical response (105 ,106 ). Yet, most studies were performed at doses
achieving more than 50% occupancy, and the minimal level of occupancy required for therapeutic response remains undefined. Two
studies performed with low doses of relatively selective D2 receptor antagonists (haloperidol and raclopride) suggested that 50% to
60% occupancy was required to observe a rapid clinical response (107 ,108 ). (c) Clozapine, at clinically therapeutic doses, achieved
only 40% to 60% D2 receptor occupancy (104 ,106 ,109 ), which, in conjunction with its anticholinergic properties, accounts for its
low liability for extrapyramidal symptoms (EPSs). (d) Occupancy of 5-HT2A receptors by “5-HT2A/D2 balanced antagonists” such as
risperidone does not confer protection against EPS, because the threshold of D2 receptor occupancy associated with EPS is not
markedly different between these drugs and drugs devoid of 5-HT2A antagonism (110 ,111 ,112 and 113 ). (e) Studies with quetiapine
suggested that, at least with this agent, transient high occupancy of D2 receptors might be sufficient to elicit clinical response
(114 ,115 ).

An interesting question relates to putative differences in degree of occupancy achieved by atypical antipsychotic drugs in striatal
and extrastriatal areas. Pilowsky et al. (116 ) reported lower occupancy of striatal D2 receptors compared to temporal cortex D2
receptors in seven patients treated with clozapine, using the high-affinity SPECT ligand [123I]epidipride. In contrast, typical
antipsychotics were reported to achieve similar occupancy in striatal and extrastriatal areas, as measured with [11C]FLB 457 (117 ) or
[123I]epidipride (118 ). It should be noted, however, that these very high affinity ligands do not allow accurate determination of D2
receptor availability in the striatum. In contrast, [18F]fallypride enables accurate determination of D2 receptor availability in both
striatal and extrastriatal areas (119 ), and preliminary PET experiments in primates with [18F]fallypride indicate that clozapine and
risperidone achieve similar D2 receptor occupancy in striatal and extrastriatal regions (120 ). Finally, it is important to point out
that the most robust evidence relative to the site of therapeutic effect of antipsychotic drugs in rodents points toward the nucleus
accumbens (121 ,122 ), whereas the imaging studies reviewed above contrasted striatal versus mesotemporal D2 receptor binding.
Improved resolution of PET cameras currently allows dissociating signals from ventral and dorsal striatum (123 ,124 ), and it is now
feasible to specifically study the clinical correlates of D2 receptor occupancy in ventral striatum in humans.

Another unresolved question is the discrepant values of D2 receptor occupancy obtained with [11C]raclopride versus [11C]NMSP. The
haloperidol plasma concentration associated with 50% inhibition of [11C]NMSP binding (3 to 5 mg/mL) (125 ) is ten times higher than
that associated with 50% inhibition of [11C]raclopride binding (0.32 ng/mL) (126 ). Quetiapine, at a dose of 750 mg, decreased
[11C]raclopride-specific binding by 51%, but failed to affect [11C]NMSP-specific binding (127 ). These observations contribute to the
debate regarding differences between benzamides and butyrophenones binding to D2 receptors.
 P.842

Future Developments
Despite the remarkable achievements of the last decade, imaging neurosignaling processes with PET and SPECT in schizophrenia are
still limited by the relative low number of probes available. For example, despite major research efforts, direct measurement of
parameters of glutamate transmission are still not available. Radiotracers enabling evaluation of second messengers and
intracellular pathways are only beginning to emerge (128 ). A growing collaboration between academic centers and industry
currently holds the promise of increasing access to molecules for evaluation as candidate radiotracers.

Studies of the DA systems in schizophrenia illustrates how dynamic measurement of neurotransmission can be more informative than
simple measurement of receptor density. With the exception of the cholinergic system (129 ,130 ), the paradigm used with
[11C]raclopride and [123I]IBZM has been difficult to extend to other neuroreceptor systems, maybe because the fundamental
mechanisms underlying acute change in in vivo binding following transmitter fluctuations are still not perfectly understood (66 ).
Additional research is warranted to better characterize the factors that confer vulnerability of radiotracers in vivo binding to
functional status of neurotransmission.

Finally, a general limitation of these radionuclide-based techniques is the technical sophistication required as well as the high cost
of these investigations. However, the growing success of PET in oncology results in a larger availability of PET cameras for
neuropsychiatric clinical and basic research. This growing availability should be associated with a vigorous research effort toward
the development of more F-18 based probes, since the relatively longer half-life of F-18 compared to C-11 does not require that
these ligands be radiolabeled locally. For SPECT, the development of technetium-based neuroreceptor ligands (131 ) will further
enhance the availability of these techniques to the nuclear medicine community.

MAGNETIC RESONANCE SPECTROSCOPY

Part of "59 - Neurochemical and Neuropharmacological Imaging in Schizophrenia "

Magnetic resonance spectroscopy (MRS) is a chemical assay technique. It is the only clinically available method for the direct
measurement of chemical moieties in the living brain. MRS is based on the same physical principles as magnetic resonance imaging
(MRI), which involves characterizing atoms and molecules based on how they interact with a magnetic field. This interaction occurs
because the nuclei of atoms with an odd number of nucleons (i.e., protons and neutrons), such as 1H, 19F, 13C, 7Li, 23Na, have angular
momentum, so-called spin, which generates a small magnetic field around the nucleus. The spin properties of specific atoms and of
the specific molecules that they compose are unique and are exploited in an MRS experiment.

When a strong external magnetic field is applied to a tissue (e.g., the main magnet of an MR scanner), nuclei with spin align
themselves with the external field and assume an equilibrium state of net magnetization, which is proportional to the strength of
the field and the spin properties of the nucleus. An MRS experiment involves four steps, analogous to an MRI procedure. First,
specific nuclei are excited with a brief “pulse” of a radiofrequency (RF) magnetic field supplied by an RF transmitter coil. This
excitation causes magnetized spins to transiently assume a higher energy state, from which they “relax” to a lower energy state of
equilibrium magnetization. Because the energy states are quantitized, only specific RFs will excite the nucleus to another state and
only these frequencies will be emitted during relaxation (“resonance”). These resonant frequencies are unique for each atom, and
vary in proportion to the strength of the external field. The motion of spins in the process of returning to equilibrium (“relaxation”)
induces a current in a receiver coil, which represents the MR signal.

The second step involves spatially encoding the signal so that its origin can be mapped to a particular locale in the field of view.
This is accomplished with the application of linear magnetic gradients that add localizing characteristics to the signal. The third step
involves translating the signal acquired over time into a representation of its component frequencies and amplitudes, the so-called
Fourier transformation. The fourth step involves the mathematical and statistical analyses of the data.

In MRI, the signal used to reconstruct images is from hydrogen atoms (1H), which are found in many molecules, but most abundantly
in water and lipids. Although the signal from hydrogen contains frequencies corresponding to many different molecules, the water
and lipids signals dominate and the signals from hydrogen in other molecules are ignored. MRS, however, is based on resolving these
other molecular signals. These molecules are identifiable because of the phenomenon called “chemical shift.” The electron cloud
that surrounds the nucleus of an atom partially shields the nucleus from the external magnetic field. This shielding effect will cause
the nucleus to experience a slightly different external field, and thus to resonate at a slightly shifted frequency. Because the degree
of shielding varies from one molecule to another, depending on the electron sharing of the chemical bonds in the molecule, the
exact shift in the resonant frequency of a target nucleus (e.g., 1H, 31P) will reflect its chemical environment. In addition to electron
shielding, complex interactions between neighboring spins (“j-coupling”) also may affect the resonant frequency of a nucleus in
certain molecules. A Fourier transformation of the emitted signal resolves the various components of the signal into a spectrum
having peaks of specific frequency and amplitude, each peak corresponding to a different chemical environment of the target
nucleus. The area under the peak is directly proportional to the concentration of spins having that frequency, and thus of that
specific chemical
 P.843

moiety. It is customary to label peaks in the spectrum based on their relative displacement (“chemical shift”) in parts per million
from a standard peak (e.g., in the proton spectra, water is assigned a value of 4.75), with higher frequencies to the left. The use of
relative shift units allows for peaks to be identified regardless of the strength of the magnet. This also makes it possible to compare
in vivo and in vitro data to establish the chemical identity of each peak.

In schizophrenia research, MRS has been used to approach four general topics: (a) assays of brain chemicals, (b) evidence of tissue
pathology, (c) functional analysis of specific neuronal populations, and (d) drug effects. Before considering the results of these
efforts, it is important to consider some general methodologic issues that are germane to the MRS literature.

MRS Methodology
MRS is a methodologically complex technique that requires many quality control steps, both at the front end in data collection and
at the back end in data analysis. It is not a turnkey technique, and careful scrutiny of the raw data is essential in every experiment.
There are no absolute standards for the quality of spectral data, e.g., for the sharpness of a peak, or the degree of noise in the
baseline. Nevertheless, spectra derived from a particular atom (e.g., 1H or 19F) have a characteristic pattern, with sharp peaks at
defined relative frequencies, appropriate separation of peaks, acceptable signal to noise, and a stable baseline. Because the goal of
MRS is to assay the concentrations of chemicals that are in the range of three to five orders of magnitude less abundant than water,
it is necessary to make compromises to maximize the signal-to-noise ratio (SNR) in the data. One of the first decisions to be made in
an MRS experiment is how to sample from the brain. Most MRS studies in the schizophrenia literature have sampled from relatively
large (5 to 80 mL) single volume elements (“voxels”), though so-called MRS imaging (MRSI) approaches have also been utilized. The
single-voxel approach benefits from enhanced signal-to-noise (STN) and reduced scanner time (5 to 20 minutes per voxel), but it
suffers from imprecise and limited anatomic localization, contamination from signal outside of the voxel, and, most importantly,
partial volume averaging. Large voxels contain multiple tissue compartments, including cerebrospinal fluid (CSF), white matter, gray
matter, and vascular tissue, which have different concentrations of chemical constituents. Thus, the signals from a voxel are the
average of signals from all of these compartments. Although there are statistical procedures aimed at “segmenting” these
compartments within a voxel, such procedures are rough approximations (132 ). One of the fundamental difficulties in comparing
data from single-voxel studies between subject groups and across studies in the literature is uncertainty about the comparability of
voxel characteristics. 1H-MRSI studies generally employ smaller voxels (<1.5 mL) and have the advantage of being more anatomically
precise. This means that regions based on known anatomy, rather than arbitrary voxel placement, can be compared. There also are
less partial volume effects. However, because of the smaller voxels, imaging studies have reduced SNR and require longer scanner
times (20 to 60 minutes for a multislice study).

Because the amplitudes and frequencies of peaks in a spectrum are critically dependent on the magnetic field surrounding a target
nucleus, magnetic field uniformity within a voxel and between voxels is essential. Field homogeneity is routinely honed with
“shimming” procedures. It is also possible to make additional corrections based on field mapping, which corrects for frequency shifts
between voxels. Most studies in the literature have used shim procedures supplied by the manufacturer of the scanner, and it is
doubtful that these procedures have been optimal and comparable across studies (133 ). Coil design is another important parameter
in data acquisition and STN. Small surface coils have been used in many of the phosphorus spectroscopy studies, because they
improve SNR. However, surface coils introduce potential variance in studies that compare signals from specific locales across
individuals. This is because as transmitters, surface coils excite spins with a gradient of intensity from the surface, and again as a
receiver, the sensitivity drops off across the volume of activated tissue. Clearly, differences in coil placement and in head geometry
may contribute to variations in the data.

There has been much discussion in the MRS literature about absolute quantification of chemical concentrations. In practice, this is
almost impossible to do. References in the schizophrenia MRS literature to studies that have employed absolute quantification are
inaccurate, as all studies have employed some normalization procedure, often based on measures acquired in normal subjects. To
accurately measure absolute concentration of a chemical peak, it is necessary to control for all the variables that will affect the
amplitude of the peak from a given voxel. These include factors that affect field homogeneity, relaxation times of the molecule(s)
responsible for the peak, transmission efficiency and reception sensitivity of the RF coil (“coil loading”), and partial volume effects.
These characteristics vary across the brain within an individual and between individuals even in the same locale in the brain.
Moreover, any attempt to control for these factors assumes that the voxel remains in place, i.e., that the subject does not move
during the scan, which is difficult for many individuals. Thus, even with the help of absolute standards for calibration, it is very
difficult to correct for differences in these parameters (134 ). All studies to date have employed some normalization routine,
whether to noise in the baseline, to the total observed signal, to another metabolite in the spectra, or to a signal from a molecule in
the spectra that is thought to be metabolically neutral (e.g., water in the 1H spectra). Although each of these normalization
procedures involves assumptions and trade-offs, they attempt to control for unavoidable variations
 P.844

in the local field, in coil loading, in relaxation times, and, to a lesser extent, for partial volume averaging effects. A further
discussion of technical issues involved in collecting and analyzing MRS data is available in several reviews of the topic (135 ,136 and
137 ).

31
P Spectroscopy: Assays Of Phospholipids And High Energy Phosphates
The first in vivo MRS study of schizophrenia was of 31P spectra. At clinical MR field strength (1.5 to 4.0 tesla), the 31P spectrum
contains several peaks across a wide chemical shift range [approximately 30 parts per million (ppm)]. The major metabolite peaks
represent resonances for (a) phosphomonoester (PME) compounds (at 6.5 ppm), which includes phosphocreatine,
phosphorylethanolamine; (b) phosphodiesters (PDE, 2.6 ppm), which include glycerolphosphocholine, glycerolphosphoethanolamine,
and various membrane phospholipids; (c) phosphate residues on adenosine triphosphate (ATP) (-16.3 ppm for beta, -7.8 for alpha,
and -2.7 for gamma); (d) inorganic phosphate at 4.9 ppm; and (e) phosphocreatine, which, as the chemical shift standard, has a
resonance set at 0 ppm. PME concentrations, which are thought to reflect in part membrane precursors, increase with tissue growth,
including in early brain development and gliomas, but also with tissue destruction, e.g., in Alzheimer's disease and HIV. In white
matter, however, PMEs are metabolites of both synthesis and breakdown of sphingomyelin. PDEs are thought to reflect in part
products of membrane breakdown and have been shown to be decreased in certain brain tumors and to increase shortly after birth.
However, both PME and PDE peaks include other phosphorylated proteins associated with cell organelles and with membrane
phospholipids that are not clearly related to membrane turnover. This fact is underscored by studies of patients with Huntington's
disease (138 ) and of at least some patients with Alzheimer's disease (139 ), which have observed no abnormalities in these
metabolites. Thus, changes in PME and PDE peaks are not easily interpreted. The phosphocreatine (PCr), ATP, and Pi peaks confer
information about the state of high-energy phosphate metabolism and pH in the tissue. It also should be noted that because 31P
spectra are acquired with large voxels (i.e., 15 to 80 cm3), the relative contributions of various tissue components (i.e., gray matter,
white matter, neurons, glia, endothelia) to the signal is uncertain.

O'Callaghan and colleagues (140 ) reported the first 31P spectroscopy study in schizophrenia, on a sample of 18 patients and 10
control subjects. They used a surface coil to acquire data from 87-cm3 voxels arbitrarily placed in both temporal lobes. There were
no differences found in any of the peaks. Pettegrew et al. (141 ) reported a highly cited study of 11 first-episode patients, using a
surface coil placed over the front of the head. They reported that their voxel of 20 cm3 sampled the dorsolateral prefrontal cortex,
though its exact location is unclear, as subsequent reports stated that both right and left prefrontal cortices were sampled (142 ).
With only a single 20-cm3 voxel, this would indicate a more midline localization. The investigators reported that patients had
decreased PMEs and Pi, and increased PDEs and ATP. They interpreted their findings to reflect greater membrane turnover and less
energy utilization, results that they speculated were consistent with hypotheses about excessive synaptic pruning and decreased
frontal lobe metabolism.

Because of these initial reports, over fifteen 31P studies of patients with schizophrenia have appeared in the literature. Although
several studies have reported partial replication of the findings of Pettegrew et al. (141 ,142 ), particularly with respect to a
reduced PME peak, the majority of the reports have failed to do so. The data with respect to high-energy phosphate peaks are
especially inconsistent and generally negative. Studies that have looked outside the frontal lobe, at parietal and temporal lobes and
at the basal ganglia, also have yielded most often negative or inconsistent results. Several recent reviews provide tabulated
summaries of this literature (143 ,144 ). The reasons for the inconsistencies are unclear. The usual explanations—differences in
patient sample (e.g., medicated or unmedicated, acute or chronic), differences in acquisition parameters (e.g., surface or volume
coil, single voxel, or multivoxel)—seem inadequate, as both positive and negative reports have appeared regardless of the
characteristics of the samples and in the context of a variety of acquisition parameters. A more likely explanation for the
inconsistencies involves the low sensitivity and reproducibility of 31P spectroscopy and problems in achieving a standardized
placement of a region of interest (ROI) or of an acquisition plane. There are very limited data about the reliability of 31P
measurements in patients, with one study reporting a coefficient of variation of 30% across two scans (145 ). Because it is impossible
to accurately register single voxels or single planes to an anatomic reference, there is unavoidable error in the localization process,
which is further compounded by subject motion during the scan. Large voxel sizes also introduce error in terms of partial volume
effects. The small differences that have been reported in most of the positive studies, on the order of 10% to 25%, would seem
especially sensitive to such methodologic difficulties.

Two recent studies are particularly noteworthy in their efforts to control for localization variance and to improve the sensitivity of
the spectral data. Volz et al. (146 ) acquired a plane of 31P spectra, composed of 30 voxels of 19 cm3 each, and drew ROIs on an
anatomic reference scan that was acquired in a coplanar orientation, on 11 medication-free patients, including seven acute patients
who were medication naive. Though the planes are not strictly registered in space, because subject motion may have tilted their
relative orientations during the scans, the authors' approach to ROI
 P.845

placement is more reliable than that in earlier studies. With the exception of one mesial prefrontal voxel having a decreased PDE
peak and one voxel localized to the basal ganglia having a decreased PME peak, no other cerebral differences in phospholipids were
found. Four prefrontal voxels having decreases in ATP and PCr also were found. The authors argued that their data suggested
decreased membrane catabolism, but, given the number of voxels analyzed, chance results cannot be excluded. In an earlier study
of chronic, medicated patients, they also found a decreased prefrontal PDE peak, but the high-energy phosphate data were in the
opposite direction (147 ). Finally, Potwarka et al. (148 ), using signal enhancement techniques that reduce the effects of coupling
between phosphorus and proton spins, were able to separate structural membrane phospholipids from other constituents of the PME
and PDE peaks, with 50-cc voxels acquired as part of a plane. Although the authors found no decrease in PMEs as reported by
Pettegrew et al., they did find increases in the frontal PDE peak, but not related to membrane breakdown products such as
glycerophosphocholine (GPC) and glycerolphosphoethanolamine (GPE), as suggested by Pettegrew et al. and others. Rather, the
difference was reflected in the membrane phospholipid components of the peak. These differences were not found in motor or
occipital cortices, providing some evidence of internal validity. This study also found no differences between patients and controls
in total PMEs, again in contrast to the Pettegrew et al. study, but a component of the PME peak, phosphocholine, was reduced.
Potwarka et al. (148 ) proposed that their data implicated membrane abnormalities selectively in DLPFC, perhaps involving
presynaptic vesicular phospholipids. However, to confuse the story even further, Bluml et al. (149 ), using a similar proton
decoupled 31P MRS approach, reported increases in GPC and GPE acquired with a large (97-cc) voxel in the middle of the cerebrum.

Several studies have attempted to link 31P data to clinical characteristics of patients, but these also have been inconsistent. For
example, Deicken et al. (150 ) reported a correlation between prefrontal PME signals and performance on the WCST, suggesting that
prefrontal membrane abnormalities were reflected in prefrontal function. However, in the same patient sample, Deicken et al. did
not find an abnormality of PME signals. Volz et al. (147 ) could not find a correlation between any 31P signals and WCST performance.
Potwarka et al. (148 ) found a correlation between an ATP peak in right prefrontal cortex and negative symptom ratings, but similar
relationships were not found in other studies (151 ).

It is difficult to arrive at a synthetic analysis of the 31P data in schizophrenia. Technical error is probably the critical factor in the
variable results that have been reported. It is doubtful that the small differences between patients and controls could escape
corruption by the many methodologic limitations of the current techniques. Future studies using higher field magnets, with better
sensitivity and resolution, combined with signal enhancement and peak separation procedures may lead to more reliable methods.

Proton Spectroscopy: Assays of Cells and Cellular Metabolism

Proton spectroscopy has been a more widely applied technique in schizophrenia research and the results are much more consistent.
A variety of chemicals in the proton spectrum can be assayed with clinical magnets, including several amino acids, membrane and
myelin metabolites, and several high-energy substrates. Although the sensitivity of proton spectroscopy is approximately 20 times
that of phosphorus, allowing for much better resolution, the metabolites of interest need to be resolved in a smaller chemical shift
range (less than 10 ppm), in the presence of large concentrations of brain water (approximately 104 times greater concentration
than the other metabolites) and mobile lipids from the skull and scalp. Until recently, most 1H MRS techniques used special
procedures to suppress the signal from water and lipids, procedures that can affect other signals. With the availability of analogue
to digital converters having greater dynamic range, it is now possible to acquire the water signal and still resolve the other
metabolites (152 ). In addition to preservation of neighboring signals, this approach also has the advantage of making the water
signal available as an internal standard for normalization and potentially for tissue segmentation.

The proton spectrum is characterized by several relatively large and distinct peaks and several complexes of smaller overlapping
peaks. The metabolite signals acquired with 1H MRS vary depending on the echo time of the pulse sequence used for the acquisition.
Many of the resolvable elements have short T2 (e.g., myoinositol, glutamate, glutamine, and GABA) and emit no observable signal
with longer echo times. On the other hand, long echo time acquisitions produce signals from several compounds that are very
distinctly resolved. The long echo time metabolite spectrum is dominated by a peak at approximately 2 ppm corresponding to the
methyl group (CH3) of several N-acetyl containing compounds, principally N-acetylaspartate (NAA) and to a small degree, N-acetyl
aspartate glutamate (NAAG) and possibly N-acetylneuraminic acid (NANA) (134 ). NAA is an intracellular neuronal marker, found
almost exclusively in mature neurons and their processes (153 ), with the highest concentrations in pyramidal glutamate neurons
(154 ). NAA is the second most abundant amino acid in the brain (155 ). Its concentration is higher in gray matter than in white
matter (156 ), and NAA signals increase during childhood, remaining relatively stable throughout adult life (156 ,157 and 158 ). The
exact implications of changes in NAA signals is uncertain, as its cellular function is still unclear. It is synthesized in mitochondria
from glutamate and either pyruvate or 3-hydroxybutyrate via L-aspartate-N-amino
 P.846

transferase and also is a by-product of NAAladase catabolism of NAAG, which occurs within glia (159 ). Whether NAA signals are
absolutely specific to neurons is unclear. Mature astroglia do not contain NAA, though small concentrations have been reported in
oligodendroglial cultures (160 ). NAA is a nonspecific though highly sensitive marker of neuronal pathology. Virtually all neurologic
conditions involving neuronal pathology that have been studied, including multiple sclerosis, motor neuron disease, Alzheimer's
disease, Huntington's disease, cerebellar degenerations, multiple sclerosis, epilepsy, and various encephalopathies, show changes in
NAA signals in the regions of brain pathology. Moreover, NAA changes are sensitive measures of dynamic neuropathologic processes
(161 ,162 and 163 ), for example, correlating over time with cognitive change in Alzheimer's disease (164 ) and with the number of
trinucleotide repeats in Huntington's disease (165 ). Although early studies interpreted NAA findings as indicative of cell loss, recent
data have established that NAA reductions can reverse following various forms of brain damage and can change with clinical
improvement and treatment (161 ,162 ,166 ,167 and 168 ). This has led to speculation that NAA reductions occur as a manifestation
of changes in neuronal volume or in NAA concentrations within a neuron, perhaps reflecting reduced mitochondrial energy
metabolism (159 ) or a change in the abundance and patterns of neuronal connectivity (169 ,170 ). It is interesting to note that in
various conditions associated with tissue volume loss and reduced NAA signals (e.g., epilepsy, Alzheimer's disease, schizophrenia),
these two parameters are not tightly correlated. The only condition in which NAA concentrations are increased is Canavan's disease,
which involves a mutation in a gene on chromosome 17 controlling the synthesis of N-acetyl-L-aspartate amidohydrolase
(aspartoacylase), the enzyme that breaks down NAA.

Two other prominent peaks are seen in the long echo time proton spectrum. At 3.2 ppm is a peak corresponding to the
trimethylamine group of various choline (CHO)-containing compounds, mostly membrane phospholipids. This signal reflects the
concentrations of several phospholipid moieties, including glycerophosphocholine, phosphocholine, and phosphatidylcholine. In
pathologic conditions associated with membrane turnover or gliosis (e.g., Alzheimer's disease, gliomas, epilepsy), the CHO peak
tends to be elevated. At 3.0 ppm, a peak corresponding to creatine and phosphocreatine (CRE) appears. These metabolites
participate as energy buffers in many energy-consuming processes in the brain, but consistent changes in the CRE signal are
generally found only in the presence of tissue loss.

At short echo time, several other peaks are observed, in addition to the peaks that persist into the long echo time spectra. The most
studied of these is the myoinositol peak (3.6 ppm) and a peak complex (called tGlx) at around 2.2 to 2.4 and 3.75 ppm
corresponding to overlapping signals from glutamate, glutamine, and GABA. Myoinositol is a hexol present in high concentrations in
human brain, and accounts for most of the myoinositol peak, though other complex inositol phosphates also contribute to the signal.
Some of these inositol phosphates may represent second-messenger signaling molecules that may vary with the state of cellular
activity. The myoinositol peak, however, tends to change in conditions associated with active membrane turnover and gliosis, and is
consistently increased in Alzheimer's disease. The glutamate and GABA peaks include soluble forms of these amino acids involved
both in neurotransmission and in peptide synthesis. Glutamine is an intermediary in glial-based recycling of the carbon skeletons of
these amino acids, and has been proposed as a more sensitive marker of turnover of the glutamate amino acid pool.

Evidence from H MRS of Neuronal Pathology

1

Nasrallah et al. (171 ) reported the first 1H MRS study of both mesial temporal lobes in schizophrenia, an investigation of 11 chronic
patients and 11 controls, using a 12-cm3 voxel. They found decreases in NAA peaks in both voxels, with the difference on the right
significant at the .05 level, and on the left at the .06 level. Since the report of Nasrallah et al., over 20 1H MRS studies of patients
with schizophrenia have appeared. Most of them have addressed metabolite changes, primarily NAA, in the frontal and temporal
lobes. Table 59.3 summarizes studies of frontal and temporal lobe NAA signals. Several recent reviews describe these reports in
greater detail (143 ,144 ,172 ). Although most of these studies have involved single voxel data, with the inherent methodologic
issues described above, several recent relatively high resolution (approximately 1-mL voxel) multivoxel and multislice imaging
studies (MRSI) have also been reported. Reliability data for NAA are much superior to those of other metabolites in either the proton
or the phosphorus spectra; for example, the coefficient of variation for repeat studies in patients using an MRSI technique is on the
order of 10% (173 ). It is interesting in this regard that all of the MRSI studies to date that have examined cortical regions in the
frontal and temporal lobes have reported reduced NAA signals in these regions (174 ,175 ,176 ,177 ,178 and 179 ). These studies are
exemplified by a series of reports from Bertolino and colleagues (174 ,175 ,176 and 177 ) using a technique that acquires over 700
1.4-mL voxels in approximately 25 minutes. The technique allows for registration of spectroscopic and anatomic images, for reliable
and anatomically correct ROI definition, and for relatively diminished partial volume effects. The studies of Bertolino et al. included
chronic medicated patients, unmedicated and several neuroleptic-naive patients, and childhood-onset patients. In each of these
samples, NAA signals were reduced selectively and bilaterally in dorsolateral prefrontal and perihippocampal cortices. Using a
similar technical approach in studies of chronic patients, Deicken et al.
 P.847

replicated these results (178 ,179 ), and also found reduced NAA signals in cingulated cortex (180 ) and in thalamus (181 ), which
were not found in the Bertolino et al. studies. The only imaging study that reported no decrease in gray matter NAA did not
regionally parcellate the cortex, and reported only an average measure for the entire cortex of the top half of the brain (182 ).
Indeed, this result is consistent with the findings of Bertolino et al., in which NAA levels throughout most regions of brain were not
different between patients and controls. Although most of the single voxel studies also found reductions in NAA signals in frontal and
temporal lobes, the few negative studies (183 ,184 ,185 ,186 and 187 ) may reflect methodologic variance related to small sample
sizes or to voxel placement, etc. The single voxel studies even of NAA signals also tend to have much less reliable data [e.g., a
coefficient of variation (cv) in a negative study by Kegeles et al. (187 ) of greater than 30%]. It is clear from this literature that
reduced NAA peaks are found in the frontal and temporal lobes of many patients with schizophrenia (see table for a summary).

TABLE 59.3. 1H MRS STUDIES OF FRONTAL AND TEMPORAL LOBE NAA SIGNALS IN
SCHIZOPHRENIA

There have been few studies of other peaks in the proton spectra, and the results have been inconclusive. Because of interest in
glutamate and cortical function in schizophrenia, several groups have attempted to measure the tGlx peak, using quantitation
methods based on a priori knowledge of the relative contributions of the metabolite components, but the reliability of the method is
limited [e.g., cv >50% (183 )]. Stanley et al. (184 ) found increased glutamine signals in chronically treated patients, but no
differences in acute patients. Bartha et al. (183 ) reported increased glutamine in cingulated cortex of a group of ten patients.
Rakow et al. (188 ) reported decreased tGlx in dorsolateral prefrontal cortex and Kegeles et al. (187 ) reported no changes in mesial
temporal lobe. The data with choline and creatine peaks have generally been negative, and the occasional positive result has been
inconsistent across studies.
 P.848

There has been considerable interest in trying to understand the meaning of NAA decreases in patients with schizophrenia. The NAA
changes have consistently been shown not to correlate with stage of illness, with medication status, and with length of illness.
Moreover, hippocampal and prefrontal volume do not appear to correlate either. In the Bertolino et al. (175 ) study of unmedicated
patients, several of the subjects had been chronically psychotic and untreated for over 10 years, and no relationship between NAA
signals and length of untreated psychosis was found. These data suggest that NAA reductions are not the result of a linearly
progressive pathologic process. They also provide evidence against the notion that chronic untreated psychosis is “neurotoxic.”
Because postmortem studies do not provide convincing evidence of neuronal loss or of neuronal degeneration in the areas where
NAA signals are consistently reduced (i.e., hippocampal formation and prefrontal cortices) (189 ), the NAA changes probably reflect
more subtle aspects of neuronal biology (see below). As such, they are unique in vivo evidence of cellular changes probably
restricted to neurons in the schizophrenic brain. Although NAA reductions are found in both medicated and unmedicated patients,
the assumption that NAA changes are independent of medical treatment may be incorrect. Bertolino et al. (167 ) recently reported
that NAA signals in dorsolateral prefrontal cortex increase slightly but significantly after only several weeks of neuroleptic treatment.
This slight increase is further evidence that NAA levels may reflect dynamic neuronal events, and is consistent with evidence that
other pharmacologic treatments can alter NAA signals [e.g., lithium (190 )]. Although NAA changes clearly occur independent of
neuroleptic treatment, the neuroleptic effect illustrates that physiologic factors may contribute to the variations in NAA signals.

NAA Signals and Abnormalities of Specific Functional Systems

Because NAA signals originate perhaps exclusively in neurons, principally glutamate neurons, it is possible to probe the functional
connectivity of cortical glutamate neurons with NAA as a surrogate marker of the integrity or activity of such connectivity. This has
been done in animals and in human studies. In animals, optic nerve injury reduces NAA concentrations in the lateral geniculate
(191 ), and prefrontal cortical injury reduces NAA signals in basal ganglia, suggestive of loss of afferent terminals or transynaptic
cellular changes (169 ). Bertolino et al. (170 ) measured NAA signals in DLPFC of adult monkeys that had undergone mesial temporal
lobe removals as neonates and in monkeys that had undergone temporal lobe removals as adults. Reduced NAA signals were found
only in the animals with neonatal removals, suggesting that NAA could reflect more complex plastic neuronal modifications, perhaps
at the level of local circuit architecture, than simply loss of afferent terminals. Similar results have also been observed in rodents
(192 ).

This evidence of relationships between NAA signals and connections between neurons led to a series of studies aimed at testing
hypotheses about the centrality of prefrontal connectivity in the pathophysiology of schizophrenia (193 ). Bertolino et al.
(63 ,194 ,195 ) used NAA signals as a marker of glutamate projection neuronal function/integrity in predicting the activity of
distributed neuronal systems implicated in positive and negative symptoms. NAA signals selectively in DLPFC were found to predict
the availability of DA receptors in the striatum in patients with schizophrenia, assayed with radionuclide imaging (194 ). Specifically,
lower DLPFC NAA predicted greater availability of DA receptors in an unstimulated, resting state, speculated to reflect less DA
release and cell firing. The same measure, i.e., low NAA, also predicted the exaggerated response to amphetamine found in
striatum with radioreceptor imaging (63 ). These data suggested that NAA in DLPFC predicted the steady-state and stimulus-induced
responses of dopamine neurons in the ventral brainstem. Similar relationships were not found for any other cortical regions or in
normal controls, implicating the neuronal pathology associated with the illness as instrumental in constraining these relationships.
Although DA release was inferred indirectly from radioligand binding availability, the assumptions were confirmed in studies of
monkeys undergoing in vivo microdialysis, in which NAA concentrations in DLPFC directly predicted DA release in the striatum (194 ).

NAA signals in DLPFC also were found in patients with schizophrenia to selectively predict the activation of the distributed working
memory cortical network, studied both with PET (195 ) and with functional MRI (fMRI) (196 ). These data suggest that prefrontal
glutamate neurons, by virtue of their intracortical connectivities, modulate the capacity and efficiency of distributed cortical
networks involved in working memory. Working memory deficits have been consistently linked to other aspects of the negative
symptoms of schizophrenia, and in fact NAA signals in DLPFC also specifically predict negative symptoms ratings in patients (197 ).

These various clinical studies of phenomena predicted by NAA signals in DLPFC converge on a tantalizing interpretation of these
various findings—that NAA reductions reflect subtle cellular pathology of intrinsic DLPFC neurons and their local circuitry that, by
virtue of intracortical and corticofugal projections, modify the activity of distributed cortical networks and of DA neurons,
implicated in the negative and positive symptoms of schizophrenia, respectively. Thus, DLPFC neurons appear to be an effector
neuronal population that is associated with the manifest biology of the illness. It is interesting to note that NAA signals in
hippocampal formation, the other region consistently implicated in studies of patients with schizophrenia, do not show these
predictable relationships. A study by Callicott et al. (177 )
 P.849

may shed some light on this apparent inconsistency. Callicott et al. studied 60 healthy siblings of patients with schizophrenia and
found that although NAA signals were also reduced in the hippocampal formation of the healthy siblings, changes in DLPFC were not
found. These data suggest that consistent with other evidence of hippocampal functional abnormalities in relatives of patients with
schizophrenia (e.g., memory deficits, P-50–evoked potentials), NAA changes in the hippocampal formation may reflect biology of
genetic risk. In contrast, consistent with the predictable relationships of NAA in DLPFC with other functional systems implicated in
schizophrenia, NAA changes in DLPFC reflect biology of manifest illness. The lack of a difference in frontal lobe NAA in healthy
relatives of patients with schizophrenia has recently been reported by another group as well (198 ). Therefore, these results have
added hippocampal NAA measures to the list of potential phenotypic markers of genetic risk for further exploration in genetic
studies of mental illness.

Future Developments
MRS is a rapidly evolving technology and its future applications in schizophrenia research should lead to important discoveries. New
developments in the near future will likely emerge from methodologic advances leading to improved sensitivity and resolution,
measurement of novel chemical moieties, indexing neuronal metabolism, and characterizing drug effects. The availability of high-
field human magnets (3 to 7 tesla) will substantially improve the STN of MRS and allow for improved reliability and resolution. At the
National Institute of Mental Health (NIMH), proton spectral images are currently acquired at 3 T with 0.7-mL voxels, and the cv of
repeat NAA measurements in the hippocampus is about 3%. Various hardware upgrades have also made it possible to shim individual
slices rather than slabs of tissue, and to acquire both early and late echo spectra within the same acquisition, without suppressing
the water signal. Use of techniques to control for motion effects (e.g., navigator signals) may further improve reliability and make it
possible to avoid lipid suppression approaches, as well. The improved STN and signal acquisition of new methods will result in more
sensitive and reliable acquisition of other spectral peaks. This will improve the potential reliability of phosphorus moieties in the
proton spectrum and will make calculations of the components of the tGlx peak more reliable. Preliminary results using spectral
editing approaches to the GABA peak suggest that clinically meaningful data about GABA metabolism can be derived from this peak
(199 ). Greater sensitivity and SNR also will permit spectral analyses of externally administered molecules. C13 glucose can be given
as a glucose load and the fate of the C13 carbon skeleton tracked over time as changing concentrations of moieties in the C13
spectra (200 ). This may provide near–real-time information about glucose metabolism, and also potentially about the turnover of
several neurotransmitters. Preliminary studies of fluorinated compounds, such as fluoxetine and fluphenazine, have demonstrated
the feasibility of measuring the concentrations of such compounds in brain. The potential applications of such measurements will
become clearer in the context of improved methodology. Finally, the demonstration that NAA changes with antipsychotic drug
treatment represents the first in vivo evidence of an intracellular effect of these drugs. Future studies will aim to understand the
basis for this change, as well as other factors that affect NAA signals.

ACKNOWLEDGMENTS
Part of "59 - Neurochemical and Neuropharmacological Imaging in Schizophrenia "
This work is supported by the National Institute of Mental Health (M.L., K02 MH01603-01). Dr. Weinberger has served as a consultant
for Janssen and Eli Lilly & Company.

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Section VII
Anxiety and stress disorder
Dennis Charney

Dennis Charney: Mood and Anxiety Disorder Research Program, National Institute of Mental Health, Bethesda, Maryland.

Anxiety and stress disorder - Introduction

The chapters in this section reflect the advances that have been made in our understanding of the molecular neurobiology and
neural circuits underlying fear and anxiety states and the potential for badly needed advances in the diagnosis, pathophysiology, and
treatment of anxiety disorders.

Many readers will be surprised by the data cited in the Kessler and Greenberg chapter on the economic burden of anxiety and stress
disorders. Recent surveys demonstrate that anxiety and stress disorders are the most commonly occurring of all mental disorders.
Anxiety disorders are temporally the primary disorders in many people with a lifetime history of any mental disorder. Indeed the
combined occurrence of high lifetime prevalence with an early age at onset and high chronicity makes anxiety disorders unique.

The chapter by Stein and Lang reviews in detail the course of anxiety and stress disorders over the lifetime. One point they make
that is especially important to emphasize is the critical need for further research in childhood anxiety disorders. The presence of an
anxiety disorder in childhood or adolescence is a predictor of the persistence into adulthood of not only anxiety disorders but other
psychiatric disorders as well, particularly depression. It is not known if early effective treatment of childhood and adolescent
anxiety disorders will prevent the development of psychiatric disorders later in life.

Important insights into the pathogenesis of anxiety disorders come from preclinical investigations reviewed by Davis in his chapter.
The identification of the brain structures and neural circuits involved in the generation of fear and anxiety-related behaviors are
most noteworthy. The delineation of specific neural pathways mediating conditioned and unconditioned fear can logically guide the
design and conduct of clinical studies investigating the neurobiological mechanisms of anxiety disorders such as generalized anxiety
disorder (unconditional fear) and phobic disorders (conditioned fear). Increased knowledge of the neural mechanisms and
neurotransmitters involved in extinction may offer novel therapeutic approaches. If we can discover pharmacologic methods to
enhance extinction, more effective drug treatments for conditioned fear or anxiety may be found. There is evidence of extinction
being mediated by γ-aminobutyric acid (GABA) release. This suggests the possibility of augmenting extinction-based psychotherapy
with GABA agonist drug treatment.

Determination of the genetic contribution to anxiety disorders is critically important to progress in understanding etiology and
improving treatment.

Merikangas and Pine review the evidence that the major subtypes of the anxiety disorders aggregate in families. However, the
magnitude of heritability is relatively moderate, indicating a strong environmental contribution to etiology. Unfortunately, thus far
linkage and association studies in anxiety disorders have not been fruitful. Future studies should determine if components of anxiety
syndromes are controlled by specific genes. The revolution occurring in genomics research as a consequence of sequencing the
human genome and the identification of over 1½ million single nucleotide polymorphisms (SNPs) should make discovery of anxiety-
related disease genes a reality. Strategies that are complementary to linkage analyses and utilize data from linkage studies are
indicated. Such approaches couple the genotyping of candidate functional SNPs with linkage and equilibrium mapping in
chromosomal regions implicated in linkage studies. In parallel with the genetic studies, enhanced efforts to identify endophenotypic
biological vulnerability markers are indicated. The studies cited in the chapter on temperament, anxiety sensitivity, autonomic
reactivity, psychophysiologic function, ventilatory function, neurochemical, and neuroendocrine factors are good examples of this
approach.

Bakshi and Kalin point out in their chapter the advantages

 P.858

of using putative animal endophenotypes of stress and anxiety to identify genetic abnormalities associated with anxiety disorders.
Rodent and nonhuman primate studies of mother-infant interactions are particularly compelling, given the important clinical
implications if these interactions are found to be a critical factor in future fearful disposition. Targeted mutations leading to
anxiety-like endophenotypes in transgenic mice have suggested roles for serotonin receptor subtype 1A (5-HT1A), corticotropin-
releasing hormone (CRH), GABA, neuropeptide Y, cholecystokinin, and substance P neural systems in the generation on anxiety-fear
behaviors. These studies provide clues for the discovery of new medications and pharmacogenomic approaches to treatment.
Accelerated drug development efforts focusing on corticotropin-releasing factor 1 (CRF-1) receptor antagonists and benzodiazepine
agonists with an anxioselective subunit profile are indicated. The feasibility of pharmacogenomic investigations designed to evaluate
the relationships among functional polymorphisms of the 5-HT1A receptor, benzodiazepine receptor, and GABA synthesis enzymes and
therapeutic responses to specific drugs should be explored.

It is imperative that these findings from the preclinical studies of anxiety and fear states be translated into increased knowledge of
the neural circuits and associated neural mechanisms that can account for the signs and symptoms in patients with anxiety disorders.
Rauch and Shin reviewed the neuroimaging findings relevant to anxiety and stress disorders. Their chapter emphasizes the areas of
congruence between animal studies and clinical neuroimaging investigations. For example, imaging studies in healthy subjects
support a role for the amygdala in fear conditioning and the frontal cortex in extinction. In the imaging studies of patients with
anxiety disorders, progress has been made in identifying specific neural circuits. Functional relationships among the amygdala,
hippocampus, and medial prefrontal cortex have been reported in patients with posttraumatic stress disorder (PTSD). The imaging
findings in patients with obsessive-compulsive disorder (OCD) are consistent with “pathology” in cortico-striatal-thalamo-cortical
circuitry. Unfortunately, a critical gap in knowledge exists regarding the relevant neural circuits involved in panic disorder, social
anxiety disorder, and generalized anxiety disorder. The neurochemical systems associated with anxiety and fear circuits are
reviewed in the chapter by Charney and Drevets. As predicted from the preclinical studies, abnormalities in norepinephrine,
benzodiazepine, glucocorticoid, and CRH systems have been identified in patients with anxiety disorders. However, most of the
findings reviewed should be deemed preliminary, and they require replication. None of the reported neurobiological distinctions
between patients and controls is robust enough to be of diagnostic relevance.

Tallman, Cassella, and Kehne review the mechanism of action of anxiolytic drugs and the status of new and novel therapeutic agents.
They highlight the therapeutic potential and current status of CRH antagonist drug development. They also note the potential of
developing targets for the CRF-2 receptor and other peptides, such as vasoactive intestinal peptide (VIP), involved in the regulation
of stress. In regard to benzodiazepine drug development, they note an ideal drug might have limited effects on the α1 subtype with
increased responsiveness at α2 and α3 subunits. Glutamate receptor agonists and modulators are proposed as viable targets for
anxiety disorders. For example, point mutations in the glycine-binding site of the NR1 subunit result in mice that have reduced
glycine affinity and an anxiolytic profile. Group II metabotrophic glutamate agonists are in early clinical development for the
treatment of anxiety disorders. Other novel drug targets include antagonists of AMPA receptors and antagonists of strychnine-
sensitive glycine site, both of which show anxiolytic profiles in animal models.

Ultimately, the goal of research on the neurobiological underpinnings of anxiety disorders is to lead to more effective, more rapidly
acting treatments, to achieve a more complete response, and to be able to predict responses to specific treatments. In their review,
Gorman, Kent, and Coplan highlight the extremely broad spectrum of action of norepinephrine and serotonin transport inhibitors in
anxiety disorders. These drugs are limited by a delayed onset and in complete response in many patients. This suggests that
norepinephrine and serotonin have broad modulatory effects on other neuronal systems, which are more primarily related to the
pathogenesis of anxiety disorders.

In summary, a reading of these chapters reveals that there have not been fundamental advances in our ability to diagnose anxiety
disorders based on known etiology. The mainstay of medication treatment continues to be classes of medications that have existed
for decades. There are major gaps in our knowledge of anxiety disorders in children. The mechanisms responsible for the occurrence
of anxiety disorders in childhood and adolescence leading to increased risk for other psychiatric disorders in adulthood are unknown.
However, the potential for progress is great. A multidisciplinary team approach utilizing the findings from preclinical investigations
on neural circuitry, neurochemistry, and genetics to inform clinical investigations of genetic vulnerability, environmental risk factors,
neuroimaging, pharmacogenomics, and novel drug design and testing will be a pathway to discovery.
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60
Anxiety and Stress Disorders: Course Over the Lifetime

Murray B. Stein

Ariel J. Lang

Murray B. Stein and Ariel J. Lang: Department of Psychiatry, University of California–San Diego, San Diego, California.

This chapter traces the course and trajectory of anxiety disorders across the life span. The chapter discusses these disorders in three
age groups: (a) childhood, (b) young adulthood to middle age, and (c) older adulthood.

 ANXIETY AND STRESS DISORDERS IN CHILDHOOD

 ANXIETY AND STRESS DISORDERS IN YOUNG TO MIDDLE-AGE ADULTS
 ANXIETY AND STRESS DISORDERS IN OLDER ADULTS
 SUMMARY
 ACKNOWLEDGMENTS

ANXIETY AND STRESS DISORDERS IN CHILDHOOD

Part of "60 - Anxiety and Stress Disorders: Course Over the Lifetime "

Temperamental and Environmental Precursors

There are a number of factors that relate to the development of anxiety in general. Although this remains a controversial area,
current evidence suggests that anxiety does not appear to be specifically heritable; what clusters in families is a more general
predisposition to mood or anxiety disorders. Evidence of this comes from twin studies (1 ) as well as from studies of the incidence of
disorders in the families of anxious patients (2 ). Specific phobia, however, may be an exception to this general heritability; family
members with specific phobias tend to be associated with increased risk only for specific phobias (3 ).

Any biological predisposition is likely moderated by environmental factors. For example, Beidel and Turner (2 ) found that parental
psychopathology was a risk factor for the development of disorder only among the lower socioeconomic status (STS) portion of their
sample. It has been suggested that environmental factors play a significant role in the manifestation of specific psychopathology (1 ).
Anxiety in particular is believed to be related to a combination of negative affect, a sense of lack of control over situations or
environments, and attentional self-focus. Early experiences of being unable to influence or control situations, therefore, may lead
to the development of anxiety (4 ).

Specific patterns within the family may lead to increased risk of development of childhood anxiety. Expressed emotion (EE), which is
an interactional style composed of emotional overinvolvement and high levels of criticism, has been associated with an increased
likelihood of childhood anxiety disorders (5 ) and with long-term functioning in those with anxiety disorders (6 ). Parents may also
influence a child's anxiety by providing positive (e.g., the child gets attention) or negative (e.g., the child is allowed to avoid
anxiety-provoking situations) reinforcement for expressions of anxiety, by providing inadequate affection and by excessive
control/overprotection (4 ).

Can it be said that some people are born anxious? The answer is a qualified yes. The last decade or so of research by Jerome Kagan
and colleagues has led to the identification of a temperament, “behavioral inhibition” (BI), that appears to be related to the
subsequent onset of anxiety disorders. BI involves reacting to unfamiliar or novel situations with behavioral restraint and physiologic
arousal (5 ). When confronted with an unfamiliar person or object, a BI child will withdraw, cling, be reluctant to interact, show
emotional distress, and stop other activities. BI has also been associated with physiologic differences, such as high and stable heart
rate, increased salivary cortisol and urinary catecholamines, pupillary dilation, and laryngeal muscle tension (7 ). These findings
have led to the hypothesis that BI is related to a low threshold for arousal in the amygdala and hypothalamus (8 ). This
characteristic, which appears to be present in 10% to 15% of children, has been identified in children as young as 14 months, has
been shown to persist throughout childhood (9 ), and is more commonly found in offspring of anxious parents (8 ). The inhibited
temperament has been associated with risk of developing an anxiety disorder, most commonly social phobia (10 ).

Some have suggested that childhood anxiety and depression are so closely related that they are best considered as part of the same
construct, often referred to as internalizing disorder. This lack of differentiation appears to be characteristic of younger children,
with increased specificity developing over time (11 ,12 ). At least by middle childhood, there is support for the set of anxiety-
related diagnoses in the
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Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). Spence (12 ) conducted a confirmatory factor
analysis with data from children of 8 to 12 years. The best model included six correlated factors—panic-agoraphobia, social phobia,
separation anxiety, obsessive-compulsive problems, generalized anxiety, and fear of physical injury (including dogs, dentists, heights,
doctors)—and a single higher-order factor reflecting overall anxiety.

There is a clear need for further research in childhood anxiety. Estimates of prevalence and recovery vary widely because of a lack
of standardization of criteria, assessment instruments, and methodology (e.g., clinician rating vs. self-report, child vs. parent or
teacher report). A few general conclusions can be drawn about childhood internalizing disorders. Internalizing symptoms appear to
remain fairly stable over time (13 ,14 ). Among boys, internalizing symptoms are not only predictive of later internalizing symptoms
but also of subsequent externalizing problems (15 ). Although there may be high rates of recovery associated with a particular
anxiety disorder, children who recover are at increased risk of developing other psychiatric diagnoses, most commonly other anxiety
disorders or depressive disorders (16 ). Similarly, the presence of an anxiety or depressive disorder [overanxious disorder
(OAD)/generalized anxiety disorder (GAD), panic, major depression] in adolescence is nonspecifically predictive of anxious or
depressive disorders in adulthood (17 ).

The remainder of this section discusses disorder-specific information, including clinical manifestations, prevalence, and course.

Generalized Anxiety Disorder (GAD)

GAD is characterized by excessive anxiety or worry, which is difficult to control and is accompanied by symptoms of tension and
physiologic arousal (18 ). The GAD diagnosis is currently used in place of overanxious disorder (OAD), which was eliminated with the
publication of the DSM-IV. Children and adolescents with GAD most frequently worry about future events, personal safety, and social
evaluation, and often present with multiple somatic complaints, such as headaches and stomachaches (19 ).

There is little information about the prevalence of GAD in children and adolescents because the diagnosis of OAD was used
previously. Prevalence estimates of OAD tended to be quite variable, 2% to 19% (19 ), and often very high, partially because
functional impairment was not necessary for the diagnosis (20 ). Recent estimates of the prevalence of GAD are in the range of 2.1%
to 10.8% (21 ). It appears to be more prevalent in older children and in girls (19 ). Onset typically falls between 10.8 and 13.4 years
of age (4 ).

Information about course is not yet available for the GAD diagnosis, but some extrapolation from OAD is possible. Cohen et al. (22 )
looked at three different statistical measures of persistence. They found that OAD was very stable in a subset of children and
younger adolescents (ages 11 to 16), but that there was a substantial amount of new onset among older adolescents (ages 17 to 19).
Their conclusion was that the disorder may be trait-like for those who exhibit symptoms early and that the development of the
disorder in others may be triggered in adolescence. Cantwell and Baker (23 ) also found considerable stability; 25% of children who
had been diagnosed with OAD approximately 4 years earlier had recovered (although the group size, eight, limits the usefulness of
this estimate). In contrast, Last et al. (16 ) reported that 80% of their OAD sample had recovered 3 to 4 years later; however, 25%
had developed another anxiety disorder and 25% had developed a depressive disorder.

GAD/OAD is a frequently co-occurring disorder. Of those with a primary diagnosis of OAD, there is often an additional diagnosis of
separation anxiety disorder (37% to 44%), social phobia (4% to 57%), simple phobia (9% to 43%) or a depressive disorder (1% to 69%)
(24 ). There are a number of potential reasons for the high rates of comorbidity, including true covariation of distinct disorders, the
presence of a single underlying construct, overlap of diagnostic categories, and measurement error (12 ,25 ).

Obsessive-Compulsive Disorder (OCD)

OCD is recognizable by the presence of obsessions and compulsions, which are distressing or cause marked interference in one's life
(18 ). Among young children, the disorder often presents with excessive and rigid ritualized behavior. For children in general,
compulsions alone are more common than obsessions alone (26 ). The most common compulsions include washing/cleaning,
repeating/redoing, and checking, and the most common obsessions include germs/contaminants and fear of harm to the self or to
another (26 ). OCD symptoms change over time in 90% of children (4 ).

OCD is relatively uncommon, with estimated prevalences of approximately 0.3% for children and 0.35% to 1.9% for adolescents
(27 ,28 ). Mean age of onset is approximately 10 years. Information about the course of OCD is variable and may be best described as
chronic but fluctuating (4 ). Leonard et al. (6 ) followed children and adolescents who had been part of National Institute of Mental
Health (NIMH) treatment studies. During the 2- to 7-year follow-up period, the patients on average received two different modalities
of treatment (medication, behavioral therapy, other individual therapy, and family therapy), with 96% having had additional
psychopharmacologic treatment and 46% some form of behavioral therapy. In spite of ongoing treatment, at follow-up 43% met the
criteria for OCD, 18% had subclinical OCD, and 28% had OC features. Of the 11% who were symptom-free, only three (of 54) patients
had no symptoms and were not on current medications. However, treatment was associated with decreased functional impairment.
Last et al. (16 ), on the other hand, found
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a 75% recovery rate in their sample. Other estimates of continued OCD at follow-up (1.5 to 7 years) range from 31% to 68% (16 ).

Comorbidity is a frequent problem with OCD. The most common co-occurring conditions include other anxiety disorders (38%), tic
disorders (24% to 30%), mood disorders (26% to 29%), and specific developmental disabilities (24%) (26 ). Leonard et al. (6 ) found
that both a lifetime history of tic disorder and current affective disorder at baseline were associated with poorer outcome.

Panic Disorder (with and without Agoraphobia) (PD)

PD involves recurrent and unexpected panic attacks, which cause significant distress for the affected individual. This frequently
leads to avoidance of various situations for fear of developing symptoms and being unable to escape (18 ). Young children tend to
articulate their panic-related fears in a different way than do adolescents or adults by virtue of their developmental level; they are
more likely to express concerns about sudden somatic symptoms and less likely to describe fears of dying, losing control, or going
crazy (4 ).

PD is uncommonly reported in children, to the point that there has been some debate as to whether it exists before puberty.
Evidence of the existence of PD in children comes from both retrospective reports of adults with PD and from case reports (29 ).
There is no epidemiologic study to date, and it is likely that many cases go undetected because of the predominance of somatic
symptoms in presentation. Prevalence of PD in adolescents is low, 0.7% girls and 0.4% boys (0.6 overall) according to one large high
school sample (28 ). The rate of panic attacks is expectedly higher, 11.6% for at least one full attack and an additional 3.2% for at
least one limited symptom attack (30 ). PD appears to be two to three times more common in females (29 ). Hayward et al. (31 )
examined the relationship between the occurrence of panic attacks and sexual development in girls and found a positive relationship.
There were no reported panic attacks among the least sexually mature girls, and a rate of 8% among those who were most
developed. The authors proposed a number of theories for this association, including hormonal changes, psychosocial factors, and
emergence of the ability to think abstractly, but further work is necessary to draw any definitive conclusions.

In the one study of the course of early PD, 30% continued to have PD and 30% had another psychiatric disorder 3 to 4 years later, but
the generalizablity of this result is questionable because of the small size of the study population (ten) (16 ). Retrospective reports
suggest that earlier onset is associated with poorer outcomes, including greater functional impairment and increased incidence of
alcohol abuse and suicidality (32 ). Available information suggests that there is a high rate of comorbidity in adolescents with PD,
particularly with affective disorders; again, this should be interpreted with caution because it is based on a single, small (N = 28)
study of adolescents (33 ).

Posttraumatic Stress Disorder (PTSD)

To meet the criteria for a diagnosis of PTSD, a person must have been exposed to a traumatic event and as a result is exhibiting
symptoms of reexperiencing, numbing/avoidance, and arousal (18 ). A recent confirmatory factor analytic study supported the
presence of these three basic clusters of symptoms in children and adolescents, although it found that arousal is often manifested as
general somatic complaints (34 ). PTSD presentations that are specific to children include reenactment of the trauma in play,
physical attempts (e.g., covering eyes or ears) to avoid memories of the trauma, reduced interest in activities, behavioral regression
(e.g., thumb-sucking, enuresis), and sleep disturbance (35 ).

Diagnosis of PTSD depends on exposure to a traumatic stressor. Each year, 6% to 7% of Americans are exposed to traumatic events
(36 ), but the incidence is much greater in certain subpopulations. For example, studies of urban youth report exposure rates of up
to 75% (37 ). Not everyone who is exposed to trauma goes on to develop PTSD. Estimates vary tremendously depending on the type
of trauma and the elapsed time between the event and assessment. In Saigh et al.'s (37 ) review of the literature, they found PTSD
prevalence among exposed youth to be 0% to 70.8% for crime-related events, 8.3% to 75% for war, and 0% to 95% for disasters.
Overall, it appears that exposed children may be somewhat more likely to develop PTSD than are exposed adults (36 ). PTSD is more
common in those who have been exposed to more severe trauma (34 ).

The course of PTSD in children over the short- or long-term has not been well studied, but it appears that prognostically important
factors are whether the trauma involves a single occurrence or is repeated, and whether it involves abuse. Although the evidence is
not entirely consistent, it appears that a single exposure is less likely to lead to long-term symptomatology (36 ).

Comorbidity is generally high, particularly with other anxiety disorders (7.7% to 41.6%) and affective disorders (16.7% to 85%), but
there is also substantial co-occurrence of attention-deficit/hyperactivity disorder (5.8% to 34.6%), conduct disorder (0% to 26.9%),
and oppositional defiant disorder (25%) (37 ). Additional disorders may be integrally related to the trauma, such as fears about
safety of the self or loved ones or grief about loss (35 ). Other psychopathology may also be a function of other factors, such as a
disrupted or disorganized childhood or engagement in risky behaviors, which increase the risk for both psychopathology and
traumatic exposure (38 ).

Separation Anxiety Disorder (SAD)

SAD is the only current anxiety disorder that is uniquely diagnosed in children and adolescents. The hallmark feature
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of this disorder is excessive concern about separation from attachment figures. This is frequently manifested as distress at
separation and excessive worry that harm will befall the attachment figure or that some negative event will lead to separation (18 ).
These children frequently avoid going to school, fear being left alone or sleeping alone, and exhibit a panic-like physiologic response
to separation (32 ).

Prevalence estimates for SAD are 2.0% to 5.4% for children and 1.3% to 4.6% for adolescents, with some evidence of higher rates in
girls, those of lower SES, and those with less educated parents (21 ,32 ). The onset of SAD is usually early and associated with a
major stressor (4 ). Of nine children with SAD followed by Cantwell and Baker (23 ), only one was still diagnosable 4 to 5 years later;
this was the highest rate of recovery of any of the disorders that they followed. Similarly, Last et al. (16 ) found an approximately
96% recovery rate among their 24 children and adolescents with SAD, although 25% had developed another disorder, most commonly
depressive, 3 to 4 years later. SAD frequently co-occurs with other disorders, most often other anxiety disorders (OAD, 23% to 33%;
specific phobias, 12.5% to 27%; social phobia, 8%) (24 ) or a depressive disorder (approximately one-third) (32 ).

SAD has been suggested to be a childhood manifestation of PD. Evidence that has been cited in support of this idea includes the
symptomatic similarity between a panic attack and the response to separation in a child with SAD; the frequency of a history of SAD
in panic patients; the clustering of SAD, PD, and depressive disorders in families; and the similarities in effective pharmacologic
treatments for the two conditions (39 ,40 and 41 ). Documented cases of panic episodes unrelated to separation, however, argue
against this hypothesis (29 ). Nonetheless, SAD appears to be a risk factor for the later development of PD, at least among females
(4 ).

Specific Phobia
A specific phobia is diagnosed if a child consistently displays significant and excessive fear in response to a specific object or
situation (18 ). The most common fears among children are heights, small animals, doctors/dentists, dark, loud noises, and
thunder/lightening (19 ). The prevalence of this disorder is in the range of 0.3% to 9.1%, with somewhat higher rates in girls and
younger children (19 ,21 ,42 ).

Unlike the other anxiety disorders reviewed here, children with specific phobias remain a fairly distinct group. Last et al. (16 ) found
that those with specific phobias were least likely to recover within 3 to 4 years (69.2%) but also were least likely to show onset of a
different disorder within the follow-up period. Similarly, Pine et al. (17 ) found that simple phobias in adolescence were related only
to adult simple phobias.

Social Phobia
Social phobia involves “marked and persistent fear of one or more performance or social situations in which the person is exposed to
unfamiliar people or to possible scrutiny by others” (18 ). The anxious response in such situations is associated with cognitions
involving concerns about being humiliated or embarrassed. Childhood social phobia is associated with significant impairment and
distress, and frequently leads to extensive phobic avoidance and deficient social skill development (43 ).

Although there are few good epidemiologic studies of social phobia in childhood, data from community studies in adolescents
suggest that it is quite common (1% to 2%), with a noticeable jump in prevalence rates sometime between ages 12 to 13 and ages 14
to 17 (44 ). One longitudinal study suggested that many cases of social phobia in childhood remit within 3 to 4 years (86.4%) (16 ).
However, when social phobia is present in adolescence, it is a strong predictor of social phobia in adulthood (17 ). These data, taken
together, suggest that social phobia in childhood may be a more transitory phenomenon than social phobia in adolescence. If these
findings are confirmed in future studies, they may suggest critical developmental time frames during which preventative efforts may
be applied.

ANXIETY AND STRESS DISORDERS IN YOUNG TO MIDDLE-AGE ADULTS

Part of "60 - Anxiety and Stress Disorders: Course Over the Lifetime "

Epidemiology
Several epidemiologic studies have documented the high rate of anxiety disorders among adults in the general population. In reports
from the Epidemiologic Catchment Area (ECA) Study, anxiety disorders were found to occur as a lifetime diagnosis in 14.6% of the
adult U.S. population aged 18 years or older (45 ). More recently, the National Comorbidity Survey (NCS) found that 24.9% of adults
in the age group 15 to 54 years had a lifetime anxiety disorder diagnosis (46 ). The two studies used somewhat different sampling
methods, and different diagnostic interviews, probably therein explaining at least some of the variance in rates between studies
(47 ).

Comorbidity
Comorbidity among the anxiety, mood, and substance use disorders is extensive (47 ). For example, two-thirds of persons in the
community with generalized anxiety disorder in a 12-month period also had major depressive disorder during that time frame (48 ).
In a clinical sample of 85 patients with major depression, 29% met criteria for a current anxiety disorder and 34% had at least one
anxiety disorder during their lives (49 ).

What is particularly noteworthy about this relationship

 P.863

is the temporal sequencing of disorders. Certain anxiety disorders, social phobia in particular (which has a median onset of between
13 and 15 years of age), almost inevitably begin prior to the onset of the mood or substance use disorder (50 ,51 ). In one study of
depressed patients, social phobia was the most common lifetime anxiety disorder (occurring in 15% of cases) followed closely by
panic disorder with agoraphobia (in 12%) (49 ). Social phobia occurred on average 2 years prior to the onset of major depressive
disorder in these patients. Similar findings have emerged from community studies (52 ), suggesting that particular anxiety disorders
such as social phobia may be considered risk factors for the subsequent development of major depression. It remains to be
established what the mechanisms might be for this observed relationship. Does being socially anxious lead to increased isolation or
decreased self-worth, thereby leading to an increase in subsequent major depression? Is social phobia merely the earliest
manifestation of an anxiety-mood disorder diathesis? These questions will only be answered with future research that focuses
broadly on psychosocial and biological vulnerabilities for anxiety and mood disorders.

Another interesting aspect of the anxiety-depression link lies in the relationship between major depressive disorder (MDD) and PTSD.
Extensive comorbidity between PTSD and MDD is the norm in studies of various traumatized groups, including persons exposed to
combat (53 ,54 ), disasters (55 ), and intimate partner violence (56 ). Community studies also demonstrate strong ties between
these two disorders, with approximately 35% to 50% of cases of PTSD in the general population being comorbid with MDD (57 ).

Studies that have examined the temporal association between major depression and PTSD have posited several causal pathways. It
has been observed that preexisting major depressive disorder increases an individual's risk for PTSD following exposure to traumatic
events (58 ,59 ). The converse has also been observed, namely that preexisting PTSD is a risk factor for the later development of
MDD (58 ,60 ). The mechanisms for this apparent reciprocal risk have yet to be explained, but might involve a general vulnerability
to stress that can result in major depression (61 ) or PTSD in susceptible individuals.

Functioning, Quality of Life, and Cost to Society

Data have been collected in the past several years that highlight the disability and reduced quality of life associated with anxiety
disorders in young and middle-aged adults. Studies in clinical samples of patients with PD, PTSD, OCD, and social phobia all depict
these as serious conditions that rob individuals of enjoyment and pleasure and impair functioning in multiple domains (62 ).
Epidemiologic studies, where the range of severity is expected to be wider and where many milder cases are expected to be seen,
also provide persuasive evidence of the seriousness of anxiety disorders (52 ,63 ,64 ). The annual cost of anxiety disorders in the
United States was estimated at $42.3 billion in 1990, or $1,542 per sufferer (65 ). Other than simple phobia, all anxiety disorders
analyzed were associated with impairment in workplace performance. These observations, gleaned from a variety of clinical and
nonclinical perspectives, portray anxiety disorders in adults as serious mental disorders worthy (and in need) of greater societal
willingness to develop and apply better interventions to prevent or mitigate their impact on the lives of individuals.

ANXIETY AND STRESS DISORDERS IN OLDER ADULTS

Part of "60 - Anxiety and Stress Disorders: Course Over the Lifetime "

Although anxiety is among the most prevalent of psychiatric disorders in the elderly, research in this area has lagged far behind that
of depression and dementia (66 ). But in the past few years, several important studies have been conducted that provide novel
information about the prevalence, features, and course of anxiety disorders in older adults.

Epidemiology
Whereas it had previously been believed that anxiety disorders decline in prevalence with age, several possible explanations for this
finding have been put forward. It has been suggested that this might be an artifact of measurement error, owing to differences in
the way older individuals report anxiety (67 ). Previous epidemiologic studies may also have underestimated the prevalence of
anxiety disorders in the elderly by limiting participation to community-dwelling older adults, who may have lower rates of anxiety
disorder than those living in institutions (68 ).

Fortunately, data have recently become available from a new community survey that provides a more accurate and detailed
perspective on anxiety disorders in older adults (69 ). The Longitudinal Aging Study Amsterdam (LASA) is based on a random sample
of 3,107 older adults (ages 55 to 85), stratified for age and sex. The overall prevalence of anxiety disorders in the community was
estimated at 10.2%. GAD was most common in a 6-month time period (7.3%), followed by social phobia (3.1%), PD (1.0%), and OCD
(0.6%). For comparison purposes, it is noteworthy that the 6-month prevalence of major depression in the same study was 2.0%.
Thus, anxiety disorders were far more common than depressive disorders in the elderly, underscoring the point made earlier that it
is surprising that the elderly have received so little attention in the clinical and research literature to date.

This study also examined vulnerability factors for anxiety disorders in older adults (69 ). Many of the vulnerability factors for anxiety
disorders in younger adults are common to older adults (e.g., female sex, lower levels of education),
 P.864

but several unique risk factors were also encountered (e.g., having suffered extreme experiences during World War II). These
investigators were also able to show that current stresses commonly experienced by older people (e.g., recent losses in the family
and chronic physical illness) also played a part in the onset or exacerbation of anxiety disorders. Current life stresses, then, should
be evaluated as possible contributors not only to depression, but also to anxiety in the elderly.

Comorbidity
Comorbidity patterns of older adults with anxiety disorders are remarkably similar to those of younger adults. In the LASA, 48% of
those with MDD also met criteria for anxiety disorders, whereas 26% of those with anxiety disorders also met criteria for MDD (70 ).

The entity known as “anxious depression” warrants special mention in this context. Although definitions vary, anxious depression
usually refers to MDD with accompanying anxiety. Anxious depression is a particularly common presentation in the elderly (66 ).
Although anxious depression is frequently severe and impairing, its outcome is no worse than nonanxious depression when treated
appropriately (71 ).

Special Features of Anxiety Disorders in the Elderly

The fact that medical illness becomes more common with increasing age can put a special twist on the presentation and origins of
certain anxiety disorders in the elderly. First and foremost, it must be recognized that many medical illnesses (e.g., thyroid disease,
chronic obstructive pulmonary disease, and stroke, to name just a few) may be associated with de novo anxiety symptoms or with
the exacerbation of a preexisting anxiety disorder (66 ). Most “new” anxiety disorders in older life are either GAD or agoraphobia,
whereas most other anxiety syndromes seen in the elderly (e.g., PD and OCD) reflect recurrence or worsening of an anxiety disorder
that had its onset earlier in life (72 ).

Agoraphobia in older adults is usually a different phenomenon, with different etiology, from agoraphobia in younger adults. In
younger adults, agoraphobia is almost always a complication of PD (73 )—the individual comes to avoid situations that are associated
with the possible occurrence of panic or difficulty escaping should a panic attack occur. In the elderly, the new onset of agoraphobia
is rarely associated with spontaneous panic attacks, but instead is a maladaptive reaction to some form of medical illness
experience that renders the individual fearful of being unable to function safely away from home (66 ). An example is an elderly
woman who breaks her hip, and even after it has satisfactorily healed, is afraid to maneuver without help and therefore avoids
leaving the house alone.

SUMMARY
Part of "60 - Anxiety and Stress Disorders: Course Over the Lifetime "

Anxiety disorders span the full range of human existence from childhood to old age, though symptoms may vary considerably owing
to developmental differences and related factors. Anxiety disorders in children are often transitory phenomena, with the majority
showing remission by adolescence or early adulthood. Yet, in a minority, extremely shy and fearful temperament in childhood can
merge almost imperceptibly into social phobic and panic disorders in adolescence. Anxiety disorders in youth appear to be a risk
factor for the subsequent development of major depression (and, although less certain, possibly also substance use disorders) in late
adolescence and young adulthood. By adulthood, comorbidity is the rule, with most individuals experiencing multiple anxiety
disorders, or concurrent mood and anxiety disorders. For the most part, anxiety disorders are chronic, and these persist from young
adulthood into old age. But even in later life, new onset of anxiety disorders can occur, often in the context of medical illness or
other sources of life stress.

ACKNOWLEDGMENTS
Part of "60 - Anxiety and Stress Disorders: Course Over the Lifetime "

Dr. Stein has received research support from the following companies: Bristol-Myers Squibb; Eli Lilly and Company; Forrest
Laboratories; Hoffman-LaRoche Pharmaceuticals; Novartis; Parke-Davis; Pfizer; SmithKline Beecham; and Solvay Pharmaceuticals.
He is currently or has been in the past a consultant for Forrest Laboratories; Hoffmann-La Roche Pharmaceuticals; Janssen Research
Foundation; SmithKline Beecham and Solvay Pharmaceuticals. Finally, he receives or has received speaking honoraria from Bristol-
Myers Squibb; Eli Lilly and Company; Hoffmann-La Roche Pharmaceuticals; Pfizer; Pharmacia & Upjohn; SmithKline Beecham and
Solvay Pharmaceuticals.

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61
Genetic and other Vulnerability Factors for Anxiety and Stress
Disorders

Kathleen R. Merikangas

Daniel Pine

Kathleen R. Merikangas and Daniel Pine: National Institute of Mental Health, Bethesda, Md.

Despite dramatic advances in our understanding of genetics and neurobiology, the etiology of the anxiety disorders is still relatively
unknown. To date, there remain no pathognomonic markers with which a presumptive diagnosis of an anxiety disorder may be made.
This highlights the importance of the empirical epidemiologic approach to investigating the definitions and risk factors for the
expression of anxiety across the life course. Anxiety disorders are developmental conditions that often emerge during childhood and
follow varied developmental trajectories (1 ,2 ). Research on early-life vulnerability factors that predict the trajectory of anxiety
symptoms across development holds promise for elucidating mechanistic pathways in anxiety.

In evaluating the risk factors for the development of anxiety disorders, there are several issues requiring consideration. First, there
is substantial overlap between the anxiety disorders and other psychiatric disorders both concomitantly and longitudinally. Second,
manifestations of anxiety change substantially across the life course, particularly during childhood and adolescence. Therefore, a
developmental perspective is essential in evaluating links between risk factors and anxiety disorders. Third, the assessment of
anxiety requires evaluation of the context in which the individual experiences anxiety as well as the subjective response to anxiety-
inducing situations. As such, anxiety becomes a disorder when there is a mismatch between inherent threat posed by a particular
stimulus or situation and the cognitive or somatic response.

Research on vulnerability factors has undergone a relatively marked transformation in recent years, due to conceptual changes in
causal theories of mental disorders. Such conceptual changes are reflected in three major themes that organize current research on
vulnerability factors in anxiety. First, although studies through the early 1990s often emphasized the role of one or another
particular risk factor, more recent studies emphasize the manner in which multiple risk factors might interact to cause mental
syndromes, including anxiety, as part of a mechanistic pathway. For example, although dysregulation in fear conditioning has been
linked to anxiety for more than two decades (3 ), such dysregulation is now viewed as part of a larger chain of intrinsic and extrinsic
events that may ultimately culminate in an anxiety disorder (4 ). Second, as a corollary to this view, vulnerability markers are now
conceptualized as tied to families of anxiety disorders, as opposed to specific conditions. This change in perspective follows the
observation that validators of individual mental syndromes related to differential course, familial aggregation, or psychophysiology
relate more closely to families of disorders than to particular disorders. Third, marked advances over the past 20 years in
neuroscience have stimulated a closer integration of basic and clinical work on vulnerability markers in anxiety disorders. Progress in
elucidating neural circuits related to anxiety has facilitated research on vulnerability markers for anxiety disorders that integrates
data from basic and clinical science.

This chapter examines the major risk factors for the development of anxiety disorders across the life span. Particular attention is
paid to the specificity of vulnerability factors and to developmental differences in expression of the disorders themselves.

 MAGNITUDE AND DEMOGRAPHIC RISK FACTORS

 FAMILIAL AND GENETIC FACTORS
 VULNERABILITY MARKERS
 ENVIRONMENTAL EXPOSURES
 SUMMARY AND FUTURE DIRECTIONS FOR RESEARCH ON ANXIETY VULNERABILITY
 ACKNOWLEDGMENTS

MAGNITUDE AND DEMOGRAPHIC RISK FACTORS

Part of "61 - Genetic and other Vulnerability Factors for Anxiety and Stress Disorders "

Magnitude of Anxiety in General Population

The anxiety disorders are the most common psychiatric disorders both in the United States and elsewhere (2 ,5 ,6 ). The
 P.868

results of the two large-scale community-based surveys of psychiatric disorders of adults in the United States, the Epidemiological
Catchment Area study (ECA) (2 ) and the National Comorbidity Study (NCS) (5 ), reveal that the total prevalence rates of anxiety
disorders are greater than those of the affective disorders, behavior disorders, and substance use disorders. Phobias tend to be the
most common anxiety disorder, whereas panic disorder is fairly rare in the general population. There is substantial overlap both
cross-sectionally and longitudinally between the anxiety disorders and other disorders, as well as between the subtypes of anxiety
disorders themselves (6 ). On average, there is a threefold increased risk of having a second disorder compared to that of
manifesting an anxiety disorder alone across the lifetime.

Comorbidity between anxiety disorders and other psychiatric disorders has been demonstrated in both clinical and community
samples. Anxiety disorders are most strongly associated with affective disorders and with substance use disorders (6 ), though they
are generally associated with all other major classes of disorders including depression, disruptive behaviors, eating disorders, and
substance use. Comorbidity between anxiety disorders and other disorders in the Diagnostic and Statistical Manual of Mental
Disorders, third edition revised or fourth edition (DSM-IIIR or -IV) may be even more common in adolescents than in adults (7 ). A
review of comorbidity of anxiety and depression by Brady and Kendall (8 ) suggests that anxiety and depression may be part of a
developmental sequence in which anxiety is expressed earlier in life than depression. Although the association between anxiety and
depression is quite consistent, the evidence of links between anxiety disorders and behavior problems is inconclusive.

Sex Differences in Anxiety Disorders

Similar to the affective disorders, females tend to exhibit greater rates of anxiety disorders, though there is some variability
according to specific subtypes. Table 61.1 presents the sex-specific lifetime rates of the major subtypes of anxiety disorders
assessed in the ECA and NCS (5 ,6 ). Although the magnitude of the rates of anxiety disorders varies substantially between the two
studies, the sex ratio is strikingly similar: women have an approximately twofold elevation in lifetime rates of panic, generalized
anxiety disorder, agoraphobia, and simple phobia than men in both studies. In contrast, there is a nearly equal sex ratio for the
lifetime prevalence of social phobia.

TABLE 61.1. SEX-SPECIFIC LIFETIME PREVALENCE RATES OF ANXIETY DISORDERS

IN COMMUNITY SURVEYS IN THE UNITED STATES
There is increasing evidence from community-based studies that anxiety symptoms and disorders are also the most common
problems in childhood and adolescence as well. The rates of anxiety disorders in community or school-based surveys of children and
adolescents as defined by contemporary diagnostic criteria range from 0.1% to 13.3% in males and 0.4% to 28.6% in females (9 ).
Similar to the sex ratio for adults, girls tend to have more of all subtypes of anxiety disorders, irrespective of the age composition of
the sample. For example, in a recent epidemiologic study, females compared to males had greater rates of current anxiety disorders
(i.e., 12.2% vs. 8.5%), past anxiety disorders (5.2% vs. 2.7%), as well as anxiety symptom scores on a dimensional rating (mean = 1.9
vs. 0.9) (10 ). Nevertheless, Lewinsohn et al. (10 ) reported that despite the greater rates of anxiety in girls across all ages, there
was no difference between boys and girls in the average age at onset of anxiety (mean for girls = 8.0 ± 3.9; mean for boys = 8.5 ±
3.8).

Age-Specific Patterns of Expression of Anxiety Disorders

Retrospective reports of adults with anxiety disorders suggest that the onset of anxiety disorders generally occurs in childhood or
adolescence. Although there is substantial
 P.869

variation across studies, the results of prospective community-based research reveal differential peak periods of onset of specific
subtypes of anxiety: separation anxiety and specific phobias in middle childhood (i.e., ages 7 to 9); overanxious disorder in late
childhood (i.e., 10 to 13); social phobia in middle adolescence (i.e., 15 to 16); and panic attacks, sometimes progressing to panic
disorder, in late adolescence (i.e., 17 to 18) (1 ,11 ,12 ,13 and 14 ). Anxiety disorders, particularly the phobias, tend to persist
across the life course. However, there are major differences among the anxiety subtypes in terms of specificity and chronicity.
Whereas the phobic states tend to be fairly stable and nonprogressive, generalized anxiety and panic tend to be less specific and
less stable over time (1 ,15 ,16 ).

Several follow-up studies of children and adolescents have shown that anxiety symptoms and disorders in general tend to exhibit
some stability, but with substantial switching across categories of anxiety disorders over time (17 ,18 ). A recent 8-year follow-up
study of a community sample of youth ages 9 to 18 at study entry provides compelling evidence of the stability of the subtypes of
anxiety disorders (17 ). The stability of both social phobia and simple phobia was highly specific over time, whereas overanxious
disorder was associated with major depression, social phobia, and generalized anxiety in early adulthood.

Epidemiologic surveys of adults reveal that the female preponderance of anxiety disorders is present across all stages of life but is
most pronounced throughout early and mid-adulthood. The rates of anxiety disorders in males are also rather constant throughout
adult life, whereas the rates in females peak in the fourth and fifth decades of life and decrease thereafter. The increased rates in
females are present across all ages and do not diminish as the rates of anxiety decrease in late life. The importance of pure anxiety
disorders in late life was described by Beekman et al. (19 ), who found different risk factors for anxiety disorders than for either
depression or comorbid anxiety and depression in a community sample of adults over age 55.

Social Class and Ethnicity

Rates of anxiety disorders in general are greater among those at lower levels of socioeconomic status (20 ). Several community
studies have yielded greater rates of anxiety disorders, particularly phobic disorders, among African-Americans (5 ). With respect to
children, Compton et al. (14 ) found that Caucasian children were more likely to report symptoms of social phobia, whereas African-
American children had more separation anxiety symptoms. Pine et al. (1 ) reported that phobias were greater among those at lower
levels of social class. The reasons for ethnic and social class differences have not yet been evaluated systematically; however, both
methodologic factors as well as differences in exposure to stressors have been advanced as possible explanations.

FAMILIAL AND GENETIC FACTORS

Part of "61 - Genetic and other Vulnerability Factors for Anxiety and Stress Disorders "

The familial aggregation of all of the major subtypes of anxiety disorders has been well established (21 ). As reviewed below, the
results of more than a dozen controlled family studies of probands with specific subtypes of anxiety disorders converge in
demonstrating a 3- to 5-fold increased risk of anxiety disorders among first-degree relatives of affected probands compared to
controls. The importance of the role of genetic factors in the familial clustering of anxiety has been demonstrated by numerous twin
studies of anxiety symptoms and disorders (22 ,23 ). However, the relatively moderate magnitude of heritability also strongly
implicates environmental etiologic factors. Table 61.2 summarizes the results of family and twin studies of anxiety disorders.

TABLE 61.2. SUMMARY OF FAMILY AND TWIN STUDIES OF ANXIETY DISORDERS

Review of Family and Twin Studies of Anxiety Disorders in Adults

Panic Disorder
Of the subtypes of anxiety, panic disorder is the anxiety syndrome that has been shown to have the strongest degree of familial
aggregation. A recent review of family studies of
 P.870

panic disorder by Gorwood et al. (24 ) cited 13 studies that included 3,700 relatives of 780 probands with panic disorder compared
to 3,400 relatives of 720 controls. The lifetime prevalence of panic was 10.7% among relatives of panic probands compared to 1.4%
among relatives of controls, yielding a relative risk of 6.8. In addition, early-onset panic, panic associated with childhood separation
anxiety, or panic associated with respiratory symptoms has each been shown to have a higher familial loading than other varieties of
panic disorder (25 ).

Although there has been some inconsistency reported by twin studies of panic disorder (26 ), two studies applying modern diagnostic
criteria demonstrated considerably higher rates for monozygotic compared to dizygotic twins (27 ,28 ). Furthermore, current
estimates derived from the Virginia Twin Registry show panic disorder to have the highest heritability of all anxiety disorders at 44%
(29 ).

Phobic Disorders
Though there are far fewer controlled family and twin studies of the other anxiety subtypes, all of the phobic states (i.e., specific
phobia, agoraphobia) have also been shown to be familial (30 ,31 ,32 and 33 ; see refs 34 ,35 and 36 for reviews). The average
relative risk of phobic disorders in the relatives of phobics is 3.1. Stein et al. (33 ) found that the familial aggregation of social
phobia could be attributed to the generalized subtype of social phobia. Data from the Virginia Twin Study report the estimated total
heritability for phobias to be 35% (29 ,37 ).

Generalized Anxiety Disorder

There is also evidence of both the familial aggregation and heritability of generalized anxiety disorder in a limited number of studies.
The average familial odds ratio is approximately 5 (32 ,38 ), and the heritability was 0.32 among female twin pairs (37 ).

Obsessive-Compulsive Disorder
Likewise, there are also very few controlled family studies of obsessive-compulsive disorder. Two of the three studies (39 ,40 )
reported familial relative risks of 3 to 4, whereas Black et al. (41 ) found no evidence for familial aggregation. Nestadt et al. (40 )
found that both the age of onset and obsessions were associated with greater familiality. Twin studies have yielded weak evidence
for heritability of obsessive compulsive disorder (42 ,43 and 44 ).

Linkage and Association Studies

Based on indirect evidence implicating the adrenergic system in panic disorder (45 ), several linkage studies have investigated the
role of mutations in adrenergic receptor loci on chromosomes 4, 5, or 10 (46 ), but without success. Other work has similarly
excluded linkage with γ-aminobutyric acid receptor A (GABAA) genes (47 ). Reports from a genomic survey of panic disorder using 600
markers have not yielded evidence of linkage (48 ). Similarly, linkage studies have excluded the possibility that panic disorder was
due to mutations in adrenergic receptor loci on chromosomes 4, 5, or 10 (46 ), and other work has similarly excluded linkage with
GABAA receptor genes (47 ). Recent reports from a genomic survey of panic disorder using 600 markers have not yielded evidence of
linkage (48 ).

Family Studies and Phenotypic Definitions

The lack of success in identifying specific genes for anxiety disorders is not surprising given their complexity. Similar to several other
psychiatric disorders, the anxiety disorders are complicated by etiologic and phenotypic heterogeneity, a lack of valid diagnostic
thresholds, unclear boundaries between discrete anxiety subtypes, and comorbidity with other forms of psychopathology.
Impediments to estimating genetic influences in youth are demonstrated by dramatic differences in heritability according to the
informant regarding child psychopathology. For example, Eaves et al. (49 ) found that the heritability of both separation anxiety and
overanxious disorder was far greater for parent-reported rather than child-reported disorder.

The family study approach, particularly when employed with systematic community-based samples, is one of the most powerful
strategies to minimize heterogeneity because etiologic factors for the development of a particular disorder can be assumed to be
relatively homotypic within families. There is a dearth of studies that have employed within-family designs to examine either
phenotypic expression or some of the putative biological factors underlying the major anxiety disorders. For example, both Perna et
al. (50 ,51 ) and Coryell (52 ) have shown that healthy relatives of probands with panic disorder have increased sensitivity to CO2
challenge, suggesting that CO2 sensitivity may be a promising trait marker for the development of panic, as described below. Smoller
and Tsuang (36 ) discuss the value of family and twin studies in identifying phenotypes for genetic studies.

Both family and twin studies have been used to examine sources of overlap within the anxiety disorders, and between the anxiety
disorders and other syndromes including depression, eating disorders, and substance abuse. Fyer et al. (31 ,53 ) have demonstrated
the independence of familial aggregation of panic and phobias. With respect to comorbidity, whereas panic disorder, generalized
anxiety, and depression have been shown to share common familial and genetic liability (23 ,54 ,55 ), there is substantial evidence
for the independent etiology of anxiety disorders and substance use disorders (36 ,55 ,56 ). Similar results have emerged from
 P.871

studies of symptoms of anxiety and depression in youth in which both anxiety and depression was found to result from common
genetic diathesis (57 ,58 ).

In a comprehensive consideration of what may be inherited, Marks (59 ) reviews the components of anxiety that have been
investigated in both human and animal studies. Evidence from twin studies has indicated that somatic manifestations of anxiety may
lie under some degree of genetic control. These studies demonstrate that physiologic responses, such as pulse, respiration rate, and
galvanic skin response, are more alike in monozygotic than in dizygotic twin pairs. Furthermore, twin studies of personality factors
have shown high heritability of anxiety reaction. Finally, the results of animal studies have suggested that anxiety or emotionality is
under genetic control. Selective breeding experiments with mammals have demonstrated that emotional activity analogous to
anxiety is controlled by multiple genes (59 ). These findings suggest that anxiety and fear states are highly heterogeneous and that
future studies need to investigate the extent to which the components of anxiety result from common versus unique genetic factors
and the role of environmental factors, either biologic or social, in either potentiating or suppressing their expression.

High-Risk Studies of Anxiety Disorders

Given the early age of onset for anxiety disorders, studies of children of parents with anxiety have become an increasingly important
source of information on the premorbid risk factors and early forms of expression of anxiety. Increased rates of anxiety symptoms
and disorders among offspring of parents with anxiety disorders have been demonstrated by Turner et al. (60 ), Biederman et al.
(61 ), Sylvester et al. (62 ), Last et al. (63 ), Warner et al. (64 ), Beidel and Turner (65 ), Beidel (66 ), Capps et al. (67 ), Merikangas
et al. (68 ), Unnewehr et al. (69 ), and Warner et al. (70 ). Table 61.3 presents the risk of anxiety disorders among offspring of
parents with anxiety disorders compared to controls averages 3.5 (range 1.3 to 13.3), suggesting specificity of parent-child
concordance within broad subtypes of anxiety disorders.

TABLE 61.3. CONTROLLED HIGH-RISK STUDIES OF ANXIETY

However, similar to studies of adults that show common familial and genetic risk factors for anxiety and depression (27 ,71 ,72 ),
studies in children have also revealed a lack of specificity with respect to depression (60 ,64 ,65 ,73 ). Studies that employed a
comparison group of parent probands with depressive disorders have shown that rates of anxiety disorders are also increased among
the offspring of these parents (60 ,62 ,65 ,70 ); conversely, offspring of parents with anxiety disorders and depression have elevated
rates of depression when compared to those of controls (62 ) or to offspring of anxiety-disordered parents without depression (61 ).
Similar findings emerged from the family study by Last et al. (63 ), who found an increase in rates of major depression among the
adult relatives of children with anxiety. Weissman et al. (74 ) have even suggested that childhood anxiety represents one of the
earliest manifestations of familial risk for depression. These findings are usually interpreted as providing evidence for age-specific
expression of common risk factors for anxiety in childhood and depression with or without comorbid anxiety in adulthood.

The high rates of anxiety disorders among offspring of parents with anxiety suggest that there may be underlying psychological or
biological vulnerability factors for anxiety disorders in general, which may already manifest in children
 P.872

prior to puberty. Previous research has shown that children at risk for anxiety disorders throughout life are characterized by
behavioral inhibition (75 ), autonomic reactivity (66 ,76 ), somatic symptoms (60 ,77 ), social fears (60 ,62 ), enhanced startle reflex
(76 ), and respiratory sensitivity (160 ). Empirical research on each of these domains of risk is reviewed in the next section.

VULNERABILITY MARKERS
Part of "61 - Genetic and other Vulnerability Factors for Anxiety and Stress Disorders "

The current section reviews recent studies on vulnerability markers in anxiety disorders. This includes data on temperamental
factors and biological profiles. The first section reviews evidence regarding individual-level vulnerability factors, whereas the
subsequent section examines data linking exogenous or environmental factors with risk for anxiety. As noted above, both sets of
vulnerability markers operate within complex causal chains involving multiple interacting risk factors. Moreover, in such complex
chains, the boundary between intrinsic and exogenous risk factors can become blurred. For example, the effects of exogenous
factors, including life events; social rearing experiences, such as trauma or parental nurturance; and factors such as the use of illicit
substances may operate through effects on intrinsic factors, such as the regulation of neural systems that monitor danger.

Intrinsic, individual-oriented vulnerability markers for anxiety disorders can be conceived across a range of perspectives, focusing on
increasingly more specified biological systems. At the most complex or global level, specific temperamental or personality
characteristics, such as neuroticism, harm avoidance, and behavioral inhibition have been linked to risk for anxiety. At a more
specified level, vulnerability can be modeled through the assessment of cognitive function, in the form of attention and memory, or
peripheral physiologic function, as reflected in autonomic reactivity profiles, changes in the startle reflex, or changes in ventilatory
control. These cognitive and physiologic functions, in turn, reflect functional aspects of neurochemical or neuroanatomic systems
that are presumably homologous with systems linked to fear and anxiety across a range of mammalian species. Data from humans at
each of these levels is reviewed within the context of research on fear and anxiety in other species.

Temperament/Personality

Behavioral Inhibition
One of the earliest indicators of vulnerability to the development of anxiety is behavioral inhibition, characterized by increased
physiologic reactivity or behavioral withdrawal in the face of novel stimuli or challenging situations (79 ). Behavioral inhibition may
be a manifestation of a biological predisposition characterized by both overt behavioral (e.g., cessation of play, latency to interact
in the presence of unfamiliar objects and people) and physiologic indicators (e.g., low heart rate variability, accelerated heart rate,
increased salivary cortisol level, pupillary dilation, increased cortisol level). There is an increased frequency of behavioral inhibition
among children of parents with anxiety disorders compared to those of normal controls (61 ,62 ,65 ,75 ,80 ,81 and 82 ).

Few studies have evaluated the differences in manifest inhibition and approach/avoidance in both clinical and nonclinical samples,
leaving gaps in the conceptualization of the construct of inhibition. Some studies have shown that there is more stability of
behavioral inhibition across early childhood among girls than among boys (83 ). The expression of behavioral inhibition studied
prospectively may reveal patterns of anxiety symptomatology similar to those endorsed in adult populations. In a prospective study
of a large community cohort of subjects from age 3 months to 13 years, Prior et al. (84 ) found that maternal ratings of persistent
shyness and shyness in late childhood were associated with the development of anxiety disorders in adolescence.

Anxiety Sensitivity
Anxiety sensitivity is another potential sensitive and specific trait marker for the development of anxiety disorders (85 ). Anxiety
sensitivity is characterized by beliefs that anxiety sensations are indicative of harmful physiologic, psychological, or social
consequences (e.g., fainting or an impending heart attack). The misinterpretation of bodily cues that characterizes anxiety
sensitivity may lead to a self-perpetuating “fear of fear” cycle. Thus, the fear of benign arousal sensations produces anxiety, which
in turn increases the frequency and intensity of physiologic sensations, and subsequently fuels apprehension regarding the
significance of these sensations. This process may ultimately result in a full-blown panic attack.

Anxiety sensitivity is thought to represent a stable trait-like factor that is qualitatively different from general fear and anxiety (86 ).
It has been proposed that anxiety sensitivity may interact with environmental experiences (e.g., hearing misinformation about the
negative outcome of certain bodily sensations) to shape beliefs about the dangers of anxiety sensations. Thus, anxiety sensitivity
may be involved in the development of certain anxiety disorders, particularly panic disorder (87 ,88 ). Of particular interest is the
finding of the specificity of anxiety sensitivity with respect to development of anxiety disorders but not depression in a nonclinical
sample (88 ). Likewise, Pollock et al. (89 ) reported that anxiety sensitivity appears to be specific to anxiety, as it did not contribute
unique variance above self-rated anxiety symptoms in the prediction of depressive symptoms.

Anxiety sensitivity has been shown to be under genetic (90 ) and familial influence; anxiety sensitivity was found to
 P.873

constitute a potential premorbid marker for the development of anxiety disorders in high-risk but not low-risk youth (89 ).
Prospective studies of youth have also demonstrated the prognostic significance of anxiety sensitivity in predicting the development
of anxiety disorders. Based on the results of a 5-year prospective study of adolescents, Hayward et al. (91 ) concluded that anxiety
sensitivity appeared to be a specific risk factor for the development of panic attacks in adolescents. These findings from prospective
research, particularly the specificity with respect to anxiety, together with the importance of genetic and familial liability suggest
that anxiety sensitivity is an important vulnerability factor that should be examined in future studies.

Comorbid Disorders

Psychiatric
The magnitude of comorbidity in adults and adolescents with anxiety suggests that investigation of the role of other disorders in
enhancing the risk of the initial development and persistence of anxiety disorders over time may be fruitful. The difficulty in dating
onset of specific disorders, particularly from retrospective data, diminishes our ability to determine the temporal relations between
disorders. Nevertheless, some prospective studies have examined the links between anxiety disorders and earlier expression of other
forms of psychopathology. For example, whereas some studies suggest that childhood depression may presage the onset of panic
attacks, the results of a fairly large prospective study suggest a bilateral temporal association between panic attacks and depression
(91 ).

Other disorders that may enhance the risk of development of anxiety disorders include eating disorders (92 ), depression, and
substance use and abuse. With respect to substance use disorders, Rao et al. (93 ) found that anxiety disorders may comprise a
mediator of the link between depression and the subsequent development of substance use disorders in a clinical sample. The
potential mechanisms through which anxiety may be associated with smoking in adolescents were examined by Patton et al. (94 ),
who found that both anxiety and depression were associated with smoking initiation through increased susceptibility to peer
influences. Conversely, some research suggests that substance use may trigger anxiety disorders in susceptible youth. For example,
a prospective study of a community sample revealed that posttraumatic stress disorder (PTSD) may be triggered by substance abuse
in about 50% of the cases (95 ). Similarly, Johnson et al. (96 ) found that adolescent smoking predicted adult onset of panic attacks,
panic disorder, and agoraphobia (96 ). Thus, although comorbidity between anxiety and both depression and substance problems is
quite common in children and adolescents, further research on the mechanisms for links between specific disorders both across and
within genders is necessary.

Medical Symptoms/Disorders
Several studies have also suggested that there is an association between childhood medical conditions and the subsequent
development of anxiety. Kagan et al. (97 ) reported an association between allergic symptoms, particularly hay fever, and inhibited
temperament in young children. In a retrospective review of pre- and perinatal and early childhood risk factors for different forms
of psychiatric disorders in adolescence and early adulthood, Allen et al. (98 ) found that anxiety disorders in adolescents were
associated specifically with illness during the first year of life, particularly high fever. Likewise, Allen and Matthews (102 ) reported
that adolescents and young adults with anxiety disorders were more likely to have suffered from infections during early childhood
than others. The prevalence of high fevers in childhood along with other diseases associated with immune system were also elevated
among offspring of parents with anxiety disorders in the Yale High Risk Study (76 ). Kagan (101 ) proposed that the high levels of
cortisol associated with anxiety may lead to immunologic sensitivity to environmental stimuli. Taylor et al. (99 ) reported that
immunologic diseases and infections were specifically associated with emotional disorders because children with developmental or
behavioral disorders had no elevation in infections or allergic diseases. On the other hand, Cohen et al. (100 ) suggest that such
medical problems show stronger associations with depressive as opposed to anxiety disorders during adolescence. These findings
suggest that it may be fruitful to examine links between immunologic function and the development of anxiety disorders.

Prospective studies have revealed that the anxiety disorders may comprise risk factors for the development of some cardiovascular
and neurologic diseases. Haines et al. (103 ) reported that phobic anxiety was associated with ischemic heart disease, particularly
fatal ischemic events. Bovasso and Eaton (104 ) employed cardiac and respiratory symptoms and illness to subtype panic attacks and
their association with depression in a large community-based sample. They found that “respiratory panic attacks were associated
with the subsequent risk of myocardial infarction.” Likewise, phobic disorder is strongly associated with migraine, with the onset of
phobias predating that of migraine (105 ,106 ). The results of both family studies and prospective cohort studies suggest that there
may be a subtype of migraine with shared liability for anxiety and depression (105 ).

Autonomic Reactivity
Reactions to threatening stimuli among various organisms, including primates and lower mammals, involve changes in the autonomic
nervous system. These changes can be detected through an analysis of time series for heart rate, heart period variability, blood
pressure, and catecholamine levels.
 P.874

There is a long history of research in this area, and much of the initial work concerned the assessment of physiologic changes
associated with acute anxiety states. Hence, acute episodes of anxiety, both in the laboratory and in natural settings, are typically
characterized by acute changes in heart rate, blood pressure, and heart period variability (107 ). These changes result from
coordinated changes in the parasympathetic and sympathetic innervation of the cardiovascular system.

More recent work on physiologic changes during acute anxiety states has attempted to identify specific physiologic patterns
associated with one or another emotion. The identification of such emotion-specific patterns may provide insights on emotion-
specific patterns of brain activity. For example, some forms of anxiety, such as acute panic, may be characterized by marked
parasympathetic withdrawal in the face of sympathetic enhancement. Other emotions, such as anger, may be characterized by a
distinct physiologic “finger print,” reflecting the involvement of distinct brain systems across emotions (108 ,109 and 110 ). In
general, consistent associations are found across development between acute anxiety states and changes in peripheral autonomic
indices, including heart rate, blood pressure, or heart period variability. As a result, some suggest that perturbations in autonomic
regulation may index an underlying vulnerability to develop anxiety disorders. This underlying vulnerability is thought to relate to
the functioning of particular neural circuits within the brain that exert effects on both subjective internal states and physiologic
activity. Potentially relevant neural circuits have been identified through basic science studies on the neural basis of fear and
anxiety.

Despite consistent evidence of an association between acute anxiety states and changes in autonomic physiology, the degree to
which such changes index vulnerability for anxiety, as opposed to the acute state of anxiety, remains unclear. If such changes in
autonomic physiology primarily reflect downstream manifestations of relatively high degrees of acute fear, they would provide
limited advantages as vulnerability markers. On the other hand, at least some of the underlying autonomic abnormalities in panic
disorder persist after remission and may be independent of the current state. This suggests that changes in autonomic physiology
may mirror subtle person-specific differences in brain processes related to the processing of risk or to the experience of fear. As
such, autonomic indices might index vulnerability in a fashion that is more sensitive than indices derived through self-report
measures. A series of recent studies provide preliminary evidence consistent with this possibility.

Autonomic physiologic profiles have been studied among individuals who face high risk for anxiety disorders. Physiologic profiles
have been tied to at least three indicators of risk: temperamental factors, family history, and traumatic events. In terms of
temperamental factors, Kagan (111 ) noted the relationship between behavioral inhibition, which predicts later anxiety, and a
distinct autonomic physiology profile. Children with behavioral inhibition exhibit an autonomic physiology characteristic of the
profile found during acute anxiety. Specifically, behaviorally inhibited children exhibit under conditions of novelty a shift from
parasympathetic to sympathetic control of the cardiovascular system, manifest as an increase in heart rate and a reduction in high-
frequency components of the heart period variability power spectrum. Such abnormalities in autonomic physiology are viewed as
downstream reflections of perturbations based within the limbic system. In terms of family history, Bellodi et al. (112 ) found similar
temperamental and physiologic abnormalities among children of parents with panic disorder. Such data are consistent with other
studies finding high rates of behavioral inhibition among offspring of patients with anxiety disorders. Finally, in terms of traumatic
events, physiologic reactions to an acute stress may index underlying vulnerability to develop anxiety states. Consistent with this
possibility, Shalev et al. (113 ) found that enhanced cardiovascular activity in the emergency room immediately following a motor
vehicle accident predicted the development of PTSD.

Taken together, available data clearly delineate associations between acute anxiety and autonomic physiology profiles, but the
implications of this work for the study of risk remain unclear. Moreover, the underlying assumption in this work posits an effect of
perturbations in brain systems on both autonomic physiology and anxiety symptoms. As such, more work is also needed relating brain
function to autonomic physiology.

Psychophysiologic Function
Research on fear conditioning has facilitated an integration of basic and clinical work on vulnerability for anxiety. Fear conditioning
develops following the pairing of a neutral “conditioned” stimulus (CS+), such as a tone or a light, and an aversive “unconditioned”
stimulus (UCS), such as a shock, a loud noise, or an air puff. Across a range of mammalian species, including humans, fear
conditioning results from changes in a relatively simple neural circuit that involves distinct amygdala nuclei, including the
basolateral and central nucleus. Basic science research on the role of this circuit in learned fears has also called attention to the
role played by related but relatively distinct neural circuits in the responses to other forms of danger. For example, reactions to
intrinsically dangerous contexts, such as a brightly lit room for a rodent, involve a relatively extended period of vigilance. These
reactions may more intimately involve the basolateral nucleus and the bed nucleus of the stria terminalis than the central nucleus of
the amygdala. Such reactions in animals may model worry in humans as characteristically found in many anxiety disorders (114 ).
 P.875

Similarly, acute reactions to intrinsically dangerous stimuli often involve rapid changes in behavior designed to facilitate escape or
defense. Such reactions in animals may involve the hypothalamus and lower brainstem structures; such reactions in animals may be
model acute panic in humans.

Work in neuroscience delineating circuits involved in mammal's response to danger has stimulated a series of studies on risk for
anxiety in humans. Much of this work quantifies physiologic reactions to innate and learned fears with the goal of comparing
physiology across high- and low-anxiety groups. Based on skin conductance data, Eysenck and Eysenck (115 ) suggested that
abnormal habituation of conditioned fear responses confers risk for anxiety. Similarly, Raine et al. (116 ) suggest that deficiencies in
learned fear, as modeled by skin conductance, relates to low anxiety and high risk for chronic behavior problems. However, due to
methodologic advantages, more recent studies rely on startle as a physiologic index of activity in brain circuits tied to fear. Most
importantly, it has been possible to map circuits that regulate startle in more precise detail, relative to circuits that regulate skin
conductance or other indicators of autonomic response, such as heart rate. Cross-species parallels in startle regulation facilitate
integration of basic and clinical work (76 ,114 ,117 ,118 ,119 ,120 ,121 ,122 ,123 and 124 ). For example, molecular genetic studies
on fear conditioning in mice generate specific hypotheses on the genetics of risk human disorders (125 ,126 ,127 ,128 ,129 ,130 and
131 ). Fear-relevant stimuli in animals may potentiate startle through effects on genes in limbic structures, such as the amygdala,
that are involved in fear conditioning. In adult humans, distinct stimuli effect startle across emotional disorders, but abnormal
startle in some form is seen in many disorders, including phobias (132 ), PTSD (122 ,123 and 124 ), depression (133 ,134 ), and panic
disorder (135 ). Moreover, there is some evidence that startle specifically indexes risk for anxiety. Three studies found startle
abnormalities in children born to adults with an array of anxiety disorders (76 ,119 ), and a fourth study found startle abnormalities
in inhibited children, who face high risk for anxiety disorders (111 ).

In a high-risk study of offspring of parents with anxiety disorders compared to psychiatric and normal controls, the startle reflex and
its potentiation by aversive states was used as a possible vulnerability marker to anxiety disorders in adolescent offspring of parents
with anxiety disorders (122 ). Startle was found to discriminate between children at high- and low-risk for anxiety disorders, as well
as to discriminate between children at risk for anxiety compared to those at risk for alcoholism. However, different abnormalities in
startle amplitude for high-risk males and females were observed. Startle levels were elevated among high-risk females, whereas
high-risk males exhibited greater magnitude of startle potentiation during aversive anticipation. Two possible explanations for the
gender differences in the high-risk groups were suggested by the authors: (a) differential sensitivity among males and females to
explicit threat versus the broader contextual stimuli that are mediated by different neurobiologic pathways, and (b) different
developmental levels in males and females in which the vulnerability to anxiety may be physiologically expressed earlier in females.

Nevertheless, more work in this area is needed, given inconsistencies across genders and across conditions under which startle is
most discriminatory (76 ). These data are also consistent with the findings of Watson et al. (136 ). Overall, the data suggest that
startle indices may provide an important window for assessing dysfunction in limbic circuits broadly related to mood and anxiety
regulation.

Ventilatory Function
As in the area of autonomic physiology, a wealth of research delineates associations between respiratory perturbation and acute
anxiety. This association has been most convincingly demonstrated in panic disorder, where various forms of respiratory stimulation,
including lactate infusion (137 ) and CO2 inhalation, consistently produce high degrees of anxiety and more pronounced
perturbations in respiratory physiologic parameters. Of note, these associations extend beyond the specific diagnosis of panic
disorder, because enhanced sensitivity to respiratory perturbation is also found in conditions that exhibit strong familial or
phenomenologic associations with panic disorder, including limited symptom panic attacks; certain forms of situational phobias;
childhood anxiety disorders, particularly separation anxiety disorder; and high ratings on anxiety sensitivity scales.

Compared to the work on autonomic physiology, a larger body of research implicates abnormalities in respiration in risk or
vulnerability for anxiety. At least four sets of findings suggest that respiratory indices index risk for anxiety, independent of any
association between current state and respiratory function. First, asymptomatic adult relatives of patients with panic disorder
consistently exhibit enhanced subjective sensitivity to respiratory stimulation, in the form of exogenously inhaled CO2 (51 ,138 ,139 ).
Second, among patients with panic disorder, stronger family loading is found in panic patients with evidence of respiratory
dysregulation, as opposed to those with no sign of respiratory dysregulation (51 ,140 ). Third, respiratory indices linked to panic
disorder are strongly heritable, raising questions on the potential shared genetic vulnerability for panic attacks and respiratory
dysregulation. Fourth, Pine et al. (78 ) reported increased carbon dioxide sensitivity in children with anxiety disorders. Such data
are also consistent with work on respiratory disease (141 ) and smoking (96 ,142 ), which suggest that abnormalities in respiration
predispose to later anxiety. Based on this work, abnormalities in respiration appear to provide some information on the vulnerability
for anxiety states that are related to acute panic.

Despite the consistency of findings in this area, a number of questions remain. The most consistent data emerge for
 P.876

subjective indices of respiratory sensitivity, manifest as a tendency to report dyspnea during stress or during respiratory stimulation.
The mechanisms that contribute to such enhanced sensitivity remain poorly specified. At a cognitive level, such hypersensitivity may
result from an overall sensitivity to somatic sensations, consistent with data linking high degrees of anxiety sensitivity to future
panic attacks (143 ). On the other hand, enhanced sensitivity to respiratory sensations appears more closely tied to panic attacks
than sensitivity to other somatic factors; the tie between anxiety sensitivity and respiratory sensitivity also appears relatively weak
in some studies. At the physiologic level, such hypersensitivity may result from perturbations in brain systems involved in respiratory
regulation or primary as opposed to learned fear states. Unfortunately, the precise role of fear systems in both respiratory
regulation and human anxiety states also remains poorly specified.

Neurochemical and Neurohormonal Factors

As reviewed in other sections of this book, extensive data document associations between alterations in various neurochemical
factors and ongoing anxiety disorders. This includes data on the serotoninergic, noradrenergic, and GABAergic systems. Moreover,
there is some evidence to implicate neurochemical alterations in the causal chain contributing to anxiety disorders. In animal
models, genetic manipulations of serotoninergic receptors, the serotonin reputake transporter gene, and components of the GABA
complex each produce behavioral and physiologic effects reminiscent of clinical anxiety states. Similarly, clinical studies find that
acute pharmacologic manipulations in these neurochemical systems produce concomitant change in acute anxiety. For example, the
inverse GABA agonist flumazenil precipitates anxiety in patients with panic disorder, whereas GABA agonists are potent treatments
for various forms of anxiety. These findings are consistent with evidence of a deficiency in GABAergic modulation among adults with
anxiety disorders. Similarly, manipulations of the serotoninergic system, either through tryptophan depletion or treatment with
medications, also produce both acute and more chronic changes in anxiety. Finally, manipulations of the noradrenergic system
produce similar changes in both children and adults. Sallee et al. (144 ) found that the α2-agonist yohimbine elevated selectively
anxiety symptoms and was associated with blunting of growth hormone in children with anxiety disorders. Interestingly, as with the
response of children to CO2 inhalation (160 ), the response to yohimbine appeared particularly abnormal in children with separation
anxiety disorder. However, evidence of perturbed noradrenergic function in children with depression or facing high familial risk for
depression (145 ) suggest that these findings may not be specific to anxiety but rather may relate to broad risk for mood and anxiety
disorders.

Despite the consistency of these findings relating neurochemical factors to anxiety, relatively few studies have examined the
manner in which individual differences in neurochemical function predict vulnerability to anxiety. There is evidence from studies in
adult patients that some of these neurochemical abnormalities persist after remission. For example, much like symptomatic patients,
remitted patients with panic disorder exhibit abnormal secretory profiles in terms of the growth hormone and the hypothalamic-
pituitary-adrenal (HPA) axis. These neurohormonal abnormalities are thought to reflect trait-related abnormalities in neurochemical
systems involved in neurohormonal regulation. Finally, there have been numerous studies of patients and at-risk relatives using
lactate challenge to induce anxiety (146 ,147 and 148 ). The limited information provided on neural pathways by this provocation
test limits its value in informing the pathophysiology of anxiety disorders.

Although these studies raise the possibility that risk for anxiety may result at least partially from underlying neurochemical
abnormalities, other studies are needed to confirm this possibility. For example, there are almost no studies of neurochemical
function in high-risk youth, a key source of information regarding the underlying role of biological parameters in the development of
anxiety disorders. One exception is the study of Reichler et al. (77 ), who assessed several biological factors in their high-risk study
of panic disorder including lactate metabolism, mitral valve prolapse, urinary catecholamines, and monoamine oxidase. Although
none of these parameters discriminated high-risk from low-risk youth, the lack of differences may have been attributable in part to
low statistical power.

Likewise, very few studies have compared neurochemical function in asymptomatic relatives of patients with and without anxiety
disorders. Similarly, no studies have examined family loading for anxiety disorders in patients stratified in terms of their
neurochemical functioning.

Beyond this work examining monamine systems' influence on neurohormonal regulation and vulnerability for anxiety, a relatively
extensive body of work examines the precise relationship between anxiety and HPA axis regulation. Corticotropin-releasing factor
(CRF) represents a key neuropeptide in the regulation of this system. CRF infusions in animals produce behavioral and physiologic
effects in animals that bear similarities to human anxiety states. Similarly, genetic manipulations that alter CRF produce similar
effects. As such, this work suggests that an underlying dysregulation in the HPA axis, possibly centrally involving CRF, may
contribute to vulnerability for anxiety. Consistent with basic science studies, clinical research notes a relationship between acute
anxiety states and alterations in HPA axis function. For example, a variety of acute stressors induce consistent elevations of cortisol;
patients with PTSD exhibit multiple signs of HPA axis dysregulation; multiple
 P.877

other anxiety disorders exhibit other signs of HPA axis dysregulation.

Vigilance/Attention
Studies of the association between attention regulation and anxiety have revealed that adults with anxiety disorders exhibit
enhanced vigilance for threat cues, as indexed by effects of fear-related words or pictures on reaction times. These effects have
been attributed to amygdala influences on attention allocation (149 ,150 ,151 ,152 and 153 ). Enhanced attentional bias in acute
anxiety represents a particularly robust finding, noted in more than 20 studies using various paradigms across virtually all anxiety
disorders. These effects appear particularly robust in two paradigms, the emotional Stroop and the dot-probe tests. From a
theoretical perspective, this enhanced bias is considered a vulnerability marker that antedates the developmental of anxiety
disorders among adults. Consistent with this possibility, an enhanced bias for threat cues is found early in the course of anxiety
disorders, particularly among children with anxiety disorders. On the other hand, this enhanced bias is generally not found in
remitted patients (153 ), and studies have yet to document enhanced bias for threat cues in at-risk but asymptomatic individuals.

ENVIRONMENTAL EXPOSURES
Part of "61 - Genetic and other Vulnerability Factors for Anxiety and Stress Disorders "

Perinatal Exposures
There is virtually no evidence that either prenatal factors or delivery complications comprise risk factors for the development of
anxiety disorders. The results of three studies that retrospectively assessed perinatal events converged in linking such exposures to
behavioral outcomes, but not to subsequent anxiety. For example, Allen et al. (98 ) found that children who suffered from a variety
of exposures ranging from prenatal substance use to postnatal injuries were more likely to develop behavior disorders, particularly
attention deficit disorder and conduct problems, but not anxiety disorders. Likewise, the results of the Yale High-Risk Study yielded
no association between pre- and perinatal risk factors and the subsequent development of anxiety disorders (76 ).

Life Events/Stressors
The role of life experiences in the etiology of anxiety states, particularly phobias and panic disorder, has been widely studied
(154 ,155 ,156 and 157 ). Life events have often been designated a causal role in the onset of phobias, which are linked inherently
to particular events or objects. More broadly, life experiences that to some extent threaten one's notion of safety and security in
the world are often at least retrospectively perceived to trigger or precipitate the onset of anxiety disorders. In evaluating the
evidence on the causal role of life experiences, it is critical to consider separately the subtypes of anxiety disorders. Although it is
likely that life stress may exacerbate phobic and generalized anxiety states, Marks (59 ) concludes that phobic states resulting from
exposure are far more rare than those that emerge with no apparent exposure. In contrast, posttraumatic stress disorder (PTSD) is
defined as a sequela of a catastrophic life event.

The major impediment to evaluation of the causal role of life events in anxiety (or depression) is the retrospective nature of most
research addressing this issue. For example, Lteif and Mavissakalian (158 ) found that patients with panic or agoraphobia exhibited
an increased tendency to report life events in general; this suggests that studies that limit assessment of life events to those
preceding onset of a disorder may be misleading because they fail to provide comparison for the time period of onset. Moreover,
stressful life events may interact with other risk factors such as family history of depression in precipitating episodes of panic (159 ).
In one of the few prospective studies, Pine et al. (160 ) did demonstrate a predictive relationship between life events during
adolescence and both depressive as well as generalized anxiety disorder symptoms. Interestingly, the association with anxiety was
limited to females, consistent with differential vulnerability to stress across genders.

In terms of specific environmental risk factors, there has been abundant literature on the role of parenting in enhancing
vulnerability to anxiety disorders. Based on Bowlby's (161 ) theory that anxiety is a response to disruption in the mother-child
relationship, it has been postulated that maternal overprotection is related to anxiety, particularly separation anxiety. Using the
Parental Bonding Instrument of Parker et al. (162 ), several studies of clinical samples have found that adult patients with anxiety
disorders recall their parents as less caring and more overprotective than did controls (163 ). These findings have been supported in
nonclinical samples as well (164 ,165 ). However, all of these studies caution that a causal link cannot be established because of the
lack of independent assessment of parent behaviors and offspring anxiety.

Another parental behavior that may enhance risk of anxiety in offspring is parental sensitization of anxiety through enhancing
cognitive awareness of the child to specific events and situations such as bodily functions, social disapproval, the importance of
routines, and necessity for personal safety (164 ). Bennet and Stirling (164 ) found that subjects with anxiety disorders and those
with high trait anxiety reported greater maternal and paternal overprotection and increased maternal sensitization to anxiety
stimuli than controls.

Another feature of the parental relationship that has received widespread attention in recent research has been exposure to severe
childhood trauma through either separation or abuse (161 ,166 ). There is increasing animal research on the impact of early adverse
experiences on brain systems and subsequent development (167 ,168 ). Pynoos et al. (169 )
 P.878

present a comprehensive developmental life-trajectory model for evaluating the effects of childhood traumatic stress and anxiety
disorders. They propose different avenues by which dangerous circumstances, childhood traumatic experiences, and PTSD can
intersect with other anxiety disorders across the life span. The developmental perspective is critical in light of different levels of
neural response to experience at different stages of development (170 ).

SUMMARY AND FUTURE DIRECTIONS FOR RESEARCH ON ANXIETY

VULNERABILITY
Part of "61 - Genetic and other Vulnerability Factors for Anxiety and Stress Disorders "
Despite the rich array of constructs associated with anxiety (Table 61.4 ), our ability to predict those who will suffer from anxiety
disorders in adulthood is severely limited. The principles of multifinality (i.e., many outcomes of the same risk factor) and
equifinality (i.e., diverse risk profiles leading to the same endpoint) apply to many of the risk pathways investigated herein (171 ).
Although we distinguish between intrinsic and extrinsic risk factors for the development of anxiety disorders, there is increasing
evidence that there is a bidirectional association between the factors subsumed under these two domains. Only a small proportion
of those with known vulnerability factors truly develop anxiety disorders in adulthood, despite the vast majority of those with
adulthood anxiety reporting onset in childhood and early adolescence.

TABLE 61.4. VULNERABILITY FACTORS FOR ANXIETY DISORDERS

The major impediments to identifying specific risk factors for anxiety are exclusive reliance on retrospective data, blurred
boundaries between normal and pathologic anxiety, difficulty distinguishing between risk factors and early manifestations of anxiety,
limited interdisciplinary conceptualization of models of risk and pathogenesis, lack of evidence of the specificity of risk factors with
respect to anxiety disorders or subtypes thereof, and limited tools for direct measurement of brain function. Moreover, many of the
risk factors have been shown to operate differently according to gender and age, as well as the specific subtype of anxiety.
Elucidation of the different risk profiles will provide valuable information on classification, etiology, treatment, and prevention.
Future research should do the following:

 Establish more accurate and developmentally sensitive methods of assessment of anxiety, with a focus on developing
objective measures of the components of anxiety.
 Apply within-family design to minimize etiologic heterogeneity and to refine diagnostic boundaries and thresholds.
 Investigate specificity of putative markers with respect to other psychiatric disorders and the longitudinal stability of
specific subtypes of anxiety disorders.
 Develop research on hormonally mediated neurobiological function in order to understand gender differences predisposing
women to experience decreased resiliency to fear-provoking stimuli.
 Examine mechanisms for associations between panic attacks with extrinsic exposures (i.e., substance use), developmental
periods (i.e. pubertal development), and cessation in later life.

ACKNOWLEDGMENTS
Part of "61 - Genetic and other Vulnerability Factors for Anxiety and Stress Disorders "
This work was supported primarily by grant DA05348 and in part by grants AA07080, AA09978, DA09055, MH36197, Research Scientist
Development Awards K02 DA00293 (to Dr. Merikangas), from Alcohol, Drug Abuse, and the Mental Health Administration of the
United States Public Health Service.

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62
Animal Models and Endophenotypes of Anxiety and Stress Disorders

Vaishali P. Bakshi

Ned H. Kalin

Vaishali P. Bakshi and Ned H. Kalin: Department of Psychiatry, University of Wisconsin at Madison, Wisconsin Psychiatric Institute
and Clinics, Madison, Wisconsin.

 ANIMAL MODELS OF PSYCHIATRIC ILLNESS

 PUTATIVE ANIMAL ENDOPHENOTYPES OF STRESS AND ANXIETY: STUDIES OF FEARFUL TEMPERAMENT
 INDIVIDUAL DIFFERENCES IN DEFENSIVE BEHAVIORS: NATURALLY OCCURRING FEARFUL ENDOPHENOTYPES
 MATERNAL DEPRIVATION: AN ENVIRONMENTAL MANIPULATION THAT CAN LEAD TO FEARFUL ENDOPHENOTYPES IN PRIMATES
AND RODENTS
 TARGETED MUTATIONS LEADING TO ANXIETY-LIKE ENDOPHENOTYPES: STUDIES OF TRANSGENIC MICE
 FUTURE DIRECTIONS

ANIMAL MODELS OF PSYCHIATRIC ILLNESS

Part of "62 - Animal Models and Endophenotypes of Anxiety and Stress Disorders "

Animal Models: Types Of Validity

An important criterion for developing animal models to study psychopathology involves establishing the validity of the model as a
true representation of the process being studied. Generally, three types of validity are applied to animal models: face validity,
construct validity, and predictive validity (1 ,2 and 3 ). Face validity refers to the outward similarity in appearance between the
model and the illness. Construct validity, on the other hand, does not exclusively involve outward tangible signs of the modeled
illness. Rather, it refers to the internal mechanism or state that underlies the illness. Finally, predictive validity refers to the ability
of the animal model to identify therapeutic treatments for the illness. It should be noted that the different types of validity can be
independent of each other; an animal model can possess predictive and construct validity without possessing face validity. Ideally,
an animal model should possess both construct and predictive validity so that it may be used to understand the mechanisms and
etiology of the disorder and also to identify promising treatments for the disorder.

Endophenotype Approach
Species differences in the manifestation of a particular internal state can cloud the usefulness of face validity in animal models. In
addition, when considering a complex psychiatric illness, it is likely that several different symptom clusters contribute to the final
pathologic condition; these different sets of symptoms may have different underlying substrates and thus may be ameliorated by
different treatments. Therefore, it is difficult to come up with an animal model for an illness that meets the aforementioned
criteria and also models the pathologic syndrome in its entirety. An alternative approach that has been used involves the modeling
of discrete symptom clusters and physiologic alterations rather than the whole syndrome, with the assumption that what causes the
symptoms contributes mechanistically to the illness. This general approach has involved the use of endophenotypes that may be
related to a particular psychiatric disorder. The term endophenotype refers to a set of behavioral and/or physiologic characteristics
that accompany a basic process that is altered in relation to the illness that is being studied (4 ). It is important to note that this
more narrowly defined endophenotype approach does not necessarily have to capture specific symptoms that are a part of the
clinical diagnosis, but rather may focus on a core process or function that is abnormal in the clinical population under study and that
is thought to be related to the manifestation of the illness. For example, in the case of anxiety-related disorders, investigators have
focused on studying the genetic, physiologic, and neurochemical correlates of fearful or anxious endophenotypes because a core
aspect of anxiety-related disorders involves the aberrant expression of fearful responses to neutral or mildly stressful contexts (5 ).
Thus, by identifying animals that display fearful endophenotypes, it is possible to study the neural substrates that contribute to this
basic process that may underlie the development and expression of anxiety-related psychopathology.

Using endophenotypes that are based on core and basic processes rather than the entire illness offers certain advantages. Because
the whole illness is not being modeled, the endophenotype approach affords greater possibility for construct and predictive validity
in the model, and can incorporate species-specific manifestations of the core process being modeled. This approach may also make
screening for genetic abnormalities associated with the disorder more fruitful, because the genetic factors associated with a very
discrete
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process (which could be mediated by a small number of genes) rather than an entire syndrome (which is likely caused by a complex
set of interactions between multiple genes) is being studied (4 ,6 ). Moreover, heterogeneity within a diagnostic category could
potentially dilute the strength of a sample population (i.e., not all patients with anxiety disorders are identical in their clinical
presentation), and diminish the chances of identifying genes that contribute to the illness. Ideally, one might be able to generate
several different endophenotypes for a particular disorder, and then study the genetic underpinnings of each of these separate core
processes in order to identify a set of genes that might be implicated in that particular disorder. The definition and use of
endophenotypes in animal models of psychiatric illness is a developing area. This chapter presents some promising candidates of
animal models of fearful and anxious endophenotypes, and outlines some of the preliminary genetic factors that have been
identified to contribute to the manifestation of these endophenotypes.

PUTATIVE ANIMAL ENDOPHENOTYPES OF STRESS AND ANXIETY:

STUDIES OF FEARFUL TEMPERAMENT
Part of "62 - Animal Models and Endophenotypes of Anxiety and Stress Disorders "

Defensive Behaviors
In an attempt to understand the basic neural mechanisms underlying psychiatric conditions involving fear and anxiety, several
groups have focused on identifying the neural substrates of defensive behaviors in animals. Defensive behaviors are exhibited by a
wide array of species including rats, nonhuman primates, and humans in response to perceived threats from the environment, and
are essential components of an organism's behavioral repertoire that ensure its protection and survival. Because organisms display
defensive behaviors in reaction to threat, it is thought that the aberrant expression of defensive behaviors may represent a good
example of a fearful endophenotype that would have relevance to stress and anxiety-related disorders. Although the specific
behavioral responses that compose defensive behaviors are dependent on the environmental context and vary from species to
species, a common element that unites this cross-species phenomenon is that defensive behaviors represent an organism's
behavioral response to fear. Because defensive behaviors are expressed in response to an immediate threat, they characteristically
supersede and interrupt the expression of other normal homeostatic behaviors such as feeding and reproduction that may be ongoing
at the time of the perceived threat (7 ,8 ,9 ,10 and 11 ). One defensive response pattern expressed by many species is to inhibit all
body movements and assume an immobile or freezing posture. This phenomenon of behavioral inhibition is effective in preventing
detection and attack by predators (12 ,13 ), and may have special relevance for understanding psychopathology.

In nonhuman primates, defensive behaviors are composed of a constellation of responses that include vocalizations, freezing, fleeing,
or defensive hostility and aggression. The particular set of responses that is emitted depends on, among other variables, the nature
of the perceived threat (14 ,15 ). Studies of defensive behaviors in rhesus monkeys may provide valuable information that could aid
in the understanding of fear and anxiety-related psychopathology in humans, because extreme fearful or defensive responses occur
in dispositionally fearful humans who have an increased risk to develop psychopathology (16 ).

Psychiatric illnesses such as anxiety disorders and depression might involve the aberrant expression of defensive behaviors. In other
words, pathologic anxiety could be conceptualized as the inappropriate expression of defensive or fear-related behaviors, consisting
of either an exaggerated or overly fearful response to an appropriate context, or a fearful response to an inappropriate or neutral
context. Although appropriate levels of defensive behaviors in response to environmental threats are adaptive and ensure survival,
the overly intense or context-inappropriate display of fearrelated defensive behaviors may represent a liability that interferes with
normal behavior and would likely contribute to certain forms of fear-related psychopathology. Thus, inappropriate or exaggerated
expression of defensive behaviors may represent an important animal endophenotype of anxiety. An understanding of the specific
neural substrates underlying the expression and regulation of defensive behaviors may therefore ultimately shed insight into the
processes that become dysregulated in stress-related psychopathology. In defining animal endophenotypes relevant to anxiety,
specific symptoms of a particular type of anxiety disorder are not being modeled, but rather the general phenomenon of
hyperreactivity to mildly stressful stimuli is studied. The approach of modeling anxiety by studying defensive behaviors in animals
has been described previously for rodent models (17 ,18 ). In the following sections, both primate and rodent analogues of stress
hyperresponsiveness are described, with a particular emphasis on models of either the overly intense but context-appropriate
expression of defensive behaviors or the normal but context inappropriate expression of defensive behaviors. Initially, various
behavioral paradigms that have been used to measure an animal's level of defensive behavior are described, and subsequently,
specific examples of fearful endophenotypes that have been identified using these tests are discussed.

Measuring Defensive Behaviors in Nonhuman Primates: Human Intruder Paradigm

One laboratory paradigm that has been developed to identify animals with fearful dispositions characterizes monkeys' fearful
behavioral responses to a human intruder. In the human intruder paradigm (HIP), the monkey is placed by itself in a test cage where
it remains for 30 to 40 minutes
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while its behavior is recorded on videotape. A human intruder then enters the test area, representing a potential predatorial threat
to the animal (14 ,15 ,19 ). The test session consists of three consecutive brief conditions: alone (“A,” animal left alone in cage); no
eye contact (“NEC,” animal presented with the facial profile of a human standing 2.5 m away); stare (“ST,” animal presented with a
human who faces it and engages it in direct eye contact). Typically, animals respond to the A condition by increasing their levels of
locomotion and by emitting frequent coo vocalizations, which have been likened to the human cry and function to signal the infant's
location and facilitate maternal retrieval (20 ,21 ). The NEC condition causes a reduction in cooing and an increase in behavioral
inhibition, which functions to help the monkey remain inconspicuous in the face of a predator and is often manifested as hiding
behind the food bin and freezing. The ST condition elicits aggressive (open-mouth threats, lunges, cage shaking, barking
vocalizations) and submissive (lip smacking, fear-grimacing) behaviors that represent adaptive responses to the perceived threat of
the staring experimenter. The different test conditions (A, NEC, ST) reliably elicit responses in young or adult laboratory-reared
monkeys or in feral animals (14 ,19 ). Moreover, these context-specific defensive responses are not dependent on the gender of the
intruder, and can also be elicited by showing the animal a videotape of the intruder (Kalin et al., unpublished data).

Behavioral Tests Used to Measure Fearful Endophenotype in Rodents

To identify fearful endophenotypes in rodents, a variety of behavioral paradigms have been employed. All of the paradigms in some
manner provide an assessment of the rodent's level of defensive behavior, which is essentially thought to be an index of its level of
fearfulness or anxiousness. The behavioral tests measure one of four general categories of stress-related behavior: approach-
avoidance conflicts, conditioned fear, aggression, and punished responding conflicts. Detailed descriptions and protocols for these
tests can be found in a recent review by File and colleagues (22 ).

Approach-Avoidance Conflicts
Briefly, all of the paradigms presented in this section measure the animal's ratio of approach versus avoidance behaviors by
presenting a choice between an environment that is safe (usually a dark, enclosed, small space) and an environment that seems
novel but risky (usually bright, wide open, large spaces). The entries into and amount of time spent in the safe environment relative
to the risky environment are used as an index of the animal's stress level (an increase in exploratory behaviors toward and into the
risky environment indicate a relatively low level of stress). A number of paradigms including the elevated plus maze (composed of
safer closed, dark arms versus riskier open bright arms), the open field (consisting of a darker wall-bordered peripheral portion
versus a brighter open center section), a light-dark transition box (consisting of an exploratorium divided into two halves, one that
is dark and one that is bright), and a defensive withdrawal apparatus (composed of a small dark chamber that is inside of a brightly
lit open field) have been frequently used and validated as paradigms that are sensitive to detecting shifts in an animal's approach-
avoidance–based conflict (23 ).

Conditioned Fear
Behavioral tests that measure conditioned fear utilize basic principles of Skinnerian conditioning. Two frequently used paradigms to
assess fear conditioning are conditioned freezing and fear-potentiated startle. Conditioned freezing is evaluated using a two-step
procedure. First, during the training or conditioning phase, a stressful unconditioned stimulus (UCS, such as a foot shock) that elicits
freezing is paired with a neutral stimulus that subsequently becomes a conditioned stimulus (CS). On the test day, the amount of
freezing in response to the CS is assessed; animals that have not undergone the CS-UCS pairing do not normally freeze when the CS
is presented, but animals that have learned to associate the CS with a foot shock show marked levels of freezing simply in response
to this stimulus. The CS can either be a context (i.e., the environment in which the shock is delivered) or a discrete cue (e.g., a
tone or light). The level of conditioned freezing is thought to correspond to the level of fear or anxiety that the animal is
experiencing due to anticipation of a threat (24 ). In the case of fear-potentiated startle, the unconditioned startle response to a
sudden stimulus (e.g., a loud noise burst) is measured in the presence and in the absence of a CS that has been paired previously
with shock. The startle response is markedly increased when the startling stimulus occurs in the presence of the CS; this relative
increase in startle magnitude is quantified, and serves as an index of the level of fear (thought to be elicited by a discrete cue as
the CS) or anxiety (thought to be elicited by a contextual CS) that the animal may be experiencing (25 ).

Aggression and Social Behavior

Aggressive behaviors are emitted as part of the behavioral repertoire an animal displays when it encounters a threatening situation.
The study of defensive aggressive behaviors has been summarized and reviewed by a number of investigators (26 ,27 and 28 ).
Briefly, aggressive behaviors can be studied using a resident intruder paradigm, in which the offensive/agonistic responses (e.g.,
upright postures, attacks) of a male resident or the defensive responses (e.g., submissive posture, flight, freezing) of a male
intruder are measured. Other
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stress-related paradigms involve the study of affiliative behaviors and include the social interaction test in which approach toward
and contact between two rats is measured (e.g., sniffing or grooming each other).

Punished Responding Conflict

The basic principle of punished responding tests is to present the animal with a situation in which a particular behavioral response
results in both a rewarding outcome and an aversive outcome. The extent to which the animal exhibits the behavioral response
during the conflict schedule is used as an index of its level of stress. For example, in the classic Geller-Seifter conflict test (29 ),
rats are trained to press a lever for a food reward. Gradually, the bar press is also paired with a mild foot shock, and a stable rate of
responding is established under the conflict schedule. Drugs can then be administered and evaluated for their ability to increase
responding under the punished schedule. For example, benzodiazepines have been found to increase bar-pressing during the conflict
schedule, putatively by decreasing the stress or anxiety induced by the aversive stimulus. Similarly, in the Vogel punished drinking
paradigm (30 ), thirsty rats with access to a water bottle are periodically given mild electric shocks through the spout of the bottle;
the extent to which licking is decreased is used as an index of stress.

INDIVIDUAL DIFFERENCES IN DEFENSIVE BEHAVIORS: NATURALLY

OCCURRING FEARFUL ENDOPHENOTYPES
Part of "62 - Animal Models and Endophenotypes of Anxiety and Stress Disorders "

Primates
Several lines of evidence support the notion that an individual's level of defensive responding is a relatively stable trait
characteristic (which in part may be derived from the nature of early postnatal maternal interactions, see below). Extreme
individual differences detected early in life may be predictive of future psychopathology. For example, extremely inhibited children
are at greater risk to develop anxiety and depressive disorders and are more likely to have parents that suffer from anxiety disorders
(31 ,32 ,33 and 34 ). Moreover, behavioral inhibition in childhood (based on retrospective self-reports) is highly associated with
anxiety in adulthood (35 ). Some of the physiologic correlates that have been observed in extremely inhibited children are elevated
levels of the stress-related hormone cortisol (36 ) and greater sympathetic nervous system activity (37 ). In nonhuman primates,
individual differences in defensive behaviors have been studied in an attempt to elucidate the neuroendocrine and neurobiological
concomitants of extreme behavioral inhibition and to characterize a primate analogue of an anxiety-related endophenotype.

Marked individual differences among rhesus monkeys have been noted with regard to the intensity of context-specific defensive
responses. These defensive responses have been characterized using the HIP (see previous section). For example, some monkeys
tend to coo frequently during the A condition (in which the animal is isolated), whereas other same-aged animals engage in little or
no cooing. Large individual differences have also been observed in the duration of NEC-induced freezing (in the presence of a human
profile) and ST-induced hostility (in response to direct eye contact with the human intruder). Some animals freeze the entire length
of the test period, whereas at the other extreme some never freeze and act relatively undisturbed by the human intruder. These
individual differences in fear-related responses seen in the laboratory are similar to those that have been observed in rhesus
monkeys who inhabit Cayo Santiago, a 45-acre island with approximately 1,000 free-ranging monkeys (Kalin et al., unpublished data).
Importantly, it has been found that monkeys' individual differences in defensive responses are relatively stable over time, suggesting
that the intensity of defensive behavior that is displayed reflects a trait rather than a state characteristic. It was initially
demonstrated that the duration of NECinduced freezing behavior remained stable in 12 animals tested twice with an interval of 4
months (r = .94). Using a larger sample size, the stability of NEC-induced freezing was confirmed; ST-induced hostility was also
found to be relatively stable (Kalin et al., unpublished data). Interestingly, significant correlations between the magnitude of the
different types of defensive responses were not observed within an animal. Thus, monkeys that exhibited extreme levels of NEC-
induced freezing did not necessarily display extreme levels of ST-induced hostility. This lack of correlation between different types
of defensive responses suggests that cooing, freezing, and defensive hostility represent different and somewhat unrelated
characteristics of animals' defensive styles. Pharmacologic data also support this notion. For example, manipulations of the opiate
system affect A (alone condition)–induced cooing without affecting threat-induced freezing or hostility. Conversely, benzodiazepines
reduce the threat-related behaviors, but have little effect on A-induced cooing (14 ).

Finally, to identify some of the mechanisms underlying these individual differences in defensive responding, the relationships
between the stress-related hormone cortisol or asymmetric frontal EEG activity and individual differences in fearful behavior were
examined. Thus, in 28 mother-infant pairs, it was found that in both mothers and infants freezing duration was significantly and
positively correlated with baseline (nonstressed) cortisol levels (38 ). These data are consistent with findings from human studies
demonstrating that extremely inhibited children have elevated levels of salivary cortisol (36 ,37 ), and is also consistent with
findings in rodents that corticosterone (the rodent analogue of cortisol) is required for rat pups to develop the ability to freeze when
threatened (39 ).

Extremely fearful monkeys (as identified by the HIP) also exhibit characteristic EEG patterns. In adult humans,
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asymmetric right frontal brain activity has been associated with negative emotional responses (40 ). Our studies in rhesus monkeys
have demonstrated similarities in this measure between monkeys and humans (41 ). Thus, it has been found that dispositionally
fearful monkeys have extreme right frontal brain activity, paralleling the pattern of extreme right frontal activity in humans who
suffer from anxiety-related disorders. In addition, it was found that individual differences in asymmetric frontal activity in
nonhuman primates in the 4- to 8-Hz range are a stable characteristic of an animal (41 ,42 ). Furthermore, a significant positive
correlation between relative right asymmetric frontal activity and basal cortisol levels in 50 one-year-old animals was found. As
predicted, the more right frontal an animal was, the higher was its cortisol level. An extreme groups analysis revealed that extreme
right compared to extreme left frontal animals had greater cortisol concentrations as well as increased defensive responses, such as
freezing and hostility. The association between extreme right frontal activity and increased cortisol appeared to be long-lasting
because the right frontal animals continued to demonstrate elevated cortisol levels at 3 years of age. These results are the first to
link individual differences in asymmetric frontal activity with circulating levels of cortisol. This finding is important because both
factors have been independently associated with fearful temperamental styles.

It has recently been found that cerebrospinal fluid (CSF) levels of corticotropin-releasing hormone (CRH), a peptide that mediates
stress responses, are significantly elevated in monkeys that display exaggerated defensive responses to threatening stimuli (5 ). As
stated before, these extreme individual differences in defensive behaviors are stable over time. Moreover, it was found that CSF
CRH levels are also stable over time in rhesus monkeys. Finally, when comparing monkeys with extreme right frontal activity (that
display exaggerated fearful responses) to those with extreme left frontal activity (that display low levels of fearful behaviors), the
right frontal group was found to consistently have increased CSF CRH levels over a period of 4 years (5 ). Thus, it appears that
extreme fearful behavioral responses in nonhuman primates are associated with increased levels of stress hormones such as cortisol
and brain CRH, and also with extreme right frontal brain activity versus left frontal brain activity, a profile that has been found in
humans suffering from stress-related psychopathology (43 ). Taken together, these findings suggest that in primates, a fearful
endophenotype can be conceptualized as a constellation of hormonal, electrophysiologic, and behavioral characteristics. Studying
species-specific defensive behaviors and their neuroendocrine and physiologic correlates offers a powerful approach for identifying
animal correlates of anxiety.

Rodents
Extreme individual differences in the expression of stress-related defensive behaviors have also been noted in rodent species. The
examination of naturally occurring genetic variations with regard to stress reactivity may have important implications for the
elucidation of individual differences in sensitivity to stressful situations. One example of naturally occurring individual differences
comes from the study of different rodent strains with regard to their level of stress-like behavioral responding to environmental
stimuli. Because of the important role of the CRH system in regulating defensive behaviors induced by stressful or threatening
situations, attention has been focused on identifying rat or mouse strains that display differential stress reactivity and different
baseline levels of CRH gene expression. For example, it has been found that baseline levels of CRH messenger RNA (mRNA) are
significantly higher in the amygdala of fawn-hooded rats compared to either Sprague-Dawleys or Wistars (44 ,45 ). Fawn-hooded rats
have also been reported to exhibit exaggerated behavioral responses to stress such as enhanced freezing, leading to the suggestion
that this strain may have utility as a model for endogenous stress-related CRH overexpression and anxiety. Strain differences, which
essentially reflect differential genetic makeups, have also been found to influence the effects of acute environmental stressors on
regulating CRH system gene expression. Thus, the stress of whole-body restraint produces a much larger increase in CRH mRNA
levels within the hypothalamus of Fisher rats than in Wistars or Sprague-Dawleys (46 ,47 ). Similarly, the spontaneously hypertensive
and borderline hypertensive strains of rats have increased basal and stress-induced levels of hypothalamic CRH mRNA compared to
the Wistar and Sprague-Dawley strains (48 ,49 and 50 ).

In mice, it has been shown that the BALB/c strain is hyperresponsive to a variety of stressors compared to the C57BL/6 strain;
BALB/c mice exhibit significantly higher avoidance of aversive areas in a light-dark transition test and an open field (51 ,52 ). These
mice also show high levels of neophobia (53 ). Recent genetic mapping studies in these strains have revealed that these behavioral
differences may be associated with differential levels of γ-aminobutyric acid receptor A (GABAA) expression between the strains. For
example, it has been found that BALB/c mice have significantly lower levels of benzodiazepine binding sites in the amygdala
compared to C57BL/6 mice (54 ). As described below, alterations in the expression of GABAA receptors have been found to lead to
increased anxiety-like behaviors in genetically modified mice (see CRH System Transgenic Mice ).

Taken together, these findings indicate that different rodent strains, as a consequence of their distinct genetic makeups, display
different baseline levels of gene expression within various systems that are known to regulate the expression of stress-induced
defensive behaviors. The study of various rodent strains may thus help to identify the neurogenetic differences that contribute to
individual differences in stress susceptibility, and thereby further characterize the interaction between genes and environmental
 P.888

conditions in the etiology of anxiety. Although such information is useful, it remains to be determined whether or not the specific
genetic differences identified above actually underlie the different behavioral effects. It is probable that a number of genes in
addition to those described above are differentially expressed across different rodent strains. Which other genes differ across strains,
and of these, which ones contribute to the behavioral profile? It is also unclear whether the differential gene expression patterns
are the cause or the result of the different phenotypes observed in the separate strains. Future studies in which behavioral
phenotypes are assessed after the application of novel gene targeting techniques to selectively disrupt or restore gene function in
these rodent strains will aid in clarifying these issues.

MATERNAL DEPRIVATION: AN ENVIRONMENTAL MANIPULATION THAT

CAN LEAD TO FEARFUL ENDOPHENOTYPES IN PRIMATES AND RODENTS
Part of "62 - Animal Models and Endophenotypes of Anxiety and Stress Disorders "

Converging lines of evidence from a number of species point to the importance of the early postnatal period, and in particular the
bond between mother and infant, in the development of normal defensive behaviors and the putative emotional states underlying
these behaviors. It has been observed that children who were placed in nurseries that lacked adequate social stimulation developed
a syndrome of “protest, despair, and detachment” that may be analogous to an increase in defensive responses (55 ). Furthermore,
recent reports suggest that children reared without appropriate nurturance can display neuroendocrinologic alterations and may
develop long-term behavioral and emotional difficulties including an increased risk for stress-related psychiatric illness (56 ,57 ).

Perhaps the most significant environmental factor during the early development of mammals is the interaction between the infant
and its mother. As described above, separation of an infant from its mother during this early developmental phase represents a
significant stressor that markedly and negatively affects the subsequent emotional development of the infant (55 ,57 ). In fact,
disruption of normal attachment behavior at critical developmental phases can, in a number of species, lead to marked and
persistent disturbances in behaviors and brain systems that are thought to participate in the regulation of fear-related responses;
this disruption may ultimately contribute to an individual's propensity to develop exaggerated or inappropriate defensive responses.

Altered maternal-infant interactions can lead to anxiety endophenotypes in nonhuman primates and rodents, thus identifying an
environmental manipulation that can be used to create animal models of increased stress-related functioning. Indeed, a large body
of work in monkeys and rats indicates that a number of deleterious and long-lasting effects are produced as a result of separating
infants from their mothers prior to weaning. The notion that perturbations in the early postnatal environment might have enduring
neuroendocrine, neurochemical, and behavioral effects was originally put forth several decades ago by Levine (58 ). It has since
been demonstrated that a likely source of these alterations is a disruption of the interaction between mothers and pups (59 ,60 ).

Nonhuman Primates
The classic studies by Harlow and colleagues (20 ,61 ,62 ) of the effects of maternal separation in primates found that in addition to
life-supporting nourishment, physical contact and comfort are necessary for primates' normal social and emotional development.
During the first months of life, the attachment between mother and infant is intense, and as a consequence the infant remains in
close proximity to its mother (61 ,63 ). Long-term maternal separation can result in profound alterations in stress-related behavioral
responses in the separated offspring. Monkeys that have been separated from their mothers for prolonged periods during this time
exhibit symptoms of enhanced defensive or fear-related behavioral responses into adulthood and appear socially withdrawn, a
phenomenon that has led to the suggestion that the behavioral and neuroendocrine sequelae of maternal separation might provide a
model for some of the dysfunction that is observed in anxiety disorders and depression (64 ,65 ,66 ,67 and 68 ).

Furthermore, neuroendocrine studies in rhesus monkeys indicate that an infant's stress hormone levels are negatively correlated
with the number of offspring the mother had, suggesting that when mothers are less experienced, cortisol levels in their (early born)
infants are high; elevated cortisol levels also correspond to increased fearful behavioral responses in the infants (38 ). Cortisol has
been found to play an important role in mediating the development of defensive responses (69 ); thus, factors that were expected
to affect infant primate cortisol concentrations were examined. It was found that maternal cortisol levels were moderately
correlated with those of their infants (38 ). Interestingly, it was also found that maternal parity was negatively correlated with
infant cortisol levels such that the current infants of mothers that previously had more offspring were likely to have lower cortisol
levels. This finding indicates that a mother's past infant rearing and/or pregnancy experience may contribute to individual
differences in infant baseline cortisol levels, and provides further support for the notion that the mother-infant interactions may be
a critical factor in determining the future fearful disposition of the offspring (38 ). Although the precise mechanism for this
interaction remains to be determined, it is likely that mothers with little rearing experience would interact differently with their
infants than mothers with more experience.

Evidence for the notion that long-lasting dysregulation

 P.889

of the CRH system may in part underlie the harmful consequences of early developmental stressors has been provided in a study of
nonhuman primates that were exposed to adverse rearing conditions during infancy. Coplan and colleagues (70 ,71 ) found that CSF
levels of CRH are basally and chronically elevated in adult bonnet macaques whose mothers were exposed for 3 months to an
unpredictable variable foraging demand (VFD), in comparison to mothers confronted with either a high but predictable or low but
predictable foraging demand. Infants reared by VFD-exposed mothers have been found to subsequently display abnormal affiliative
social behaviors in adulthood (72 ). These findings are consistent with the recent results from this lab that indicate that CSF CRH
levels are elevated in dispositionally fearful monkeys, and that this CRH elevation is a stable trait-like characteristic of fearful
endophenotype (5 ).

Rats
These aforementioned findings in nonhuman primates support the notion that mother-infant interactions may be a critical factor in
determining the future fearful disposition of the offspring. Maternal separation has also been found to produce long-term changes in
defensive behaviors into adulthood in rats. Using the maternal separation paradigm in rats, investigators have also been able to
begin to elucidate some of the alterations in gene expression that take place in response to this early life stressor.

Interestingly, the nature of the separation determines the direction of the long-term changes, as has been reviewed in detail
recently (73 ,74 and 75 ). Thus, brief periods of separation (3 to 15 minutes per bout, once a day, for roughly 2 weeks) from the
mother result in a profile indicative of diminished anxiety, whereas more protracted separations (3 hours or more) have the opposite
effect, resulting in increased stress-like responses. In an elegant series of studies by Plotsky and Meaney (76 ), the long-term effects
of these different types of maternal separation have been described, and the behavioral and neuroendocrine mechanisms underlying
these long-term effects have been characterized. It was initially found that rat pups that underwent very short periods of separation
(termed “handling”) from their mothers had decreased basal levels of hypothalamic CRH mRNA and median eminence CRH
immunoreactivity as adults compared to undisturbed control rats. As adults, these “handled” pups also displayed significantly lower
elevations of stress-induced corticosterone levels and blunted CRH release from the median eminence relative to controls. It has
since been found that the mechanism underlying this reduction in stress-related functioning in handled rat pups involves the type of
maternal behavior that is displayed after the pups are returned to the mother (77 ), confirming earlier hypotheses that maternal
behavior is the critical component in the developmental milieu of the infant (58 ). A brief removal of rat pups from the dam results
in a significant increase in the amount of licking, grooming, and arched-back nursing (LG-ABN) that the mother lavishes on the pups
when they are returned; the total amount of time spent nursing and contacting the offspring is not affected, but rather the quality
of the interaction between mother and pup is altered. In nonseparated pups, individual differences in LG-ABN predict hypothalamic-
pituitary-adrenal (HPA) axis responsivity in adulthood such that mothers that engage in high levels of LG-ABN have offspring that, as
adults, show reduced HPA axis activation in response to stress and have decreased levels of CRH mRNA in the paraventricular
nucleus (PVN) of the hypothalamus (77 ). Pups that are born to mothers that naturally exhibit high levels of LG-ABN grow up into
adults that display low-anxiety–like behaviors (increased exploration of novel environments) and compared to low–LG-ABN offspring,
have decreased levels of CRH receptors in brain regions such as the locus coeruleus that are thought to mediate stress responses
(78 ). Taken together, these findings indicate that increased nurturing physical contact from the mother can lead to a toned-down
stress-responsive system in the offspring.

In contrast, longer periods of maternal separation seem to have the opposite effect on stress-related functioning later in life. Rat
pups that are separated from the mother for 3 hours or longer (investigators have often used a 24-hour separation) show in
adulthood increased CRH system gene expression, exaggerated HPA axis responses to stress, and increased stress-like behaviors in
paradigms such as the elevated plus maze (76 ,79 ,80 ). Other intense stressors such as an endotoxin insult during the perinatal
stage are also able to produce marked elevations in basal CRH gene expression and lead to an exaggerated stress-induced HPA axis
response in adulthood (81 ). It has accordingly been hypothesized that the perinatal environment plays a critical role in
“programming” or “setting” the animal's stress coping system (perhaps through alterations in CRH system gene expression) for the
remainder of its life (73 ,74 and 75 ). Maternally separated rats also show alterations in other systems that are known to regulate
stress-related behaviors and that are consistent with an increased fearful endophenotype. For example, maternal separation
increases the release of norepinephrine into the PVN of the hypothalamus in response to restraint stress; stress-induced plasma
adrenocorticotropic hormone (ACTH) levels were also elevated in maternally deprived rats (82 ). Early life stressors such as maternal
separation may therefore play an important role in determining the eventual stress-related endophenotype that is exhibited in
adulthood. Moreover, the aforementioned studies provide an example of how the animal endophenotype approach can be applied to
investigating molecular correlates of anxiety-related conditions.

It should be mentioned that prenatal stress can also produce alterations in indices of stress-induced responding in adulthood. For
example, in rats, disturbing the prenatal environment by stressing the mother can lead to increases in
 P.890

CRH gene expression in the fetal PVN, increases in CRH content in the amygdala of adult offspring, and potentiation of stress-like
behavioral responses in these rats whose mothers had undergone stress during pregnancy (83 ,84 and 85 ). These findings further
support the notion that mother-infant interactions may be a critical factor in determining the future fearful disposition of the
offspring.

TARGETED MUTATIONS LEADING TO ANXIETY-LIKE ENDOPHENOTYPES:

STUDIES OF TRANSGENIC MICE
Part of "62 - Animal Models and Endophenotypes of Anxiety and Stress Disorders "

A perhaps more direct approach for studying the genetic underpinnings of a particular animal endophenotype is to characterize the
change in an organism's interaction with its environment following either overexpression or underexpression of a particular gene
product. Transgenic and knockout mice are thus now widely used in the ongoing effort to understand the contributions of specific
genes to psychopathology. The detailed methodology for the generation of these animals and their use in neuroscience research has
been reviewed (86 ). Briefly, genetic alterations are introduced in the embryonic stage such that the mouse develops with the
mutation, thereby putatively providing a model for congenital abnormalities that may contribute to anomalous functioning and the
expression of a particular endophenotype.

Using this strategy, a variety of components within the CRH, serotonin (5-hydroxytryptamine, 5-HT), and GABA systems have been
successfully targeted and studied for their roles in mediating stress-related behavioral effects (87 ,88 and 89 ). It should be noted
that in addition to the aforementioned systems, there are several other important central regulators of stress and anxiety-related
processes. The norepinephrine system has long been implicated in the modulation of anxiety states. Several recent reviews detail
the preclinical and clinical evidence for the involvement of norepinephrine (NE) in anxiety-related disorders such as panic and
posttraumatic stress disorder (90 ,91 ). Indeed, β-adrenergic receptor antagonists and α2-receptor agonists are effective in the
treatment of certain anxiety-related symptoms in humans. In addition, recent preclinical evidence indicates that a variety of central
nervous system (CNS) peptides including cholecystokinin (CCK), neuropeptide Y (NPY), and substance P/neurokinins participate in
the regulation of anxiety-like behaviors (92 ,93 and 94 ).

Although these peptide and neurotransmitter systems undoubtedly play a role in stress- and anxiety-related behaviors, the focus of
the following section will be the CRH, 5-HT, and GABA systems because these three systems are perhaps the most thoroughly studied
with transgenic models. Although the transgenic/knockout approach has provided valuable new information about the genetic
regulation of stress-related fearful endophenotypes, it should be kept in mind that there are a number of important caveats
regarding the interpretation of findings from transgenic animal studies (described at the end of this section). As outlined below,
alterations of discrete genes within each of these systems results in an anxiety-like murine endophenotype, characterized by the
increased expression of certain aspects of rodent defensive behaviors.

The CRH System

CRH and the related endogenous peptide agonist urocortin (95 ) bind to the two cloned CRH receptors, designated CRH1 and CRH2
(96 ,97 and 98 ), and to the CRH binding protein (CRH-BP) (99 ). The CRH-BP has been postulated to function as an endogenous
buffer for the actions of the CRH family of ligands at their receptors (100 ). CRH, its receptors, and its binding-protein are expressed
in key structures of the HPA axis, and thereby participate in mounting the neuroendocrine response to environmental perturbations.
The various elements of the CRH system are widely and heterogeneously expressed in cortical, limbic, and brainstem structures and
these regions are thought to regulate behavioral responses to stress.

CRH System Transgenic Mice

Given that central infusion of CRH results in enhanced fear-related defensive behaviors, CRH-overexpressing mice were predicted to
display increased stress-like behavioral responses. Indeed, these mice have been found to have several behavioral effects associated
with acute CRH administration. CRH overexpressing mice exhibit a profile that is consistent with increased levels of stress, such as
reduced baseline and stress-induced exploration of a novel environment, and decreased activity and time spent in the open arms of
an elevated plus maze (101 ,102 ). These effects are potently blocked by administration of the CRH receptor antagonist α-helical
CRH. CRH transgenic mice also show a profound decrease in sexual behaviors and significant deficits in learning; higher order
functions such as these are typically abolished when a situation is found threatening and defensive behaviors are recruited
(103 ,104 ). Thus, CRH overexpressing transgenic mice may represent a genetically engineered model of a murine anxiety-like
endophenotype.

Consistent with the notion that heightened CRH transmission elicits stress-like behaviors is the finding that CRH-BP knockout mice
show increased stress-like behaviors. CRH-BP knockout mice displayed decreases in open arm entries and open arm time in an
elevated plus maze, and showed a decrease in the number of exits from a safe box in a defensive withdrawal/open field paradigm
(105 ). These results indicate a heightened level of neophobia in these mice. Moreover, CRH-BP knockouts have reduced body weight
gain over several weeks (105 ,106 ), which is also syntonic
 P.891

with increased basal CRH activity. The behavioral profile of CRH-BP knockouts is similar to that which is seen with exogenous CRH
administration (107 ), and supports the notion that removal of the CRH-BP may lead to increased basal stress responses due to
increased CRH tone. Thus, as with the CRH-overexpressing mice, CRH-BP knockouts may represent another rodent anxiety-like
endophenotype. In terms of the predictive validity of these models, CRH receptor antagonists have been found to block the stress-
like behavioral profile observed in these animals; one recent clinical study indicates that CRH1 receptor antagonists may indeed
prove to be effective anxiolytics or antidepressants (108 ).

Interestingly, deletion of the CRH gene does not appear to decrease stress-like behaviors, as might be predicted from the
aforementioned work. Although certain endocrinologic deficits are observed in CRH knockout mice, stress-related behavioral
function in these animals remains relatively unaffected as assessed by multiple stress-related paradigms (109 ,110 ,111 ,112 ,113
and 114 ). This sparing of normal stress responsivity may be due to compensatory increases in the expression of other CRH system
ligands such as urocortin. Deletion of the CRH1 receptor gene, however, does appear to consistently result in a putative reduction in
anxiety (115 ,116 and 117 ). For example, CRH1 knockout mice show increased exploration of the open arms on an elevated plus
maze and spend more time in the brightly lit compartment of a dark-light transition box than do wild-type controls. Moreover, CRH1
knockout mice appear to be immune to the anxiogenic effects of ethanol withdrawal (117 ). Studies of CRH2 receptor knockout mice,
on the other hand, indicate that these mice display a less consistent behavioral profile than the CRH1 knockout mice (118 ,119 and
120 ). Part of the behavioral profile of CRH2 knockouts is suggestive of increased stress-like responding, but other aspects of the
behavioral profile indicate either no alteration of stress-related responding (118 ,119 ), or a decrease in anxiety-like behaviors (120 ).
The observed increases in anxiety-like behaviors in these genetically altered mice may be due to increased levels of brain CRH
and/or urocortin; in two of the three studies, an elevation of baseline CRH or urocortin mRNA levels in the CNS was seen in CRH2
knockout mice (118 ,119 ). Thus, the endophenotype displayed by CRH2 knockout mice may actually be indirectly due to a
compensatory alteration induced by the mutation rather than simply due to a lack of CRH2 receptor expression. It should be noted
that acute blockade of CRH2 receptors results in a decrease in stress-induced defensive behaviors; thus the behavioral profile of
these animals is opposite to that of mice that are missing the CRH2 receptor (121 ,122 ). Thus, the timing of the gene deletion may
critically influence the nature of the behavioral phenotype that ensues. Future studies utilizing novel inducible-knockout
technologies may help in clarifying the developmental versus acute role of various genes in the development of anxiety-related
endophenotypes (123 ).

Clinically Effective CRH System Drugs for Stress-Related Disorders

A large body of preclinical literature indicates that CRH is a critical modulator of stress and anxiety-like behaviors in nonhuman
primates and rodents (107 ,124 ). Based on the ability of CRH1 receptor-selective antagonists to block many of the behavioral
effects of stress or CRH administration, these antagonists have been proposed as potentially therapeutic agents for the treatment of
stress-related psychiatric conditions including anxiety and depression (125 ). Pharmacologic analysis of stress-induced primate
defensive responses has also revealed that the CRH system is a critical modulator of this index of anxiety-related behavior. For
example, administration of CRH into the cerebral ventricles of nonhuman primates results in a constellation of behavioral responses
that closely resemble the defensive responses that are exhibited upon presentation of a stressor (124 ). Consistent with the notion
that increased levels of CRH are associated with increased anxiety-like responding are the recent findings that small-molecule CRH1
receptor antagonists block the expression of some behavioral, physiologic, and neuroendocrine responses to stressors in rhesus
monkeys (126 ,127 ). The first report of an open-label clinical trial with a CRH1 antagonist was recently published, and revealed a
significant effect of this compound in ameliorating symptoms of depression and anxiety (108 ). Although further research is needed
to firmly establish the utility of CRH1 antagonists as psychotherapeutic agents and also to determine the possible side effects
associated with their use, these preliminary data support the notion that these compounds represent an important new class of
drugs that may offer great promise for the treatment of illnesses associated with increased anxiety and stress.

The 5-HT System

Serotonin is a member of the monoamine family of transmitters that also include dopamine and norepinephrine. As is typical for the
monoamines, cell bodies for this neurotransmitter are found in discrete nuclei within the midbrain (dorsal and medial raphe nuclei)
and send widespread 5-HT–containing projections throughout the brain (128 ). 5-HT produces its effects through at least 15 different
5-HT receptors that are differentially distributed throughout the CNS; the principal mode of 5-HT inactivation is cellular reuptake
via terminal transporter proteins (129 ). The 5-HT system has long been implicated in the regulation of mood states and anxiety, and
selective serotonin reuptake inhibitors (SSRIs) constitute a major class of antidepressants that have anxiolytic effects. As outlined
below, 5-HT transmission also plays a critical role in the regulation of anxiety-like
 P.892

behaviors. Given the plethora of 5-HT receptors and the paucity of highly selective ligands for these multiple target sites, several
investigators have employed murine gene targeting strategies to elucidate the roles of specific 5-HT receptors in the regulation of
stress and anxiety.

5-HT System Transgenic Mice

Studies of targeted gene deletions within the 5-HT system have revealed an important role for this system in the regulation of stress
and anxiety-related behaviors in mice. The behavioral sequelae of disrupting 5-HT receptor gene expression have been elegantly
summarized in several review articles (89 ,130 ,131 and 132 ). Perhaps the best-characterized 5-HT mutant mice are the 5-HT1A and
5-HT1B receptor knockouts. Mice with a mutation in the 5-HT1A receptor gene have been found to display increased stress-like
behaviors in multiple tests of approach-avoidance conflicts. These animals show decreased entries into and time spent in the more
aversive region in paradigms such as the open field, elevated plus maze, and the elevated zero maze; thus 5-HT1A knockout mice
avoid the center of an open field, the open arms of a plus maze, and the unenclosed regions of a zero maze (133 ,134 and 135 ). It
is worth noting that this “increased anxiety” pattern of results was found consistently across three different research labs,
indicating its robustness and reproducibility. Consistent with this profile is the finding that these mice also exhibit decreased
activity in the presence of and approach toward a novel object (135 ). This increase in stress-like responding is not accompanied by
changes in overall locomotor activity or motor and spatial coordination, as assessed in photocell cages and a RotaRod apparatus.
Curiously, 5-HT1A knockout mice display increased mobility in response to an acute stressor such as forced swimming or tail
suspension (133 ,134 and 135 ). Taken together, these findings indicate that 5-HT1A knockout mice may represent another animal
endophenotype of increased anxiety.

The constitutive knockout of the 5-HT1A receptor does not seem to lead to compensatory alterations in the expression of serotonin or
its transporter, or to changes in catecholamine levels in several brain regions (135 ). Interestingly, a recent report indicates that this
mutation alters GABA system expression and function (136 ). It has been found that anxiety-like behaviors in 5-HT1A knockout mice
are relatively unaffected by benzodiazepine treatment. Analysis of brain tissue from these animals indicates that GABAA receptor
binding is reduced and that the expression of α1 and α2 subunits of the GABAA receptor are decreased in the amygdala. The anxiolytic
actions of benzodiazepines may in part be mediated by GABAA receptors within the amygdala; the profile of results in 5-HT1A
knockout mice has led to the intriguing speculation that the anxiety-like endophenotype in these mice may actually in part derive
from a decrease in the expression and function of the GABAA receptor (136 ). This proposed mechanism is consistent with the
increase in stress-related behaviors that are seen in certain transgenic mice with mutations in the GABAA receptor (see below).
Further work is necessary to determine the precise mechanisms through which the developmental interruption of 5-HT1A gene
expression results in the observed anxiety-like endophenotype.

In contrast to 5-HT1A knockout mice, mice that lack the 5-HT1B receptor show decreased anxiety-like behaviors in several tests of
approach-avoidance conflicts. 5-HT1B knockout mice spend more time in the center of an open field and more readily explore novel
objects than their wild-type controls; this profile is opposite from that of 5-HT1A knockout mice, and is suggestive of diminished
neophobia (89 ,137 ). Consistent with this pattern of results is the finding that as pups, 5-HT1B mice emit fewer ultrasonic
vocalizations when separated from their mothers; separationinduced vocalizations are thought to provide a measure of anxiety and
distress in pups (138 ,139 ). It is interesting to note, however, that no changes in contextual or cue-induced conditioned freezing are
observed in 5-HT1B mutant mice, suggesting that approach-avoidance conflicts and conditioned fear may be differentially modulated
by the 5-HT system. The other main behavioral effect of constitutive 5-HT1B receptor deletion is a marked increase in aggressive
behavior (89 ,140 ,141 ). Given that aggressive behaviors represent an important part of an organism's response to threat, 5-HT1B
knockout mice may also provide valuable information on the neural and genetic factors associated with stress and anxiety-related
functioning (89 ,142 ).

It should be noted that mice with null mutations of other 5-HT receptor subtypes have also been generated, but these animals have
not been found to display as robust an anxiety-related behavioral profile as the 5-HT1A or 5-HT1B knockout mice. It has been found
that 5-HT5A receptor knockout mice show increased exploratory activity in the presence of novelty, but do not differ from wild-type
controls with regard to avoidance behaviors from an aversive environment such as the open arms of a plus maze, or the center of an
open field (143 ). These knockout mice also do not respond differently from control subjects in tests of startle reactivity or in
burying a probe that delivered a brief electric shock. Thus, these animals appear to have yet a different behavioral profile from that
of the 5-HT1A or 5-HT1B knockout mice. An initial report indicates that 5-HT6 receptor deficient mice may exhibit increased avoidance
of aversive environments; although these preliminary findings are interesting, further work is needed to fully characterize the
phenotype of these mutant mice (144 ,145 ). Mice lacking either the 5-HT2A or 5-HT2C receptors have also been created; to the best
of our knowledge, the stress-related behavioral functioning of these animals has yet to be reported (146 ,147 ).
 P.893

Clinically Effective 5-HT System Drugs for Stress-Related Disorders

As mentioned above, one of the most commonly prescribed and effective classes of drugs that is used in the treatment of depression
and anxiety is the SSRIs, which block the reuptake of 5-HT by its transporter and thereby increase serotoninergic transmission. Based
on the findings of preclinical studies including those obtained from 5-HT receptor knockout mice, 5-HT1A agonists have been
developed for the treatment of anxiety. The clinical utility of this class of compounds, however, remains to be determined. As these
transgenic approaches develop and become more refined, they will undoubtedly aid in clarifying the roles of the many other 5-HT
receptor subtypes in processes related to stress and anxiety and will aid in drug development.

The GABA System

The primary inhibitory neurotransmitter in the CNS is GABA; GABA-synthesizing cells are distributed throughout the brain (128 ). The
actions of GABA are mediated by two major classes of receptors, GABAA and GABAB, both of which modulate the activity of ion
channels. The principal mode of inactivation of GABA transmission is the presynaptic reuptake of GABA by its transporter protein.
Although both types of GABA receptors are widely distributed through the CNS, several important differences exist between the two.
Relevant to psychopharmacology is the finding that traditional anxiolytics (benzodiazepines) do not bind to GABAB receptors, but
rather mediate their effects through GABAA receptors. GABAA receptors consist of a chloride channel formed by the pentameric
arrangement of at least 18 different protein subunits (α1–6, β1–4, γ1-3, δ, ε, π, ρ1–3), thus allowing for considerable heterogeneity of the
GABAA receptor isoforms (148 ). Typically, benzodiazepine-responsive GABAA receptors consist of α, β, and γ subunits; in addition to
the benzodiazepine site, these receptors also contain distinct sites for the binding of GABA, barbiturates, and ethanol. These various
regions act as allosteric regulators of GABA-induced chloride channel opening. Although psychotherapeutic effects such as anxiolysis
are achieved through facilitation of GABA transmission at this receptor, drugs that act as GABAA receptor agonists also produce
several deleterious side effects. The extent to which differences in GABAA receptor subunit composition might contribute to possible
dissociations between the beneficial and negative effects of these compounds is currently being investigated.

GABA System Transgenic Mice

The synthesis of GABA is regulated by two isoforms of the enzyme glutamate decarboxylase (GAD), GAD67, and the shorter form
GAD65 (149 ). Whereas GAD67 is thought to maintain basal GABA levels, GAD65 is thought to regulate the synthesis of GABA at nerve
terminals in response to high GABA demand (150 ). Given the important role of GABA in inhibitory neurotransmission associated with
anxiolysis, several investigators have evaluated the behavioral profile of genetically altered mice that lack the GAD65 gene. Two
separate groups have reported that GAD65-/- mice display an increase in stress-like behaviors in numerous behavioral paradigms
(151 ,152 ). GAD65 knockout mice had fewer entries into and time spent in the center of an open field or the open areas of an
elevated zero maze (similar to an elevated plus maze), indicating that they were more avoidant of inherently aversive areas.
Similarly, these mice had lower levels of activity in the bright portion of a light-dark transition box. It should be mentioned that
GAD65-/- mice also displayed an elevation in the occurrence of spontaneous and stress-induced seizures, and that these mice had a
dramatically increased mortality rate starting at 4 to 5 weeks after birth (151 ). Thus, although the behavioral profile of GAD65
knockout mice is suggestive of increased anxiety-like responses, it is possible that these effects are secondary to the occurrence of
seizures and to the factors leading to early lethality. The usefulness of this knockout as a model for anxiety-related deficits may
therefore be limited. Given that benzodiazepines and barbiturates act as positive modulators of GABA transmission at the GABAA
receptor by enhancing GABA-induced chloride channel opening, it is of interest to note that GAD65-/- mice were not sensitive to the
effects of either benzodiazepines or barbiturates, but did respond to the direct GABAA agonist muscimol, which binds directly to the
GABA site of the GABAA receptor and increases opening of the chloride channel in the absence of GABA (152 ). This pharmacologic
profile is consistent with the finding that GABA synthesis is blocked by the GAD65 null mutation, but that GABAA receptor binding is
unaffected by this change. Furthermore, this mutation does not seem to alter the functioning of GABA receptors because direct
agonists stimulate the receptor but indirect modulators of GABA do not.

In an attempt to delineate the roles of the various GABAA receptor subunits in the regulation of stress- and anxiety-related behaviors,
investigators have generated mutant mice with alterations in the expression of specific GABAA receptor subunits. It was initially
reported that deletion of the γ2 subunit led to a selective (94%) reduction in the expression of benzodiazepine sites in the CNS
without alterations in the level of GABA sites or changes in the expression of other GABAA receptor subunits (153 ). Thus, γ2 knockout
mice possessed functional GABAA receptors that responded normally to GABA site ligands or barbiturates, but did not respond to
benzodiazepines; these findings led to the conclusion that the γ2 subunit is not necessary for the formation of functional GABAA
receptors, but is required to create
 P.894

the benzodiazepine-responsive site of those receptors. Mice that were homozygous for the mutation, however, did not live past
weaning in this study. In mice carrying only one copy of the functional γ2 gene, a 20% reduction in benzodiazepine sites was
observed, but these mice did not show overt developmental deficits. In a recent study, a detailed characterization of the behavioral
profile of these animals was carried out. Heterozygotes displayed a decrease in the number of entries into and amount of time spent
in the open arms of an elevated plus maze and the bright compartment of a light-dark box. These animals also exhibited a decrease
in the exploration of novel areas, and an increase in certain forms of fear conditioning that are thought to be mediated by the
hippocampus. Finally, γ2 heterozygotes were found to react to partially conditioned stimuli (only weakly paired with aversive
consequences) as if they were full and potent predictors of threat; compared to wild-types, which showed low levels of defensive
behaviors to the partially conditioned stimulus, heterozygotes displayed high levels of conditioned freezing to the partial
conditioned stimulus that were identical to those displayed by all animals in response to the full conditioned stimulus. This profile
has been proposed to be a model for the tendency to interpret neutral situations as threatening that is seen in anxiety patients.
Taken together, the results from this extensive behavioral profile indicate that γ2+/- mice have increased neophobia and stress-like
responses and may thus provide a model for increased anxiety-like behaviors (154 ,155 and 156 ). Interestingly, all of the elevations
in stress-like behaviors in γ2 heterozygotes were blocked by the benzodiazepine diazepam, suggesting that this animal model may
also have good predictive validity for identifying clinically effective anxiolytics.

It is also extremely important to mention the α1 subunit transgenic mice, whose behavioral profiles have been thoroughly and
insightfully reviewed in recent articles (157 ,158 ). In these mice, a single amino acid is altered (histidine replaced by arginine at
the 101 position of the peptide) in the α1 subunit of the GABAA receptor complex. This subtle change does not produce any overt
alterations in baseline responses to stress in the genetically altered mice; these animals behave similarly to wild-type controls in
tests such as the elevated plus maze and the fear-potentiated startle paradigm, a measure of conditioned fear (159 ,160 ). Thus,
under drug-free, normal conditions, these animals do not display a behavioral pattern that is consistent with an anxiety-like
endophenotype. When these mice are treated with conventional benzodiazepines, however, they react very differently to the drug
than their wild-type counterparts. Mice with the mutation in the α1 subunit display a normal reduction of stress-induced anxiety-like
behaviors after benzodiazepine treatment, but fail to display some of the more deleterious side effects associated with this class of
drugs such as sedation, amnesia, and ataxia. These results indicate that the anxiolytic effects of benzodiazepines can be separated
from the negative side effects of these compounds, and that the α1 subunit of the GABAA receptor is likely to mediate some of these
potentially harmful properties of benzodiazepines. Interestingly, McKernan and colleagues (160 ) demonstrate that a novel
benzodiazepine-site ligand that binds to GABAA receptors containing α2, α3, or α5 subunits but avoids receptors with the α1 subunit
produces a behavioral profile that is identical to that of the α1 subunit knockout mice; in normal mice, this compound decreases
murine anxiety-like behaviors without eliciting sedation or ataxia (160 ).

Clinically Effective GABA System Drugs for Stress-Related Disorders

As stated above, the most widely used GABA system-based drugs for the treatment of anxiety are the benzodiazepines, which
facilitate GABA transmission through the GABAA receptor. As outlined in the previous section, the search for novel compounds that
may act selectively at specific GABAA subunits is ongoing, with the ultimate hope of discovering ligands that produce anxiolysis but
do not cause some of the serious side effects that are commonly associated with benzodiazepines. As demonstrated by McKernan
and colleagues (160 ), drugs that selectively target certain GABAA receptor subunits may hold great promise for the treatment of
anxiety without harmful side effects. This development would represent a major breakthrough in the pharmacotherapy of anxiety-
related disorders. The use of targeted genetic alterations in identifying the roles of various GABAA subunits will undoubtedly aid in
this effort to create “designer drugs” for the treatment of anxiety (158 ).

General Issues and Caveats of Transgenic Animal Studies

As mentioned above, mice carrying certain mutations within either the CRH, the 5-HT, or the GABA system display an anxiety-like
endophenotype. It appears that these genetically engineered mouse models also have some predictive validity; the stress-like
endophenotype observed in at least two of the aforementioned models is normalized by administration of a clinically effective
antianxiety agent that acts within the system that was genetically targeted. It remains to be determined, however, the extent to
which these genetically altered models serve to identify potential antianxiety agents from different chemical classes. For example,
do benzodiazepines reduce stress-like effects of CRH overexpressers? The extent to which the stress-like endophenotype in these
animals is altered by compounds that act on systems that were not directly targeted by the genetic mutation will aid in determining
the generalizability and utility of these models as predictors of novel anxiolytic agents. If one assumes that these animals provide a
model of inherent trait-like anxiety, they can serve as a powerful tool for
 P.895

screening new potential anxiolytics. These models do provide a sound approach to study the long-term effects of congenital
abnormalities in these neurotransmitter and neuropeptide systems.

Several broad issues should be considered when interpreting studies utilizing genetically altered mice. Generally, the hypotheses
regarding the behavioral profiles of transgenic mice are based on earlier findings from psychopharmacologic studies. For example,
within the CRH field, the prediction that CRH overexpressers would display increased anxiety-like behaviors was based on the
observation that CRH administration produces stress-like behaviors in rodents and primates (107 ,124 ). When the outcome of the
transgenic studies agrees with the psychopharmacology-based prediction, the findings are taken as a confirmation of that
hypothesized mechanism of action. When the outcome of the transgenic studies disagrees with the predicted phenotype, however,
concerns about possible developmental confounds are raised. One of the most commonly cited drawbacks of the
transgenic/knockout strategy is that the gene of interest is altered from the embryonic stage, therefore possibly influencing other
genes involved in the normal development of the animal. Thus, it is difficult to tease apart the effects of under- or overexpression
of that gene on the endpoints under study from effects due to compensatory or downstream developmental changes that may have
occurred as a result of the mutation (86 ,87 ,161 ). Therefore, the transgenic/knockout approach provides an excellent method for
modeling a congenital abnormality that leads to a disease state, but this approach may be less useful for identifying the discrete
functions of a specific gene product because of the problems of interpretation that arise from the developmental confound. Indeed,
with regard to all of the studies discussed in this section on genetically altered mice, it will be important in future studies to
delineate the compensatory alterations that occur in response to the congenital mutation, and that may indirectly contribute to the
adult endophenotypes that are reported for these animals. Future studies utilizing novel inducible-knockout strategies will
circumvent the developmental issue; inducible knockouts may thus become a valuable tool for exploring the functions of discrete
gene products for which no selective ligands are available (123 ).

It should also be noted that there is a large literature concerning the use of antisense oligonucleotide infusions to knock down the
expression of particular gene products that may be related to fearful endophenotypes. The antisense oligonucleotide approach,
however, has been plagued with a number of issues regarding toxicity, and may therefore not represent the optimal method for
studying gene function in vivo (162 ).

FUTURE DIRECTIONS
Part of "62 - Animal Models and Endophenotypes of Anxiety and Stress Disorders "

Although the studies summarized in this chapter have contributed a great deal of knowledge about some of the genetic contributions
to the development of stress and anxiety-like endophenotypes in animals, further information is needed to understand the precise
nature of gene–environment interactions in stress regulation. It is likely that a particular stressor results in alterations of gene
expression in myriad systems and that the overall response to stress involves the coordination of gene activation and/or suppression
within these various systems. Novel high-throughput technologies have recently been developed that enable the expression of
thousands of genes to be assayed at once. “Gene chips” and “DNA arrays” are two powerful new tools for analyzing complex
multilocus genetic interactions associated with a particular environmental perturbation or disease state (163 ,164 ). This approach
and its application to psychiatry research have been discussed comprehensively in a recent review article (165 ). Briefly, gene chip
and DNA array technology involve the hybridization of gene transcripts from a tissue sample onto a glass slide or filter that contains
up to 10,000 different nucleotide sequences. The amount and pattern of the signal hybridized to the array are then assessed; this
method thus permits a rapid analysis of changes in the expression of multiple genes. This technology can also be used to identify
single nucleotide polymorphisms in a particular gene by comparing the hybridization patterns of samples from different candidate
populations on chips that contain multiple copies of the gene of interest, each copy differing from the previous one by just one base
in the sequence. Theoretically, depending on the size of the gene, it would be possible to carry out a base-by-base examination of
the entire gene on a single gene chip. However, it is important to realize that although a broad approach can be taken with this
technology, it may not be sensitive enough to detect small but functionally important changes in gene expression. This technology
can be applied to preclinial and clinical questions regarding the complex genetic control of stress and anxiety by examining event-
related gene expression changes and also baseline differences in gene sequences (polymorphisms) that might contribute to
differential stress responsivity (165 ). This technique, along with the recent completion of the Human Genome Project, not only
raises the potential to simultaneously profile multiple gene expression systems at once, but also holds great promise for the
identification of completely novel genes in stress regulation and anxiety.

A greater challenge, however, is the elucidation of the functional role of these new genes in processes related to stress and anxiety.
Given this daunting task, methods for more specific and long-term gene targeting will increasingly gain importance in neuroscience
research aimed at uncovering genetic dysregulation relating to psychopathology. One technique that is likely to be helpful is that of
virally mediated gene transfer. In this method, a gene of interest is cloned into viral vector (with most of the viral genome removed
to reduce toxicity and infection) and the modified vector is then infused into a particular brain region using
 P.896

standard stereotaxic procedures (see ref. 166 for review). Depending on the gene insertion and the selection of the promoter to
drive the expression of the gene, it is possible to obtain either an increase or decrease in the amount of protein resulting from the
gene of interest. This method allows for highly selective gene regulation and thus provides a valuable new tool with which to study
the effects of a particular gene product on stress-related functioning. The virally mediated gene transfer approach also has certain
advantages over the current transgenic and antisense oligonucleotide strategies: it can be administered to the animal at any time or
into any brain region, it results in a fairly robust and long-lasting up- or down-regulation of the gene, and it can be used to insert
several genes at once in the same animal. Thus, the viral gene transfer approach completely avoids the issue of developmental
confounds, which are perhaps the most commonly cited problems that plague current transgenic and knockout approaches. A few
groups have already reported successful long-term up-regulation or down-regulation of discrete gene products related to
neuroscience research applications; the behavioral effects associated with this technique appear to be quite robust and do not
appear to be associated with the high level of toxicity that has been reported with antisense oligonucleotides (167 ,168 and 169 ).
Thus, these methods may provide valuable new strategies to more rapidly uncover the neurogenetic basis for stress-related
psychopathology.
On the clinical side, human genomic studies are indicating the existence of polymorphisms in the regulatory region of the gene
encoding CRH (170 ,171 and 172 ). As careful analysis of genes for the other elements of the CRH system progresses, it will be
interesting to see if particular mutations can be associated with stress-related disease states. This method has been applied
successfully to study the role of the serotonin (5-HT) system in anxiety disorders; reports of polymorphisms in the gene encoding the
5-HT transporter have been made in patients with anxiety-related traits (173 ,174 ,175 and 176 ). Clinically, one challenge will be
to develop more discrete definitions of anxiety-related dysfunction that will optimize the screening of patient populations for
abnormalities in genes that are believed to be related to stress and anxiety (177 ). Moreover, gene chip technology applied to
animal analogues of stress endophenotypes may provide a rapid and comprehensive method for identifying novel gene candidates for
stress-related disorders. Using these methods, it may be possible in the near future to have even greater crosstalk between animal
studies and clinical findings. These combined efforts will undoubtedly facilitate our understanding of the interactions between
environmental and genetic contributions to anxiety and stress-related disorders.

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63
Neurobiological Basis of Anxiety Disorders

Dennis S. Charney

Wayne C. Drevets

Dennis S. Charney: Mood and Anxiety Disorder Research Program, National Institute of Mental Health, Bethesda, Maryland.

W. C. Drevets: Section on Mood and Anxiety Disorders Imaging, Molecular Imaging Branch, National Institute of Mental Health,
Bethesda, Maryland.

The 1990s witnessed tremendous progress in the acquisition of knowledge about the molecular, cellular, and anatomic correlates of
fear and anxiety. Advances in neuropharmacology and molecular biology have enabled elucidation of multiple chemical
neurotransmitter systems that play roles in fear and anxiety behavior. The anatomic circuits where these transmitters participate in
mediating and modulating fear and anxiety are also being illuminated through improvements in neurotoxic techniques, which have
enhanced the selectivity of lesion analyses in experimental animals, and by advances in neuroimaging technology, which have
permitted mapping of the neurophysiologic correlates of emotion in humans. The findings of these investigations have informed the
design and interpretation of clinical neuroscience approaches aimed at investigating how dysfunction within these neurochemical
and anatomic systems may result in psychiatric conditions such as panic, posttraumatic stress, and phobic disorders. This chapter
reviews the preclinical and clinical data regarding the neural mechanisms underlying normal and pathologic anxiety and discusses
their implications for guiding development of novel treatments for anxiety disorders.

 NEUROANATOMIC CIRCUITS SUPPORTING FEAR AND ANXIETY

 FUNCTIONAL ANATOMIC CORRELATES OF SPECIFIC ANXIETY DISORDERS
 NEUROCHEMICAL BASIS OF FEAR AND ANXIETY
 CONCLUDING REMARKS

NEUROANATOMIC CIRCUITS SUPPORTING FEAR AND ANXIETY

Part of "63 - Neurobiological Basis of Anxiety Disorders "

Fear and anxiety normally comprise adaptive responses to threat or stress. These emotional-behavioral sets may arise in response to
exteroceptive visual, auditory, olfactory, or somatosensory stimuli or to interoceptive input through the viscera and the endocrine
and autonomic nervous systems. Anxiety may also be produced by cognitive processes mediating the anticipation, interpretation, or
recollection of perceived stressors and threats.

Emotional processing in general can be divided into evaluative, expressive, and experiential components (1 ). Evaluation of the
emotional salience of a stimulus involves appraisal of its valence (e.g., appetitive versus aversive), its relationship with previous
conditioning and behavioral reinforcement experiences, and the context in which it arises (2 ,3 ). Emotional expression conveys the
range of behavioral, endocrine, and autonomic manifestations of the emotional response, whereas emotional experience describes
the subjective feeling accompanying the response. To optimize their capacity for guiding behavior, all these aspects of emotional
processing are modulated by complex neurobiological systems that prevent them from becoming persistent, excessive, inappropriate
to reinforcement contingencies, or otherwise maladaptive.

The emotional processes pertaining to fear and anxiety that have been most extensively studied (largely because of their
amenability to experimental manipulation) have involved pavlovian fear conditioning and fear-potentiated startle (4 ,5 ). These
types of “fear learning” have been shown to comprise experience-dependent forms of neural plasticity in an extended anatomic
network that centers around the critical involvement of the amygdala (1 ,6 ). The structures that function in concert with the
amygdala during fear learning include other mesiotemporal cortical structures, the sensory thalamus and cortices, the orbital and
medial prefrontal cortex (mPFC), the anterior insula, the hypothalamus, and multiple brainstem nuclei (1 ,5 ,7 ). Much of this
network appears to participate in the general process of associating a conditioned stimulus (CS) or operant behavior with an
emotionally salient unconditioned stimulus (US) (see Fig. 63.1 on p. 905) (5 ,8 ,9 ,10 and 11 ).

FIGURE 63.1. The innervation of the

parasympathetic nervous system from limbic

structures is thought to mediate visceral

symptoms associated with anxiety.

Role of the Amygdala in Fear Learning and Expression

The anatomic systems supporting fear learning are organized to permit both rapid responses to simple perceptual
 P.902

elements of potentially threatening stimuli and longerlatency responses to more highly processed information about complex sensory
stimuli and environmental contexts. The former processes depend on monosynaptic projections from the sensory thalamus to the
amygdala, whereas the latter involve projections from sensory association cortices and mesiotemporal cortical structures to the
amygdala (1 ,12 ). These neural networks also respond to visceral input received both directly through the nucleus
paragigantocellularis and the nucleus tractus solitarius (NTS) of the vagus nerve and indirectly through the locus ceruleus (LC), the
anterior insula, and the infralimbic and prelimbic cortices (4 ,7 ,13 ). Finally, neural activity within the amygdala is modulated by
cortisol, norepinephrine (NE), and other neurotransmitters and by mnemonic input related to previous conditioning and
reinforcement experiences conveyed by projections from mesiotemporal and prefrontal cortical structures (14 ,15 ,16 ,17 and 18 ).

The lateral nucleus of the amygdala (LA) comprises the primary sensory interface of the amygdala and receives synaptic input
representing sensory information from the sensory thalamus and cortex (4 ). Single neurons within the LA are responsive to auditory,
visual, and somatic stimuli, thus enabling the LA to serve as a locus of convergence for information about CS and US (19 ). Olfactory
input, in contrast, directly projects to the periamygdaloid cortex from the olfactory bulb through the olfactory tract (20 ). The
olfactory tract also sends projections to the pyriform cortex and the entorhinal cortex, areas with reciprocal connections to the
amygdala (20 ). Although the periamygdaloid cortex neurons project to deeper amygdaloid nuclei, the specific pathways conveying
olfactory information through the amygdala have not been delineated.

In addition to its role in conditioning to explicit sensory stimuli, the amygdala is involved in the development of emotional responses
to environmental context. The projections from the hippocampal formation to the amygdala through the fornix have been
specifically implicated in spatial contextual conditioning (21 ,22 ). Thus, lesioning these projections specifically prevents fear
conditioning to the chamber or the position within a maze in which aversive stimulation previously occurred (22 ,23 ,24 and 25 ).
Other structures that participate in the modulation of contextual fear include the rostral perirhinal cortex and the ventrolateral
PFC/ anterior (agranular) insula. Lesions of the latter regions reduce fear reactivity to contextual stimuli, but they do not affect CS
acquisition or response extinction (26 ). In contrast, lesions placed in the rostral perirhinal cortex after fear conditioning interfere
with the expression of conditioned fear responses elicited by visual and auditory stimuli when these stimuli are presented in
contexts that differ from the initial conditioning context (27 ). Notably, genetic studies in mice identified a quantitative trait locus
for contextual conditioning (28 ,29 ) that was associated with mouse “emotionality” in another study (30 ), although the molecular
genetic, neurochemical, and functional anatomic correlates of this trait have not been established.

The projections from sensory thalamus to the LA are thought to support rapid conditioning to simple visual and auditory features,
presumably accounting for fear responses below the level of conscious awareness (31 ). Thus, lesioning the auditory cortex before
conditioning does not prevent conditioning to single auditory tones. In contrast, projections to the LA from the primary sensory and
sensory association cortices appear to be essential for some aspects of conditioned responding to more complex sensory stimuli
(4 ,32 ). These relationships are modality specific. For example, disruption of the projections from the auditory thalamus and
auditory cortex to the LA specifically prevents acquisition of fear conditioning to auditory stimuli and fear-conditioned responses to
previous auditory CSs (33 ,34 and 35 ).

After sensory input enters the LA, the neural representation of the stimulus is distributed in parallel to various amygdaloid nuclei,
where it may be modulated by diverse functional systems, such as those mediating memories from past experiences or knowledge
about ongoing homeostatic states (36 ). The most extensive extranuclear projections of the LA are composed of reciprocal
projections to the basal and accessory basal nuclei and the central nucleus of the amygdala (CE) (37 ,38 ). Lesions of either the LA
or the CE—but not of other amygdala nuclei—disrupt fear conditioning to a tone CS, a finding suggesting that this direct projection
from LA to CE is sufficient to mediate conditioning to simple sensory features (4 ).

The projections from LA to the basal amygdaloid nuclei also participate in forming long-lasting memory traces for fear conditioning
(2 ,15 ,39 ). Functional inactivation of the lateral and basal amygdaloid nuclei before pavlovian fear conditioning interferes with
acquisition of learning, whereas inactivation immediately after conditioning has no effect on memory consolidation (40 ). The basal
nuclei have widespread intranuclear connections and also project to other amygdalar nuclei, including the CE and the LA (41 ). They
also share extensive, reciprocal projections with the orbital and mPFC (43 ). The basal nuclei are thus anatomically positioned to
modulate neuronal responses in both the LA and the PFC (42 ,43 ).

The plasticity within the amygdala that constitutes memory for conditioning experiences has been shown to involve long-term
potentiation–like associative processes (6 ). Plasticity related to fear learning also occurs in cortical areas, presumably making
possible the establishment of explicit or declarative memories about the fear-related event through interactions with the medial
temporal lobe memory system (44 ,45 ). The influence of the amygdala on cortically based memories has been most clearly
characterized with respect to late plastic components of the auditory cortex neuronal responses to a CS. Single-unit recordings
during fear conditioning indicate that some auditory cortex neurons, which before conditioning did not respond to the CS tone,
develop
 P.903

late-conditioned responses (i.e., 500 to 1,500 milliseconds after CS onset) that anticipate the US and show extinction-resistant
memory storage (46 ). These late-conditioned auditory cortical neuronal responses take more trials to learn and respond more slowly
than LA neurons within trials, and their late development is prevented by amygdala lesions. Thus, whereas rapid conditioning of fear
responses to potentially dangerous stimuli depends on plasticity in the amygdala, learning involving higher cognitive (i.e., mnemonic
and attentional) processing of fear experiences may depend on plasticity involving cortical neurons that is influenced by neural
transmission from the amygdala to the cortex.

Other auditory cortex neurons show an early (less than 50 milliseconds of stimulus onset) plastic component during fear conditioning,
in which the preexisting electrophysiologic responses of auditory cortex neurons to the CS become enhanced by conditioning (46 ).
This short-latency plasticity within the auditory cortex appears to depend on input from the auditory thalamus and is unaffected by
amygdala lesions. Nevertheless, such short-latency responses are extinguished more quickly (during repeated exposure to the CS
alone) in animals with amygdala lesions, a finding implying that the amygdala is involved in preventing extinction of these responses.

In human neuroimaging studies, hemodynamic activity in the amygdala increases during initial exposures to fear-conditioned stimuli
(47 ,48 ). However, during repeated, unreinforced exposures to the same stimulus, single-trial functional magnetic resonance
imaging (fMRI) studies show that this initial elevation of hemodynamic activity attenuates and subsequently decreases to less than
baseline (47 ). This observation suggests that synaptic input into the amygdala may be actively reduced during the extinction
process (49 ), although the level at which this suppression of afferent synaptic activity into (or within) the amygdala is being
suppressed during nonreinforced exposures to the CS has not been established.

Activation of the amygdala during an emotional event enhances the strength of long-term memory for emotional stimuli represented
in other cortical memory circuits as well (16 ,50 ,51 ). These circuits presumably involve the medial temporal lobe memory system,
which has extensive anatomic connections with the amygdala and presumably provides a neuroanatomic substrate for the
interaction between storage and explicit recall of affectively salient memories (16 ). For example, as healthy humans read stories,
the magnitude of physiologic activation in the amygdala correlates both with the negative emotional intensity and with the
subsequent recall performance of the story's content (52 ,53 ). Physiologic activity in the amygdala and the hippocampus measured
during memory encoding reportedly correlates with enhanced episodic memory for pleasant as well as aversive visual stimuli (54 ),
and the amygdala's role in modulating emotional memory may depend more generally on the degree of arousal or the behavioral
salience associated with verbally conveyed information (9 ,16 ).

Human neuroimaging and electrophysiologic and lesion analysis studies have also demonstrated that the amygdala is involved in the
recall of emotional or arousing memories (4 ,53 ,55 ). In humans, bursts of electroencephalographic activity have been recorded in
the amygdala during recollection of specific emotional events (56 ). Moreover, electrical stimulation of the amygdala can evoke
emotional experiences (especially fear or anxiety) (57 ) and the recollection of emotionally charged life events from remote memory
(58 ).

Role of the Amygdala in Organizing Emotional Expression

The amygdaloid output nuclei, especially the CE, receive convergent information from multiple amygdala regions and generate
behavioral responses that are thought to reflect the sum of neuronal activity produced by different amygdaloid nuclei (36 ). The CE
comprises the interface between the amygdala and the motor, autonomic, and neuroendocrine systems involved in expressing fear
behavior (4 ,5 ). The CE projects to nuclei in the hypothalamus, midbrain, and medulla that mediate the neuroendocrine, autonomic,
and behavioral responses associated with fear and anxiety. For example, the amygdala facilitates stress-related corticotropin-
releasing hormone (CRH) release by both intrinsic CRH-containing neurons and bisynaptic (double γ-aminobutyric acid–ergic
[GABAergic]) anatomic projections to the paraventricular nucleus (PVN) of the hypothalamus (59 ). Electrical stimulation of the CE
produces responses similar to those elicited by fear-conditioned stimuli (60 ,61 ), and lesions of the CE prevent the expression of
fear responses of various types (4 ,62 ,63 ). In contrast, lesioning of specific structures efferent to the CE, such as the lateral
hypothalamus or periaqueductal gray (PAG), produces selective deficits in cardiovascular or somatomotor behavioral fear responses,
respectively (1 ,64 ).

The amygdala also sends projections to the thalamus, the nucleus accumbens, the ventromedial caudate, and parts of the ventral
putamen that participate in organizing motor responses to threatening stimuli (65 ). For example, activation of the amygdalar
projections to the ventral striatum arrests goal-directed behavior in experimental animals (66 ), a finding suggesting a possible
neural mechanism for the cessation of motivated or reward-directed behavior during anxiety and panic. The amygdala may also
influence motor behavior by projections through the hypothalamus and PAG (1 ). For example, in experimental animals, stimulation
of the lateral PAG produces defensive behaviors, sympathetic autonomic arousal, and hypoalgesia, whereas stimulation of the
ventrolateral PAG produces social withdrawal and behavioral quiescence, as in response to deep injury or visceral pain (67 ).
 P.904

Other Roles of the Amygdala in Fear Processing

The amygdala also appears to play important roles in mediating innate fear and in processing affective elements of social
interactions (68 ). Amygdala lesions cause rats to lose their fear of cats and monkeys to lose their fear of snakes (reviewed in ref. 4 ).
In monkeys, amygdala lesions reduce aggression as well as fear and cause animals to become more submissive to dominant animals
(69 ). In humans, blood flow increases in the amygdala as subjects view faces expressing fear or sadness (70 ,71 ), and amygdala
lesions impair the ability to recognize fear or sadness in facial expression (55 ,72 ) and fear and anger in spoken language (73 ).

Bed Nucleus of the Stria Terminalis: Hypothesized Role in Anxiety

The hypothalamic and brainstem structures that mediate the expression of emotional behavior can also be activated directly by the
bed nucleus of the stria terminalis (BNST) (5 ). Anxiety-like responses elicited either by exposure to a threatening environment for
several minutes or by intraventricular administration of CRH appear to be specifically mediated by the BNST, rather than the CE (5 ).
This system is thus hypothesized to play a role in mediating anxiety during exposure to less explicit, or less well defined, sensory
cues or to contexts that occur over a longer duration.

Other Temporal Cortical Structures

The perirhinal cortex shares reciprocal anatomic connections with the amygdala (74 ), and it is thought to play a role in conveying
information about complex visual stimuli to the amygdala during presentation of fear-conditioned visual stimuli. Lesions of the
anterior perirhinal cortex, the basolateral nucleus of the amygdala, or the CE can each completely eliminate fear-potentiated
startle during exposure to some conditioned visual stimuli (75 ,76 ). In contrast, complete removal of the entire visual cortex,
insular cortex, mPFC, and posterior perirhinal cortex produces no significant effect on the magnitude of fear-potentiated startle,
and lesions of the frontal cortex only partly attenuate fear-potentiated startle. The perirhinal cortex receives input regarding
conditioned visual stimuli from the lateral geniculate nucleus, and lesions of this structure can also block fear-potentiated startle
(77 ). Finally, the anterior perirhinal cortex receives afferent projections from the visual cortices as well as from the anterior
cingulate cortex (ACC), the infralimbic cortex, and the parietal cortex (74 ), structures implicated in modulating behavioral
responses to fear-conditioned stimuli.

The temporopolar cortex has been implicated in modulating autonomic aspects of emotional responses and in processing emotionally
provocative visual stimuli. Electrical stimulation of various sites within the temporopolar cortex can alter a variety of autonomic
responses (reviewed in ref. 1 ). In humans with simple phobias or posttraumatic stress disorder (PTSD), physiologic activity increases
in the anterior temporopolar cortex during experimentally induced exacerbations of anxiety involving visual exposure to phobic
stimuli or word scripts describing traumatic events, respectively (78 ,79 ). Blood flow also increases in the anterior temporopolar
cortex of healthy humans during exposure to emotionally provocative visual stimuli, whether the stimuli convey “sad,” “disgusting,”
or “happy” content, relative both to conditions involving exposure to emotionally “neutral” visual stimuli and to conditions in which
corresponding emotional states are elicited by recall of autobiographic information (80 ,81 ). Portions of the temporopolar cortex
may thus function as sensory association areas that participate in evaluating the emotional salience of actual or anticipated stimuli
and in modulating autonomic responses to such stimuli.

Neuroendocrine and Autonomic Responses during Fear or Stress

The peripheral hormonal and autonomic responses to threat mediated by the hypothalamic-pituitary-adrenal (HPA) axis and the
sympathetic and parasympathetic autonomic nervous systems also play adaptive roles in responding to threat or stress (5 ).
Stimulation of the lateral nucleus of the hypothalamus by afferent projections from the CE of the amygdala, the BNST, or the
ventral striatum (82 ) activates the sympathetic system and produces increases in blood pressure and heart rate, sweating,
piloerection, and pupillary dilation. Stress stimulates release of CRH from the PVN of the hypothalamus and amygdala. The CRH
secretion from the PVN, in turn, increases peripheral adrenocorticotropic hormone (ACTH) levels, and this stimulates the adrenal
glands to secrete cortisol. The ACC, anterior insula, and posterior orbital cortex send anatomic projections to the hypothalamus that
participate in modulating or inhibiting cardiovascular and endocrine responses to threat and stress (1 ,43 ,83 ).

The vagus and splanchnic nerves constitute the major efferent projections of the parasympathetic nervous system to the viscera.
The vagal nuclei receive afferent projections from the lateral hypothalamus, the PVN, the LC, the amygdala, the infralimbic cortex,
and the prelimbic portion of the ACC (43 ,84 ). The splanchnic nerves receive afferent connections from the LC. The innervation of
the parasympathetic nervous system from these limbic structures is thought to mediate visceral symptoms associated with anxiety,
such as gastrointestinal and genitourinary disturbances (Fig. 63.1 ).

Role of Prefrontal Cortical Structures in Modulating Fear and Anxiety Behavior

Multiple areas of the medial and orbital PFC appear to play roles in modulating anxiety and other emotional behaviors.
P.905
 P.906

These PFC structures are thought to participate in interpreting the higher-order significance of experiential stimuli, in modifying
behavioral responses based on competing reward versus punishment contingencies, and in predicting social outcomes of behavioral
responses to emotional events (8 ,11 ,85 ,86 ). These areas share extensive, reciprocal projections with the amygdala, through
which the amygdala can modulate PFC neuronal activity and the PFC can modulate amygdala-mediated responses to emotionally
salient stimuli (17 ,18 ,42 ,43 ).

Areas within the orbital and mPFC and the anterior insula also participate in modulating peripheral responses to stress, including
heart rate, blood pressure, and glucocorticoid secretion (13 ,17 ,43 ,87 ). The neuronal activities within these areas are, in turn,
modulated by various neurotransmitter systems that are activated in response to stressors and threats. For example, the
noradrenergic, dopaminergic, and serotonergic systems play roles in enhancing vigilance, modulating goal-directed behavior, and
facilitating decision making about probabilities of punishment versus reward by modulating neuronal activity in the PFC (86 ,88 ,89
and 90 ).

Medial Prefrontal Cortex

The mPFC areas implicated in anxiety and fear-related behavior in humans and experimental animals include the infralimbic cortex,
the ACC located ventral (“subgenual”) and anterior (“pregenual”) to the genu of the corpus callosum, and a more dorsal mPFC
region that extends from the rostral ACC (BA 24, 32) toward the frontal pole (91 ). The reciprocal projections between the amygdala
and the mPFC are hypothesized to play critical roles in attenuating fear responses and extinguishing behavioral responses to fear-
conditioned stimuli that are no longer reinforced (17 ,18 ). Lesions of the ACC in rats resulted in enhanced freezing to a fear-
conditioned tone, a finding suggesting that this mPFC region may be involved in fear reduction (17 ). In addition, neurons in the rat
prelimbic cortex (thought to be homologous to subgenual PFC) reduce their spontaneous firing activity in the presence of a
conditioned, aversive tone to an extent that is inversely proportional to the magnitude of fear (42 ). This suppression of prelimbic
cortex neuronal firing activity is inversely correlated with increases in amygdala neuronal activity. Finally, lesions of the infralimbic
cortex specifically interfere with the recall of extinction processes after long delays between the acquisition of extinction learning
and reexposure to the initial CS (18 ). Extinction does not appear to occur by erasing memory traces of the CS-US association, but
rather by new learning through which the behavioral response to the CS is actively inhibited (31 ).

In humans, the pregenual ACC shows areas of elevated hemodynamic activity during a variety of anxiety states elicited in healthy or
anxiety-disordered subjects (reviewed in ref. 49 ). Electrical stimulation of this region elicits fear, panic, or a sense of foreboding in
humans and vocalization in experimental animals (reviewed in ref. 7 ). Nevertheless, physiologic activity also increases in the ACC
during the generation of positive emotions in healthy humans (92 ,93 ) and during depressive episodes in some subtypes of major
depressive disorder (MDD) (94 ,95 ).

The subgenual ACC has been implicated in healthy sadness, MDD, mania, and PTSD (90 ,96 ,97 ). In patients with familial unipolar
and bipolar depression, reductions in cerebral blood flow (CBF) and metabolism were associated with left-lateralized reductions in
the volume of the corresponding cortex (96 ,98 ,99 ). The subgenual PFC activity shows a mood state dependency in which the
metabolism is higher in the depressed than the remitted phase of MDD, consistent with the findings that blood flow increases in this
region in healthy, nondepressed humans during experimentally induced sadness (85 ,100 ,101 ) and in persons with PTSD during
internally generated imagery of past trauma (97 ).

Both the subgenual and the pregenual ACC share reciprocal anatomic connections with areas implicated in emotional behavior such
as the posterior orbital cortex, amygdala, hypothalamus, nucleus accumbens, PAG, ventral tegmental area (VTA), raphe, LC, and
NTS (Fig. 63.1 ) (102 ,103 ). Humans with mPFC lesions that include the pregenual and subgenual ACC show abnormal autonomic
responses to emotionally provocative stimuli, inability to experience emotion related to concepts, and inability to use information
regarding the probability of aversive social consequences versus reward in guiding social behavior (104 ). In rats, bilateral or right-
lateralized lesions of the ventral mPFC composed of infralimbic, prelimbic, and ACC cortices attenuate corticosterone secretion,
sympathetic autonomic responses, and gastric stress disorders during restraint stress or exposure to fear-conditioned stimuli
(17 ,83 ,105 ). In contrast, left-sided lesions of this cortical strip increase sympathetic arousal and corticosterone responses to
restraint stress (105 ). Finally, the ventral ACC contains glucocorticoid receptors that, when stimulated, inhibit stress-induced
corticosterone release in rats (87 ).

Physiologic activity also increases in more dorsal mPFC areas in healthy humans as they perform tasks that elicit emotional
responses or require emotional evaluations (81 ,106 ,107 ). During anxious anticipation of an electrical shock, CBF increases in the
rostral mPFC (vicinity of anterior BA24, BA32, and rostral BA9), and the magnitude of ΔCBF correlates inversely with changes in
anxiety ratings and heart rate (107 ). In rats, lesions of the rostral mPFC result in exaggerated heart rate responses to fear-
conditioned stimuli, and stimulation of these sites attenuates defensive behavior and cardiovascular responses evoked by amygdala
stimulation (83 ). In primates, whereas BA24 and 32 have extensive reciprocal connections with the amygdala through which they
may modulate emotional expression, the BA9 cortex has only sparse projections to the amygdala. Nevertheless, all three areas send
extensive efferent projections to the PAG and the hypothalamus through which cardiovascular
 P.907

responses associated with emotional behavior can be modulated (43 ,108 ).

In the depressed phase of MDD and bipolar disorder, metabolic activity is abnormal in the dorsomedial and dorsal anterolateral PFC
(in the vicinity of rostral BA9) (91 ,109 ). Postmortem studies of these regions have shown abnormal reductions in the size of glia and
neurons in MDD (110 ). Given the preclinical and neuroimaging evidence presented earlier, indicating that this area may modulate
anxiety, it may be hypothesized that dysfunction of this mPFC area contributes to the development of anxiety symptoms in mood
disorders.

Orbital and Anterior Insular Cortex

Other areas of the PFC that are implicated in studies of fear or anxiety in human and nonhuman primates are the posterior and
lateral orbital cortex, the anterior (agranular) insula, and the ventrolateral PFC (1 ,43 ). Physiologic activity increases in these areas
during experimentally induced anxiety states in healthy subjects and in subjects with obsessive-compulsive disorder (OCD), simple
phobia, and panic disorder (PD) (49 ,111 ). (See Chapter 65 ) The baseline metabolic activity is also abnormally elevated in these
regions in unmedicated study subjects with primary MDD (91 ) and OCD (112 ) scanned while resting with eyes closed. The elevated
activity in these areas in both MDD and OCD appears state dependent, and effective antidepressant or antiobsessional treatment
results in decreases in CBF and metabolism in the medicated-improved relative to the unmedicated-symptomatic phase (112 ,113
and 114 ).

A complex relationship exists between anxiety-depressive symptoms and physiologic activity in the orbital cortex and the
ventrolateral PFC. In MDD, whereas CBF and metabolism increase in these areas in the depressed relative to the remitted phase, the
magnitude of these measures correlates inversely with ratings of depressive ideation and severity (115 ,116 ). Similarly, posterior
orbital cortex flow increases in OCD and animal phobic subjects during exposure to phobic stimuli and in healthy subjects during
induced sadness, but this change in CBF correlates inversely with changes in obsessive thinking, anxiety, and sadness, respectively
(114 ,117 ,118 ).

These data appear consistent with electrophysiologic and lesion analysis data showing that the orbital cortex participates in
modulating behavioral and visceral responses associated with fearful, defensive, and reward-directed behavior as reinforcement
contingencies change. Nearly one-half of the orbital cortex pyramidal neurons alter their firing rates during the delay period
between stimulus and response, and this firing activity relates to the presence or absence of reinforcement (11 ). These cells are
thought to play roles in extinguishing unreinforced responses to aversive or appetitive stimuli (7 ,11 ,66 ). The posterior and lateral
orbital cortex and the amygdala send projections to each other and to overlapping portions of the striatum, hypothalamus, and PAG
through which these structures modulate each other's neural transmission (Fig. 63.1 ) (42 ,66 ,108 ,119 ). For example, the defensive
behaviors and cardiovascular responses evoked by electrical stimulation of the amygdala are attenuated or ablated by concomitant
stimulation of orbital sites, which, when stimulated alone, exert no autonomic effects (120 ).

Humans with orbital cortex lesions show impaired performance on tasks requiring application of information related to punishment
or reward, perseverate in behavioral strategies that are unreinforced, and exhibit difficulty in shifting intellectual strategies in
response to changing task demands (11 ,121 ). Likewise, monkeys with surgical lesions of the lateral orbital cortex and ventrolateral
PFC demonstrate “perseverative interference,” characterized by difficulty in learning to withhold prepotent responses to
nonreinforced stimuli as reinforcement contingencies change (122 ). Activation of the orbital cortex during anxiety or obsessional
states may thus reflect endogenous attempts to attenuate emotional expression or to interrupt unreinforced aversive thought and
emotion (91 ). Conversely, dysfunction of the orbital cortex may contribute to pathologic anxiety and obsessional states by impairing
the ability to inhibit nonreinforced or maladaptive emotional, cognitive, and behavioral responses to social interactions and sensory
or visceral stimuli.

Posterior Cingulate Cortex

Many functional imaging studies report that exposure to aversive stimuli of various types increases physiologic activity in the
retrosplenial cortex and other portions of the posterior cingulate gyrus (reviewed in ref. 123 ). Posterior cingulate cortical flow and
metabolism have also been found abnormally elevated in some studies of depressed subjects with MDD (reviewed in ref. 91 ). In
contrast, Mayberg et al. reported that script-driven sadness resulted in decreased posterior cingulate activity in healthy subjects,
and flow was decreased in depressed relative to remitted subjects with MDD, findings raising the possibility that this large region is
functionally heterogenous with respect to emotional behavior (101 ). The posterior cingulate cortex appears to serve as a sensory
association cortex and may participate in processing the affective salience of sensory stimuli. The posterior cingulate cortex sends a
major anatomic projection to the ACC, through which it may relay such information into the limbic circuitry (124 ).

FUNCTIONAL ANATOMIC CORRELATES OF SPECIFIC ANXIETY

DISORDERS
Part of "63 - Neurobiological Basis of Anxiety Disorders "

Neuroimaging studies have assessed neurophysiologic abnormalities in anxiety-disordered samples in the baseline,
 P.908

“resting” condition and during symptom provocation. These data converge with those obtained from studies of healthy subjects and
of experimental animals to implicate the limbic, paralimbic, and sensory association areas reviewed earlier in the functional
anatomy of emotional behavior. Nevertheless, the results of most of the imaging studies reviewed herein await replication, and the
data they provide do not clearly establish whether differences between anxiety-disordered and control subjects reflect physiologic
correlates of anxiety symptoms or traitlike biological abnormalities underlying the vulnerability to anxiety syndromes.

Panic Disorder
The baseline state in PD is characterized by mild to moderate levels of chronic anxiety (termed anticipatory anxiety). In this state,
abnormalities of CBF and glucose metabolism have been reported in the vicinity of the hippocampus and parahippocampal gyrus.
Reiman et al. initially reported an abnormal resting asymmetry (left less than right) of blood flow and oxygen metabolism in a region
of interest placed over the parahippocampal gyrus (125 ). Nordahl et al. similarly found that glucose metabolism measured over the
hippocampus-parahippocampal gyrus was asymmetric and concluded that this abnormality reflected an abnormal metabolic
elevation on the right side (126 ). Bisaga et al. also found abnormal metabolism in this vicinity, but with the opposite laterality (i.e.,
elevated metabolism in the left hippocampal-parahippocampal area) in lactate-sensitive PD study subjects relative to healthy
controls (127 ). In contrast, De Cristofaro et al. reported that resting perfusion, measured using single photon emission computed
tomography (SPECT) and [99mTc]hexamethylpropyleneamineoxime (HMPAO), was abnormally decreased in the hippocampus,
bilaterally, in lactate-sensitive PD study subjects relative to controls (128 ).

Each of these studies employed region-of-interest based approaches that were incapable of localizing the center of mass of the
abnormality in this region. Reanalysis of some of these data using a voxel-by-voxel approach suggested that the abnormal
radioactivity in the vicinity of the mesiotemporal cortex may actually reflect elevated metabolism in the adjacent midbrain (111 ).
This midbrain region, which may reflect the lateral PAG, has been implicated in lactate-induced panic (129 ), other acute anxiety
states (130 ), and animal models of panic attacks (67 ).

Study subjects with PD have also been imaged during panic elicited using a variety of chemical challenges. Panic attacks induced by
intravenous sodium lactate infusion were associated with regional CBF increases in the anterior insula, the anteromedial cerebellum,
and the midbrain (129 ); areas of increased CBF may also exist in the temporal polar cortex, but these findings were confounded by
corresponding increases in the adjacent facial muscles during severe anxiety (115 ). Blood flow also increased in these regions in
animal phobic subjects during exposure to phobic stimuli and in healthy subjects during the threat of a painful electrical shock,
findings suggesting that these CBF changes reflect the neurophysiologic correlates of fear processing in general (111 ,130 ).
Consistent with this hypothesis, anxiety attacks induced in healthy humans using cholecystokinin tetrapeptide (CCK-4) were also
associated with CBF increases in the insular-amygdala region and the anteromedial cerebellum (131 ).

Indirect evidence suggests that the neurophysiologic responses in the PFC during panicogen challenge may differ between PD
subjects and healthy controls. For example, panic attacks induced using CCK-4 were associated with CBF increases in the ACC in
healthy humans (131 ), but flow did not significantly change in the ACC in subjects with PD during lactate-induced panic (129 ). The
ACC was also a region where flow significantly increased in healthy subjects but not in subjects with PD during fenfluramine
challenge in a study in which fenfluramine induced panic attacks in 56% of subjects with PD but in only 11% of control subjects (132 ).
Finally, Cameron et al. found that normalized medial frontal CBF increased in healthy controls after yohimbine administration (i.e.,
after normalizing to remove effects on whole brain CBF) (133 ), whereas Woods et al. found that the relative prefrontal cortical flow
was decreased in PD relative to control subjects following yohimbine challenge (134 ).

Structural MRI studies have begun to investigate whether morphometric or morphologic abnormalities may exist in PD. Ontiveros et
al. reported qualitative abnormalities of temporal lobe structure in PD (135 ), although these findings have not been replicated.
Vythilingam et al. reported that hippocampal volume did not differ between PD and healthy control subjects (136 ).

Phobias
In simple animal phobias, phobic anxiety was imaged by acquiring blood flow scans during exposures to the feared animal. During
the initial fearful scans, flow increased in the lateral orbital-anterior insular cortex, bilaterally, the pregenual ACC, and the
anteromedial cerebellum (78 ,111 ), areas where CBF also increases in other anxiety states (see earlier). During the development of
habituation to phobic stimuli, the magnitude of the hemodynamic responses to the phobic stimulus diminished in the anterior insula
and the medial cerebellum, but it increased in the left posterior orbital cortex in an area where flow had not changed during
exposures that preceded habituation (117 ). The magnitude of the CBF increase in this latter region was inversely correlated with
the corresponding changes in heart rate and anxiety
 P.909

ratings. As discussed earlier, the posterior orbital cortex was a site where CBF increased in subjects with OCD during exposure to
phobic stimuli, with the increase in flow inversely correlated with obsessional ratings (114 ).

In social anxiety disorder, an aversive conditioning paradigm (in which the US was an aversive odor and the CS was a picture of a
human face) showed that hemodynamic activity decreased in the amygdala and the hippocampus during presentations of the CS in
healthy controls, but it increased in social phobic subjects (137 ). Interpretation of these data was confounded by the problem that
both human faces and aversively CSs normally activate the amygdala, so it remained unclear which of the stimuli produced abnormal
responses in social phobia. Nevertheless, these data appear conceptually intriguing, given the role of hippocampal-amygdalar
projections in mediating contextual fear and the possibility that deficits in the transmission of information regarding context may be
involved in the pathogenesis of phobias (21 ).

Posttraumatic Stress Disorder

PTSD is hypothesized to involve the emotional-learning circuitry associated with the amygdala, because the traumatic event
constitutes a fear-conditioning experience, and subsequent exposure to sensory, contextual, or mnemonic stimuli that recall aspects
of the event elicits psychological distress and sympathetic arousal. Potentially consistent with this expectation, some studies
demonstrated activation of the amygdala as patients with PTSD listened to auditory scripts describing the traumatic event (79 ) or to
combat sounds (in combat-related PTSD) (138 ) or generated imagery related to the traumatic event without sensory cues (139 ).
However, other studies found no significant changes in amygdala CBF as patients with PTSD listened to scripts describing the
traumatic event or viewed trauma-related pictures, and studies comparing CBF responses with trauma-related stimuli have not
shown significant differences in the amygdala between patients with PTSD and trauma-matched, non-PTSD control subjects
(97 ,139 ,140 and 141 ). The extent to which these negative findings reflect limitations in statistical sensitivity or in positron
emission tomography (PET) temporal resolution must be addressed in provocation studies involving larger subject samples and
employing fMRI instead of PET. In this regard, it is noteworthy that a preliminary fMRI study found exaggerated hemodynamic
changes in the amygdala in patients with PTSD relative to trauma-matched, non-PTSD control subjects during exposure to pictures of
fearful faces presented using a backward-masking technique (142 ). If replicated, this finding may suggest that the emotional
dysregulation associated with PTSD may involve amygdalar responses to emotional stimuli of various types.

Other limbic and paralimbic cortical structures have also been implicated in provocation studies of PTSD. In both patients with PTSD
and trauma-matched, non-PTSD control subjects, CBF increases in the posterior orbital cortex, anterior insula, and temporopolar
cortex during exposure to trauma-related stimuli, but these changes have generally not differentiated PTSD and control samples
(79 ,139 ,140 ). In contrast, the pattern of CBF changes elicited in the mPFC by traumatic stimuli may differ between PTSD and
control subjects. During exposure to trauma-related sensory stimuli, flow decreased in the left (97 ,140 ) but increased in the right
pregenual ACC in PTSD (79 ,138 ), a finding potentially consistent with the evidence reviewed earlier that the role of the mPFC in
emotional behavior is lateralized (105 ). However, CBF in the right pregenual ACC increased significantly more in non-PTSD, trauma-
matched control subjects than in patients with PTSD (139 ). Moreover, in the infralimbic cortex, CBF decreased in patients with
combat-related PTSD but increased in combat-matched, non-PTSD
 P.910

control subjects during exposure to combat-related visual and auditory stimuli (141 ).

Given evidence that the ACC and the infralimbic cortex play roles in extinguishing fear-conditioned responses (17 ,18 ), the
observation that patients with PTSD fail to activate these structures to a similar extent as traumatized, non-PTSD control subjects
during exposure to traumatic cues (139 ,141 ) suggests that neural processes mediating extinction to trauma-related stimuli may be
impaired in PTSD. Compatible with this hypothesis, PTSD samples have been shown to acquire de novo conditioned responses more
readily and to extinguish them more slowly than control samples (143 ,144 ). Such an impairment could conceivably be related to
the vulnerability to developing PTSD, because PTSD occurs in only 5% to 20% of individuals exposed to similar traumatic events.

Structural MRI studies of PTSD have identified subtle reductions in the volume of the hippocampus in PTSD samples relative to
healthy or traumatized, non-PTSD control samples (145 ,146 ,147 and 148 ). Although limitations existed in these studies in the
matching of alcohol use or abuse between PTSD and control samples, the reductions in hippocampal volume did not correlate with
the extent of alcohol exposure in the PTSD samples, and no volumetric differences were found between PTSD and control samples in
the amygdala, entire temporal lobe, caudate, whole brain, or lateral ventricles. Although the magnitude of the reduction in
hippocampal volume only ranged from 5% to 12% in the PTSD samples relative to trauma-matched controls, these abnormalities were
associated with short-term memory deficits in some studies (145 ,147 ). It remains unclear whether the difference in hippocampal
volume may reflect a result of the chronic stress associated with PTSD (e.g., from sustained exposure to elevated glucocorticoid
concentrations) or a biological antecedent that may confer risk for developing PTSD (149 ,150 ).

Obsessive-Compulsive Disorder
The anatomic circuits involved in the production of obsessions and compulsions have been elucidated by converging evidence from
functional neuroimaging studies of OCD, analysis of lesions resulting in obsessive-compulsive symptoms, and observations regarding
the neurosurgical interventions that ameliorate OCD (113 ,114 ,151 ). PET studies of OCD have shown that “resting” CBF and glucose
metabolism are abnormally increased in the orbital cortex and the caudate nucleus bilaterally in primary OCD (reviewed in ref. 112 ).
With symptom provocation by exposure to relevant phobic stimuli (e.g., skin contact with “contaminated” objects for patients with
OCD who have germ phobias), flow increased further in the orbital cortex, ACC, caudate, putamen, and thalamus (114 ). During
effective pharmacotherapy, orbital metabolism decreased toward normal, and both drug treatment and behavioral therapy were
associated with a reduction of caudate metabolism (112 ). The baseline areas of hypermetabolism in the orbital cortex and the
caudate may thus reflect physiologic concomitants of obsessive thoughts or chronic anxiety, and, conversely, the reduction in
caudate metabolism associated with effective (but not ineffective) treatment may reflect a physiologic correlate of symptom
resolution rather than a primary mechanism of treatment.

Based on the evidence reviewed earlier from electrophysiologic and lesion analysis studies indicating that the orbital cortex
participates in the correction of behavioral responses that become inappropriate as reinforcement contingencies change, posterior
orbital areas may be specifically activated as an endogenous attempt to interrupt patterns of nonreinforced thought and behavior in
OCD (11 ,91 ). Compatible with this hypothesis, the posterior orbital cortex CBF increases during symptom provocation in OCD, but
the magnitude of this increase correlates inversely with the corresponding rise in obsession ratings (r = -0.83) (114 ). In contrast,
flow also increases in an area of the right anterior orbital cortex implicated in a variety of types of mnemonic processing, and the
change in CBF in this region correlates positively with changes in obsession ratings (114 ,152 ).

The neurologic conditions associated with the development of secondary obsessions and compulsions also provide evidence that
dysfunction within circuits formed by the basal ganglia and the PFC may be related to the pathogenesis of OCD. Such conditions
involve lesions of the globus pallidus and the adjacent putamen: Sydenham chorea (a poststreptococcal autoimmune disorder
associated with neuronal atrophy in the caudate and putamen), Tourette syndrome (an idiopathic syndrome characterized by
motoric and phonic tics that may have a genetic relationship with OCD), chronic motor tic disorder, and lesions of the ventromedial
PFC (151 ,152 ,153 and 154 ). Several of these conditions are associated with complex motor tics (repetitive, coordinated,
involuntary movements occurring in patterned sequences in a spontaneous and transient manner). It is conceivable that complex tics
and obsessive thoughts may reflect homologous, aberrant neural processes manifested within the motor and cognitive-behavioral
domains, respectively, because of their origination in distinct portions of the cortical-striatal-pallidal-thalamic circuitry (113 ,155 ).

In contrast to the regional metabolic abnormalities found in primary OCD, imaging studies of obsessive-compulsive syndromes arising
in the setting of Tourette syndrome or basal ganglia lesions have not found elevated blood flow and metabolism in the caudate and
in some cases have found reduced metabolism in the orbital cortex in such subjects relative to controls (111 ,151 ). The differences
in the functional anatomic correlates of primary versus secondary OCD are consistent with a neural model in which dysfunction
arising at various points within the ventral prefrontal cortical-striatal-pallidal-thalamic circuitry may result in pathologic obsessions
and compulsions. This circuitry appears
 P.911

to be generally involved in organizing internally guided behavior toward a reward, switching of response strategies, habit formation,
and stereotypic behavior (66 ,155 ).

These circuits have also been implicated in the pathophysiology of MDD, another illness in which intrusive, distressing thoughts recur
to an extent that the ability to switch to goal-oriented, rewarding cognitive-behavioral sets is impaired (91 ). Although MDD and OCD
appear distinct in their course, prognosis, genetics, and neurochemical concomitants, substantial comorbidity exists across these
syndromes. Major depressive episodes occur in about one-half of patients with OCD, pathologic obsessions can arise in primary MDD,
and the pharmacologic interventions that ameliorate OCD can also effectively treat MDD. Moreover, the neurosurgical procedures
that are effective at reducing both obsessive-compulsive and depressive symptoms in intractable cases of OCD and MDD interrupt
white matter tracts carrying neural projections between the frontal lobe, the basal ganglia, and the thalamus (155 ). The clinical
comorbidity across these two disorders may thus reflect involvement of an overlapping neural circuitry by otherwise distinct
pathophysiologic processes.

NEUROCHEMICAL BASIS OF FEAR AND ANXIETY

Part of "63 - Neurobiological Basis of Anxiety Disorders "

The neuroanatomic circuits that support fear and anxiety behavior are modulated by a variety of chemical neurotransmitter systems.
These include the peptidergic neurotransmitters, CRH, neuropeptide Y (NPY), and substance P, the monoaminergic transmitters, NE,
serotonin (5-hydroxytryptamine or 5-HT), and dopamine (DA), and the amino acid transmitters, GABA and glutamate. The
neurotransmitter systems that have been best studied in association with responses to stress or threat involve the HPA axis and the
central noradrenergic system. These neurochemical systems subserve important adaptive functions in preparing the organism for
responding to threat or stress, by increasing vigilance, modulating memory, mobilizing energy stores, and elevating cardiovascular
function. Nevertheless, these biological responses to threat and stress can become maladaptive if they are chronically or
inappropriately activated. Additional neurochemical systems that play important roles in modulating stress responses and emotional
behavior include the central GABAergic, serotonergic, dopaminergic, opiate, and NPY systems. The preclinical and clinical literature
regarding these neurochemical concomitants of stress and fear and their potential relevance to the pathophysiology of anxiety
disorders are reviewed in the following sections.

Role of the Central Noradrenergic System in Fear and Anxiety

Exposure to stressful stimuli of various types increases central noradrenergic function. Thus, exposure to fear-conditioned stimuli,
immobilization stress, foot shock, or tail pinch increases NE turnover in the LC, the hypothalamus, the hippocampus, the amygdala,
and the cerebral cortex (156 ). The firing activity of LC neurons also increases during exposure to fear-conditioned stimuli and other
stressors or threats (157 ,158 and 159 ). For example, the firing activity of NE neurons in the cat LC increases two- to threefold
during confrontation with a dog or an aggressive cat, but it remains unchanged during exposure to other novel stimuli or to
nonaggressive cats (160 ). However, repeated exposure to severe stressors from which the animal cannot escape results in the
behavioral pattern termed learned helplessness, which is associated with depletion of NE, possibly reflecting a point at which NE
synthesis cannot keep pace with NE release (161 ,162 ).

Acquisition of fear-conditioned responses requires an intact central noradrenergic system, a finding suggesting that NE release plays
a critical role in fear learning (157 ,163 ,164 ). For at least some types of emotional learning, memory consolidation depends on
noradrenergic stimulation of β- and α1-adrenoreceptors in the basolateral nucleus of the amygdala (15 ). The activation of NE
release in such models may, in turn, depend on effects of stress hormones on noradrenergic neurons (15 ).

The responsiveness of LC neurons to future novel stressors can be enhanced by chronic exposure to some stressful experiences. In
rats, the amount of NE synthesized and released in the hippocampus and the mPFC in response to a novel stressor or to local
depolarization is increased after repeated exposure to chronic cold stress (165 ,166 and 167 ). This effect may result from a stress-
mediated alteration in the sensitivity of presynaptic α2-adrenoreceptors, which inhibit NE synthesis and release. In the native state,
administration of the α2-adrenoreceptor antagonists, idazoxan or yohimbine, increases the electrophysiologic response of LC neurons
to stressful stimuli (without altering their basal firing rates) and increases NE release and synthesis, whereas administration of the
α2-adrenoreceptor agonist, clonidine, decreases NE release and synthesis (167 ,168 ). In chronically cold-stressed rats, idazoxan
administration produces a greater increase in NE release and synthesis, and clonidine administration produces a blunted attenuation
of NE release and synthesis relative to naive rats (167 ). Consistent with these observations, Torda et al. found that cold
immobilization stress decreases the α2-adrenoreceptor density in the hippocampus and the amygdala (169 ).

The effect of chronic stress on noradrenergic responses to subsequent, novel stressors may constitute a form of “behavioral
sensitization,” a process by which single or repeated exposures to aversive stimuli or pharmacologic agents can
 P.912

increase the behavioral sensitivity to subsequent stressors (reviewed in ref. 170 ). Such phenomena are hypothesized to account for
clinical observations that patients with anxiety disorders report experiencing exaggerated sensitivity to psychosocial stress. Neural
models for the pathogenesis of anxiety disorders built on sensitization phenomena thus hold that repeated exposure to traumatic
stress comprises a risk factor for the subsequent development of anxiety disorders, particularly PTSD.

Noradrenergic Function in Anxiety Disorders

The recurrent symptoms of anxiety disorders, such as panic attacks, insomnia, exaggerated startle, and chronic sympathetic
autonomic arousal, may conceivably reflect elevated noradrenergic function (171 ,172 and 173 ). Patients with PTSD and PD show
evidence of heightened peripheral sympathetic nervous system arousal that, because of the correlation between peripheral
sympathetic activity and central noradrenergic function, is compatible with the hypothesis of increased central NE activity in these
disorders (174 ,175 ). Moreover, patients with PD, PTSD, and phobic disorders report that their hyperarousal symptoms and intrusive
memories are attenuated by alcohol, benzodiazepines (BZDs), and opiates, agents known to decrease LC neuronal firing activity, but
are exacerbated by cocaine, which increases LC neuronal firing. The risk of abuse of these substances appears increased in patients
with anxiety disorders, a finding raising the possibility that such patients are “self-medicating” anxiety symptoms with these agents.
It remains unclear, however, whether alterations in noradrenergic function play a primary, etiologic role in the pathogenesis of
anxiety disorders, or instead reflect secondary, compensatory changes in response to disorders in other systems.
PD has been specifically associated with elevations of α2-adrenoreceptor sensitivity and nocturnal urinary NE excretion (176 ),
although β-adrenoreceptor function, baseline heart rate and blood pressure, and other measures reflecting central NE secretion
have not been consistently altered in PD (see Table 63.1 ) (177 ). Altered α2-adrenoreceptor sensitivity is evidenced by findings that
administration of the α2-adrenoreceptor agonist, clonidine, results in greater hypotension and larger reductions in plasma 3-
methoxy-4-hydroxyphenylethylene glycol (MHPG) in PD relative to control subjects (178 ,179 ,180 and 181 ). In addition,
administration of the α2-adrenoreceptor antagonist, yohimbine (which stimulates NE release by antagonizing presynaptic α2-
adrenoreceptors) produces exaggerated anxiogenic and cardiovascular responses and enhanced plasma MHPG and cortisol increases
in PD relative to control subjects (133 ,172 ,173 ,182 ,183 ,184 ,185 and 186 ). Finally, yohimbine administration resulted in
reduced relative frontal cortex flow in patients with PD that did not occur in control subjects, as measured with SPECT and
[99mTc]HMPAO (134 ); it remains unclear, however, whether this difference reflected a differential physiologic sensitivity to
yohimbine or an effect of greater anxiety in the patients with PD, because all the patients with PD but only one control subject
developed increased anxiety in response to yohimbine. The sensitivity of α2-adrenoreceptors also appears increased in PTSD.
Patients with combat-related PTSD show increased behavioral, chemical, and cardiovascular responses to yohimbine, relative to
healthy controls (187 ,188 and 189 ).

TABLE 63.1. EVIDENCE OF ALTERED

CATECHOLAMINERGIC FUNCTION IN
ANXIETY DISORDERS

Considerable evidence also indicates that noradrenergic function is abnormal in PTSD (see Table 63.1 ). Women with PTSD secondary
to childhood sexual abuse showed elevated 24-hour urinary excretion of catecholamines and cortisol (190 ). In addition, men—but
not women—with PTSD resulting from a motor vehicle accident exhibited elevated urinary levels of epinephrine, NE, and cortisol 1
month after the accident and still had higher epinephrine levels 5 months later (191 ). Similarly, maltreated children with PTSD
excreted greater amounts of urinary DA, NE, and cortisol over 24 hours than controls, with the urinary catecholamine and cortisol
output positively correlated with the duration of PTSD trauma and the severity of PTSD symptoms (192 ). Exposure to traumatic
reminders (e.g., combat films or sounds) produced greater increases in plasma, epinephrine, NE, and cortisol in patients with PTSD
than in control subjects (191 ,193 ,194 ), although baseline concentrations of catecholamines are not consistently altered in combat-
related PTSD (188 ,189 ). Geracioti et al. found that cerebrospinal fluid (CSF) NE concentrations are abnormally elevated in PTSD
(195 ). Finally, platelet α2-adrenoreceptor density (196 ), platelet basal adenosine, isoproterenol, forskolin-stimulated cyclic
adenosine monophosphate signal transduction (197 ), and basal platelet monoamine oxidase activity (198 ) were decreased in PTSD,
findings hypothesized to reflect compensatory responses to chronically elevated NE release.
In study subjects with specific phobias, plasma NE and epinephrine concentrations, heart rate, blood pressure, and subjective
anxiety ratings increase in response to exposure to phobic stimuli (199 ). Subjects with social anxiety disorder show greater
increases in plasma NE during orthostatic challenge than healthy subjects or those with PD (200 ).The growth hormone response to
intravenous clonidine (a marker of central α2-adrenoreceptor function) is blunted in social anxiety disorder (201 ), although the
density of lymphocyte β-adrenoreceptors has not differed between social anxiety–disordered and control samples (202 ) (Table
63.1 ).
Finally, Gerra et al. reported that, plasma NE concentrations increased to a greater extent in male peripubertal patients with
generalized anxiety disorder than in controls in response to a psychological stress test (203 ). However, the
 P.913

pretest baseline NE concentrations did not differ between the anxious and control subjects.

Hypothalamic-Pituitary-Adrenal Axis and Corticotropin-Releasing Hormone

Exposure to acute stress of various types results in release of CRH, ACTH, and cortisol. This HPA-axis activation during acute stress
can produce a transient elevation of the plasma cortisol concentration and partial resistance to feedback inhibition of cortisol
release that persists during and shortly after the duration of the stressful stimulus. This phenomenon may involve a rapid down-
regulation of glucocorticoid receptors, because elevated glucocorticoid levels such as those elicited by acute stress decrease the
number of hippocampal glucocorticoid receptors, with a resulting increase in corticosterone secretion and feedback resistance
(204 ). After stress termination, as glucocorticoid levels decrease (presumably because the limbic drive on CRH release diminishes),
glucocorticoid-receptor density increases, and feedback sensitivity normalizes (204 ).

During some types of chronic stress, adaptive changes in ACTH and corticosterone secretion occur such that the plasma ACTH and
corticosterone concentrations achieved are lower than those seen in response to acute stress (205 ). In contrast, other types of
chronic stress are associated with enhanced corticosterone secretion in rats (206 ). Moreover, Dallman and Jones showed that the
experience of prior stress can result in augmented corticosterone responses to subsequent stress exposure (207 ). The factors that
determine whether adaptation or sensitization of glucocorticoid activity occurs after chronic stress remain poorly understood.

Some stressors experienced within critical periods of neurodevelopment exert long-term effects on HPA-axis function. In rats
exposed to either severe prenatal (in utero) stress or early maternal deprivation stress (208 ,209 ), the plasma concentrations of
corticosterone achieved in response to subsequent stressors are increased, and this tendency to show exaggerated glucocorticoid
responses to stress persists into adulthood. Early postnatal adverse experiences such as maternal separation are associated with
long-lasting alterations in the basal concentrations of hypothalamic CRH mRNA, hippocampal glucocorticoid-receptor mRNA, median
eminence CRH, and in the magnitude of stress-induced CRH, corticosterone, and ACTH release (210 ,211 and 212 ). In nonhuman
primates, adverse early experiences induced by variable maternal foraging requirements reportedly result in alterations in juvenile
and adult social behavior, such that animals are more timid, less socially interactive, and more subordinate (213 ). Adult monkeys
who were raised in such a maternal environment are also hyperresponsive to yohimbine and have elevated CRH concentrations and
decreased cortisol levels in the CSF, findings that parallel those in humans with PTSD (213 ).

Conversely, positive early-life experiences during critical developmental periods may have beneficial long-term consequences on the
ability to mount adaptive responses to stress or threat. For example, daily postnatal handling of rat pups by human experimenters
within the first few weeks of life has been shown to produce persistent (throughout life) increases in the density of type II
glucocorticoid receptors. This increase was associated with enhanced feedback sensitivity to glucocorticoid exposure and reduced
glucocorticoid-mediated hippocampal damage in later life (214 ,215 ). These effects are hypothesized to comprise a type of “stress
inoculation” induced by the mothers' repeated licking of the pups after they were handled by humans. Taken together with the data
reviewed in the preceding paragraph, these data indicate that a high degree of plasticity exists in stress-responsive neural systems
during the prenatal and early postnatal periods that “programs” future biological responses to stressful stimuli (210 ).

Regional differences in the regulation of CRH function by glucocorticoid-receptor stimulation and stress may play major roles in the
mediation of fear and anxiety (216 ). The feedback inhibition of CRH function by glucocorticoids (to suppress HPA-axis activity)
occurs at the level of the PVN of the hypothalamus, where systemically administered glucocorticoids reduce CRH expression, and the
anterior pituitary, where glucocorticoids decrease CRH receptor expression (217 ,218 ,219 and 220 ). The regulation of CRH
receptor mRNA expression shows a regional specificity that becomes altered when stress occurs concomitantly with elevated
glucocorticoid concentrations. After both short-term and long-term corticosterone (CORT) administration, the CRH receptor RNA
expression decreases in the PVN and the anterior pituitary (219 ). However, after acute or repeated immobilization stress sufficient
to produce a large increase in plasma CORT levels, the CRH mRNA expression decreases in the anterior pituitary, but increases in the
PVN. In contrast, neither CORT administration nor restraint stress alters the CRH receptor expression in the CE of the amygdala or
the BNST. Furthermore, CRH secretion is not constrained by glucocorticoids in the CE or the lateral BNST, and CRH mRNA expression
increases in these areas during systemic CORT administration (217 ,218 ,220 ). It is thus conceivable that the positive feedback of
glucocorticoids on extrahypothalamic CRH function in the amygdala or the BNST may contribute to the production of anxiety
symptoms (216 ,221 ).

Another level through which the CRH-glucocorticoid system maintains homeostasis and provides mechanisms for modulating
mechanism over stress or anxiety responses involves functional differences between CRH-receptor subtypes. The CRH1 and CRH2
receptors appear to play reciprocal roles in mediating stress responsiveness and anxiety-like behaviors (221 ). Mice genetically
deficient in CRH1-receptor expression exhibit diminished anxiety and
 P.914

stress responses to threat or stress (222 ,223 ). In contrast, mice deficient in CRH2 receptors display heightened anxiety in response
to stress (224 ,225 ). The affinity of CRH is higher for CRH1 than CRH2 receptors, a finding consistent with evidence that CRH elicits
anxiogenic effects either when exogenously administered to native animals or when endogenously released in mice genetically
altered to overexpress CRH (221 ). Also consistent with the hypothesis that CRH1-receptor stimulation facilitates anxiety responses,
oral administration of the CRH1-receptor antagonist, antalarmin, inhibits the behavioral, sympathetic autonomic, and
neuroendocrine responses (i.e., attenuating increases in the CSF CRH concentration and in the pituitary-adrenal and adrenal-
medullary activity) to acute social stress in monkeys (226 ).

Regional differences in the anatomic distribution of CRH1 and CRH2 receptors likely play a role in balancing facilitatory versus
modulatory effects of CRH-receptor stimulation on stress responses. In monkeys, the CRH1-receptor density is high in most
amygdaloid nuclei, the cingulate cortex, the PFC, the insular cortex, the parietal cortex, the dentate gyrus, and the entorhinal
cortex, and it is moderate in the CE and the LC. The CRH2-receptor density is high in the cingulate cortex, the mPFC, the CE, the
CA-1 region of the hippocampus, and the PVN and supraoptic nucleus of the hypothalamus. An important avenue of future research
will involve assessments of the homeostatic balance between CRH1- and CRH2-receptor systems in anxiety disorders.

HPA-Axis Function and CRH Release in Anxiety Disorders

The anxiety disorder for which abnormalities of CRH or HPA-axis function has been most commonly reported is PTSD. Nevertheless,
the nature of such abnormalities has been inconsistent across studies, because basal plasma or 24-hour urine cortisol concentrations
have been reported to be abnormally decreased (227 ,228 and 229 ), not different (230 ,231 ), or abnormally increased
(190 ,192 ,232 ,233 ) in PTSD samples relative to healthy or trauma-matched control samples. Differences across these studies may
reflect effects of gender, age of illness onset (i.e., childhood versus adult), trauma type or duration, or physiologic variation
relative to illness phase. For example, Hawk et al. showed that 24-hour urine cortisol concentrations were elevated in males but not
females with PTSD, and that this abnormality in males was evident at 1 month but not 6 months after the traumatic event (191 ).

The HPA-axis responses to behavioral or pharmacologic challenge have also been assessed in PTSD. During provocation of PTSD
symptoms by exposure to combat sounds, the changes in plasma cortisol and ACTH concentrations did not differ between patients
with combat-related PTSD and either healthy or combat-matched, non-PTSD control subjects (232 ). In response to dexamethasone
administration, cortisol suppression was found to be normal (234 ) or enhanced (228 ,235 ,236 ) in PTSD, with the latter result
particularly found in response to low-dose (0.25 and 0.5 mg) dexamethasone. Yehuda et al. also observed that patients with PTSD
have an increased density of glucocorticoid receptors on peripheral lymphocytes (228 ). This finding, together with the observations
that patients with PTSD show hypersensitivity to low-dose dexamethasone, led Yehuda et al. to hypothesize that an increase in
hypothalamic glucocorticoid-receptor function results in enhanced feedback sensitivity to cortisol, leading to decreased peripheral
cortisol levels (237 ). Preliminary data suggest that a reduced cortisol response after trauma exposure may predict PTSD
development, a finding raising the possibility that enhanced feedback sensitivity to cortisol may arise acutely or may even antedate
illness onset in some patients with PTSD (229 ,238 ).

The central release of CRH in PTSD was examined in two studies of CSF concentrations, both of which found abnormally increased in
chronic, combat-related PTSD (239 ,240 ). Potentially consistent with this observation, PTSD samples show a blunted ACTH response
to CRH relative to control samples (241 ,242 ). Although these observations would appear most consistent with findings that basal
cortisol secretion and excretion are abnormally increased in PTSD (190 ,192 ,232 ,233 ), they do not clearly contradict the findings
of normal or reduced peripheral cortisol concentrations in PTSD because hypothalamic and extrahypothalamic CRH secretion are
independently regulated (216 ).

Nevertheless, the studies that either identified reductions or were unable to identify elevations in peripheral cortisol concentrations
in PTSD present a challenge to the hypothesis that the reduced hippocampal volume found in MRI studies of PTSD (reviewed earlier)
are accounted for by cortisol hypersecretion (150 ). This hypothesis may still be reconciled with the peripheral cortisol measures
associated with chronic PTSD if the cortisol secretion was elevated near the time of the stressor (191 ,243 ). Longitudinal studies in
male patients who developed PTSD after motor vehicle accidents suggest that cortisol secretion is elevated 1 month after the
trauma, but it is normal when measured 6 months after the trauma (191 ). In rats, the atrophy of pyramidal cell apical dendrites
that occurs in response to stress-induced corticosterone secretion is reversible when the exposure to elevated glucocorticoid
concentrations is terminated early, but it can become irreversible if the elevated corticosterone concentration persists beyond a
critical time period (149 ). Hippocampal damage may thus conceivably occur in PTSD during a period of excessive cortisol secretion
that follows the traumatic event and is prolonged enough so that hippocampal neuronal atrophy becomes irreversible. An alternative
hypothesis for the reduction of hippocampal volume in PTSD, however, is that this abnormality antedates the
 P.915

development of PTSD and may comprise a risk factor for developing PTSD in response to traumatic stress.

In PD, the results of studies examining CRH-receptor and HPA-axis function have been less consistent (Table 63.2 ). Elevated plasma
cortisol levels were reported in one study (244 ), but not in another (245 ), and the results of studies assessing urinary free cortisol
have been similarly inconsistent (177 ,246 ). In a study of 24-hour secretion of ACTH and cortisol, PD subjects had subtle elevations
of nocturnal cortisol secretion and greater amplitude of ultraradian secretory episodes relative to control subjects (247 ), but these
findings await replication. Both normal and elevated rates of cortisol nonsuppression after dexamethasone administration have been
reported in PD (248 ). After combined dexamethasone-CRH challenge, the HPA-axis response was higher in PD subjects than in
healthy controls, but the magnitude of this abnormality was less than that seen in depressed samples (249 ,250 ). The ACTH
response to CRH was blunted in some studies (249 ,250 ), but not in others (250 ), in PD relative to control samples, although CSF
levels of CRH did not differ between PD and control samples (251 ). The extent to which pathophysiologic heterogeneity within PD
samples may account for the inconsistency of these findings remains unclear.

TABLE 63.2. EVIDENCE OF

ALTERATIONS IN CRF-HPA AXIS
FUNCTION IN ANXIETY DISORDERSa

Functional Interactions among Noradrenergic, HPA, and CRH Systems

Coordinated functional interactions between the HPA axis and the noradrenergic systems play major roles in producing adaptive
responses to stress, anxiety, or fear. The secretion of CRH increases LC neuronal firing activity and results in enhanced NE release in
a variety of cortical and subcortical regions (252 ,253 ). Conversely, NE release stimulates CRH secretion in the PVN (the nucleus
containing most of the CRH-synthesizing neurons in the hypothalamus). During chronic stress in particular, the LC is the brainstem
noradrenergic nucleus that appears preferentially to mediate NE release in the PVN (254 ). Conversely, as CRH release in the PVN
stimulates ACTH secretion from the pituitary and thereby increases cortisol secretion from the adrenal glands, the rise in plasma
cortisol concentrations acts through a negative feedback pathway to decrease both CRH and NE synthesis at the level of the PVN.
Glucocorticoid-mediated inhibition of NE-induced CRH stimulation may be evident primarily during stress, rather than under resting
conditions, as an adaptive response that restrains stress-induced neuroendocrine and cardiovascular effects mediated by the PVN
(254 ). NE, cortisol, and CRH thus appear tightly linked as a functional system that offers a homeostatic mechanism for responding
to stress.

A clinical phenomenon of anxiety disorders that may be specifically regulated by interactions between NE and glucocorticoid
secretion involves the acquisition and consolidation of traumatic memories. A characteristic feature of PTSD and PD is that
memories of the traumatic experience or the original panic attack, respectively, persist for decades and are recalled in response to
multiple stimuli or stressors. In experimental animals, alterations of both brain catecholamine and glucocorticoid levels affect the
consolidation and retrieval of emotional memories (50 ,51 ). Glucocorticoids influence memory storage by activation of
glucocorticoid receptors in the hippocampus, whereas NE effects are mediated in part through β-adrenoreceptor stimulation in the
amygdala (255 ). In humans, adrenocortical suppression blocks the memory-enhancing effects of amphetamine and epinephrine
(256 ), and propranolol impairs memory for an emotionally provocative story, but not for an emotionally “neutral” story (257 ).
These data suggest that the acute release of glucocorticoids and NE in response to trauma may modulate the encoding of traumatic
memories. It is conceivable that long-term alterations in these systems may account for memory distortions seen in PTSD, such as
the memory fragmentation, hypermnesia, and deficits in declarative memory.

Central Benzodiazepine-GABA–Receptor System

Several lines of preclinical and clinical evidence have established that BZD-receptor agonists exert anxiolytic effects
 P.916

and have suggested that BZD-receptor function may be altered in anxiety disorders. Central BZD receptors are expressed are present
throughout the brain, but they are most densely concentrated in the cortical gray matter. The BZD and GABAA receptors form parts
of the same macromolecular complex, and although they constitute distinct binding sites, they are functionally coupled and regulate
each other in an allosteric manner (258 ). Central BZD-receptor agonists potentiate and prolong the synaptic actions of the
inhibitory neurotransmitter, GABA, by increasing the frequency of GABA-mediated chloride channel openings (258 ,259 ).
Microinjection of BZD-receptor agonists in limbic and brainstem regions such as the amygdala and the PAG exert antianxiety effects
in animal models of anxiety and fear (260 ). Conversely, administration of BZD-receptor inverse agonists, such as β-carboline-3-
carboxylic acid ethylester, produces behaviors and increases in heart rate, blood pressure, plasma cortisol, and catecholamines
similar to those seen in anxiety and stress (261 ,262 ), effects that can be blocked by administration of BZD-receptor agonists (263 ).

Transgenic mouse studies have identified behavioral roles for specific GABAA-receptor subunits. The anxiolytic action of diazepam
appears absent in mice with α2 subunit point mutations, but it is present in mice with α1 or α3 subunit point mutations (264 ,265 ).
These data suggest that the anxiolytic effect of BZD agonists is at least partly mediated by the GABAA-receptor α2 subunit, which is
largely expressed in the limbic system, but not by the α3 subunit, which is predominately expressed in the reticular activating
system, or the α1 subunit, which is implicated in mediating the sedative, amnestic, and anticonvulsive effects of BZDs (265 ,266 ).
These findings hold clear implications for investigations of the pathophysiology of anxiety disorders and for the development of
anxioselective BZD-receptor agonists.

Some other agents with anxiolytic effects appear to modulate the function of the GABAA/BZD-receptor–chloride ionophore complex
by mechanisms distinct from those of the BZD agonists. The neurosteroid, allopregnenolone, exerts antianxiety effects in conflict
paradigms that serve as putative animal models of anxiety. The anticonflict effects of allopregnenolone are reversed by either
isopropylbicyclophosphate, which binds at the picrotoxinin site on the GABAA receptors, or RO15-4513 (ethyl-8-azido-5,6-dihydro-5-
methyl-6-oxo-4H-imidazo[1,5-α]-[1,4]benzodiazepine-3-carboxylate), a BZD-receptor inverse agonist that inhibits GABAA-activated
chloride flux in neuronal membranes. In contrast, administration of the BZD-receptor antagonist flumazenil (ethyl-8-fluoro-5,6-
dihydro-5-methyl-6-oxo-4H-imidazo[1,5-α]-[1,4]benzodiazepine-3-carboxylate) does not block allopregnenolone's anxiolytic-like
effects, a finding indicating that allopregnenolone does not bind at the BZD site. Allopregnenolone may thus exert anxiolytic-like
effects by stimulating the chloride channel in GABAA receptors by binding at the picrotoxinin site or at a site specific for RO15-4513.

The antianxiety effects of antidepressant drugs with primary effects on monoamine reuptake may also be partly mediated through a
GABAergic mechanism. These agents are effective for the treatment of a spectrum of anxiety disorders including social anxiety
disorder, generalized anxiety disorder, PD, and PTSD. One of the multiple secondary effects of these agents involves potentiation of
GABAergic function. For example, in rats, the effective dose of phenelzine (15 mg/kg) on the elevated plus maze administered
produces a more than twofold increase in whole-brain level GABA concentrations, whereas an ineffective dose of phenelzine (5.1
mg/kg) does not significantly alter GABA levels (267 ). Moreover, the N-acetylated metabolite of phenelzine, N-2-acetylphenelzine,
which potently inhibits monoamine oxidase but does not change whole-brain GABA concentrations, does not produce anxiolytic
effects in the elevated plus-maze test (267 ). Phenelzine's anxiolytic effects in the plus-maze model may thus depend on elevating
brain GABA concentrations, in contrast to the mechanism of the classic BZDs, which instead increase the affinity of GABAA receptors
for GABA.

Effects of Stress on Benzodiazepine-GABA Receptors A

BZD- and GABA-receptor function can be altered by exposure to stress in some brain regions. In experimental animals exposed to
inescapable stress in the form of cold swim or foot shock, the BZD-receptor binding decreases in the frontal cortex, with less
consistent reductions occurring in the hippocampus and hypothalamus, but no changes in the occipital cortex, striatum, midbrain,
thalamus, cerebellum, or pons (268 ). Chronic stress in the form of repeated foot shock or cold water swim resulted in decreased
BZD-receptor binding in the frontal cortex and hippocampus, and possibly in the cerebellum, midbrain, and striatum, but not in the
occipital cortex or pons (268 ,269 and 270 ). These reductions in BZD-receptor binding were associated with deficits in maze escape
behaviors that may have reflected alterations in mnemonic processing (269 ,270 ). Some of these stress effects may be mediated by
glucocorticoids, because chronic exposure to stress levels of CORT alters mRNA levels of multiple GABAA-receptor subunits (271 ).
Consistent with the effects of chronic stress on BZD-receptor expression, the Maudsley “genetically fearful” rat strain shows
decreased BZD-receptor density relative to other rats in several brain structures including the hippocampus (272 ).

Stressors arising early in life may also influence the development of the GABAergic system. In rats, early-life adverse experiences
such as maternal separation result in decreased GABAA-receptor concentrations in the LC and the NTS, reduced BZD-receptor sites in
the LC, the NTS, the frontal cortex, and the CE and the LA of the amygdala, and reduced mRNA levels for the γ2 subunit of the
GABAA-receptor complex in the LC, the NTS, and the amygdala (273 ). The extent to which these developmental responses to early-
life
 P.917

stress may alter the expression of fear and anxiety in adulthood remains unclear.

Benzodiazepine-GABA–Receptor Function in Anxiety Disorders

The central BZD receptor has been implicated in anxiety disorders on the basis of the anxiolytic and anxiogenic properties of BZD
agonists and inverse agonists, respectively, and by the evidence that the BZD-receptor sensitivity to BZD agonists is reduced in some
anxiety-disordered subjects (21 ,274 ,275 ). Hypotheses advanced regarding the role of GABAA-BZD–receptor function in anxiety
disorders have proposed either that changes in the GABAA-BZD macromolecular complex conformation or that alterations in the
concentration or properties of an endogenous ligand account for the pathologic anxiety symptoms seen in anxiety disorders.
However, these hypotheses have not been conclusively tested by in vivo or postmortem studies of anxiety-disordered humans.
In PD, oral (276 ) and intravenous (274 ) administration of the BZD-receptor antagonist, flumazenil, produces panic attacks and
increases anticipatory anxiety in some subjects with PD, but not in healthy controls. In addition, the sensitivity to the effects of
diazepam on saccadic eye movement velocity is abnormally reduced in PD, a finding implying that the functional sensitivity of the
GABAA-BZD supramolecular complex is attenuated in brainstem regions controlling saccadic eye movements (275 ). Subjects with PD
also show abnormally reduced sensitivity to the suppressant effects of diazepam on plasma NE, epinephrine, and heart rate (see
Table 63.3 on p. 920) (277 ).

TABLE 63.3. EVIDENCE OF

ALTERATION IN OTHER
NEUROTRANSMITTER SYSTEMS IN
ANXIETY DISORDERS

Receptor imaging studies using PET and SPECT have assessed central BZD-receptor binding in anxiety disorders. SPECT studies have
reported reduced uptake of the selective BZD-receptor radioligand, [123I]iomazenil, in the frontal (278 ,279 and 280 ), temporal
(278 ,279 ), and occipital (278 ) cortices in subjects with PD relative to control subjects. However, interpretation of these results
was limited by the absence of medication-free PD study subjects and of healthy controls (278 ,279 ) or by the dependence on
nonquantitative methods for estimating BZD-receptor binding. A SPECT-iomazenil study that quantitated BZD-receptor binding by
derivation of distribution volumes found reduced binding in the left hippocampus and precuneus in unmedicated PD relative to
healthy control samples and reported an inverse correlation between panic anxiety ratings and frontal cortex iomazenil binding
(281 ). Another SPECT-iomazenil study reported lower distribution volumes for BZD receptors in the dorsomedial PFC in PTSD
relative to control samples (281a ). These findings appeared consistent with the evidence cited earlier that stress down-regulates
BZD-receptor binding in the frontal cortex and the hippocampus of experimental animals.
Central BZD-receptor binding has also been assessed in PD using PET and [11C]flumazenil. Malizia et al. reported a global reduction in
BZD site binding in seven study subjects with PD relative to eight healthy controls, with the most prominent decreases evident in the
right orbitofrontal cortex and the right insula (areas consistently activated during normal anxiety processing) (282 ). In contrast,
Abadie et al. found no differences in the Bmax, Kd or bound/free values for [11C]flumazenil in any brain region in ten unmedicated PD
study subjects relative to healthy controls (283 ).

Dopaminergic System
Acute stress increases DA release and turnover in multiple brain areas. The dopaminergic projections to the mPFC appear
particularly sensitive to stress, because brief or low-intensity stressors (e.g., exposure to fear-conditioned stimuli) increase DA
release and turnover in the mPFC in the absence of corresponding changes in other mesotelencephalic dopaminergic projections
(284 ). For example, in rats, low-intensity electric foot shock increases tyrosine hydroxylase activity and DA turnover in the mPFC,
but not in the nucleus accumbens or the caudate-putamen (285 ). In contrast, stress of greater intensity or longer duration
additionally enhances DA release and metabolism in other areas as well (285 ). The regional sensitivity to stress appears to follow a
pattern in which dopaminergic projections to the mPFC are more sensitive to stress than the mesoaccumbens and nigrostriatal
projections, and the mesoaccumbens dopaminergic projections are more sensitive to stress than the nigrostriatal projections (284 ).
Thus far, there is little evidence that dopaminergic dysfunction plays a primary role in the pathophysiology of human anxiety
disorders. In PD, Roy-Byrne et al. found a higher plasma concentration of the DA metabolite, homovanillic acid (HVA), in patients
with high levels of anxiety and frequent panic attacks relative to controls (286 ). Patients with PD were also shown to have a greater
growth hormone response to the DA-receptor agonist, apomorphine, than depressed controls (287 ). However, Eriksson et al. found
no evidence of alterations in the CSF HVA concentrations in patients with PD or for correlations between CSF HVA and anxiety
severity or panic attack frequency (288 ). In addition, genetic studies examining associations between PD and gene polymorphisms
for the DA D4 receptor and the DA transporter have produced negative results (289 ).
In social phobia, two preliminary SPECT imaging studies involving small subject samples reported abnormal reductions in DA-
receptor binding. Tiihonen et al. found a significant reduction in β-CIT binding in the striatum in social phobic relative to healthy
control samples (290 ), presumably reflecting a reduction in DA-transporter binding. Schneier et al. reported reduced uptake of the
DA D2/D3-receptor radioligand, [123I]IBZM, in social phobic subjects relative to healthy control subjects (291 ). Both findings await
replication.
 P.918

Serotonergic System
Exposure to various stressors including restraint stress, tail shock, tail pinch, and high-level (but not low-level) foot shock results in
increased 5-HT turnover in the mPFC, nucleus accumbens, amygdala, and lateral hypothalamus in experimental animals (285 ).
During exposure to fear-conditioned stimuli, the 5-HT turnover in the mPFC appears particularly sensitive to the severity of stress,
increasing as the aversiveness of the US and the magnitude of the conditioned fear behavioral response increases (285 ). However,
exposure to repeated electric shocks sufficient to produce learned helplessness is associated with reduced in vivo release of 5-HT in
the frontal cortex (292 ), a finding possibly reflecting a state in which 5-HT synthesis is outpaced by release. Preadministration of
BZD-receptor agonists or tricyclic antidepressant drugs prevents stress-induced reductions in 5-HT release and interferes with the
acquisition of learned helplessness, whereas infusion of 5-HT into the frontal cortex after stress exposure reverses learned-
helplessness behavior (292 ,293 ). Finally, administration of 5-HT–receptor antagonists produces behavioral deficits resembling those
of the learned helplessness seen after inescapable shock during animal stress models that do not ordinarily result in learned
helplessness (293 ).

The effect of stress in activating 5-HT turnover may stimulate both anxiogenic and anxiolytic pathways within the forebrain,
depending on the region involved and the 5-HT–receptor subtype that is predominantly stimulated. For example, microinjection of
5-HT into the amygdala appears to enhance conditioned fear, whereas 5-HT injection into the PAG inhibits unconditioned fear (260 ).
Graeff et al. hypothesized that the serotonergic innervation of the amygdala and the hippocampus mediates anxiogenic effects by 5-
HT2A–receptor stimulation (260 ), whereas serotonergic innervation of hippocampal 5-HT1A receptors suppresses formation of new CS-
US associations and provides resilience to aversive events. Potentially compatible with this hypothesis, 5-HT1A–receptor knockout
mice exhibit behaviors consistent with increased anxiety and fear, and long-term administration of 5-HT1A–receptor partial agonists
exerts anxiolytic effects in generalized anxiety disorder (295 ).

Notably, stress and glucocorticoids exert major effects on the genetic expression of 5-HT1A and 5-HT2A receptors. Postsynaptic 5-HT1A–
receptor gene expression is under tonic inhibition by adrenal steroids in the hippocampus and possibly other regions where
mineralocorticoid receptors are expressed (reviewed in ref. 296 ). Thus, 5-HT1A–receptor density and mRNA levels decrease in
response to chronic stress or CORT administration and increase after adrenalectomy (296 ,297 ,298 and 299 ). The stress-induced
down-regulation of 5-HT1A–receptor expression is prevented by adrenalectomy, a finding showing the importance of circulating
adrenal steroids in mediating this effect (296 ). Although both mineralocorticoid-receptor stimulation and glucocorticoid-receptor
stimulation are involved in mediating this effect, the former is most potent, and 5-HT1A mRNA levels markedly decrease within hours
of mineralocorticoid-receptor stimulation (296 ). Conversely, 5-HT2A–receptor expression is up-regulated during chronic stress and
CORT administration, and it is down-regulated in response to adrenalectomy (298 ,300 ). In view of evidence that 5-HT1A and 5-HT2A
receptors may play reciprocal roles in mediating anxiety, it is conceivable that these corticosteroid mediated effects on 5-HT1A and
5-HT2A expression may be relevant to the pathophysiology of anxiety.

Serotonergic Function in Anxiety Disorders

The literature regarding serotonergic function in anxiety disorders is in disagreement (see Table 63.3 ). In PD, platelet 5-HT uptake
has been reported to be abnormally elevated (301 ), normal (302 ), or abnormally reduced (303 ). Platelet imipramine binding (to a
site related to the 5-HT transporter site), did not differ in PD relative to control samples (304 ,305 ). Another study reported
reduced concentrations of circulating 5-HT in PD relative to control samples (306 ), although this finding has not been replicated.

Pharmacologic challenge studies involving 5-HT have been similarly unable to establish a primary role for 5-HT in the
pathophysiology in PD. Neuroendocrine responses to challenge with the 5-HT precursors, L-tryptophan and 5-hydroxytryptophan (5-
HTP), did not differentiate PD study subjects from healthy controls (307 ,308 ). Moreover, tryptophan depletion did not prove
anxiogenic in unmedicated PD study subjects (309 ). Nevertheless, challenge with the 5-HT releasing agent, fenfluramine, produced
greater increases in anxiety, plasma prolactin, and cortisol in PD compared with control subjects (131 ,310 ). Fenfluramine
challenge also resulted in reduced CBF in the left posterior parietal-superior temporal cortex in PD study subjects relative to healthy
controls (131 ), although it was unclear whether this abnormality reflected an abnormality of serotonergic function or a physiologic
correlate of fenfluramine-induced anxiety, because more PD study subjects (56%) developed panic attacks than did control subjects
(11%).

Preliminary data regarding the sensitivity of specific 5-HT–receptor subtypes appear more promising, particularly because the
elevation of plasma ACTH and cortisol and the hypothermic responses to the 5-HT1A partial agonist, ipsapirone, were blunted in PD
relative to healthy control samples (311 ). Finally, increases in anxiety and plasma cortisol in PD relative to control samples have
been reported after oral (312 ), but not intravenous, administration of the 5-HT2–receptor agonist, m-chloromethylpiperazine (mCPP)
(313 ).

Samples with combat-related PTSD have been shown to have decreased paroxetine binding in platelets relative to controls, a finding
suggesting alterations in the 5-HT transporter (314 ).
 P.919

Southwick et al. observed that a subgroup (five of 14 subjects) with PTSD experienced panic anxiety and “flashbacks” after mCPP
challenge (189 ). Thus, a subgroup of patients with PTSD may have abnormal sensitivity to serotonergic provocation.

Cholecystokinin
CCK is an anxiogenic neuropeptide present in both the brain and the gastrointestinal tract. CCK-containing neurons are found in high
density in the cerebral cortex, amygdala, hippocampus, midbrain PAG, substantia nigra, and raphe. Iontophoretic administration of
CCK has depolarizing effects on pyramidal neurons and stimulates action potential formation in the dentate gyrus of the
hippocampus (reviewed in ref. 315 ).

The CCK-receptor agonist, CCK-4, is anxiogenic in a variety of animal models of anxiety, whereas CCK-receptor antagonists exert
anxiolytic effects in the same models (315 ). CCK has important functional interactions with other systems implicated in anxiety and
fear (noradrenergic, dopaminergic, BZD). For example, the panicogenic effect of CCK-4 in PD is attenuated by administration of the
β-adrenoreceptor antagonist, propranolol, and by long-term imipramine treatment, which down-regulates β-adrenoreceptors (316 ).

Study subjects with PD or PTSD are more sensitive to the anxiogenic effects of CCK-4 than are control subjects (317 ,318 ). For
example, Strohle et al. found that of 24 PD study subjects tested, 15 experienced a panic attack after CCK-4 administration (319 ).
Although the mechanism underlying the enhanced sensitivity to CCK-4 has not been elucidated, it is noteworthy that CSF
concentrations of CCK are lower in PD study subjects than in healthy controls (320 ).

The neuroendocrine effects associated with CCK-4 induced panic appear to differ between PD and PTSD. In PTSD, CCK-4–induced
panic was associated with a lower ACTH response in the PTSD study subjects than in healthy controls, and cortisol concentrations
increased in both the PTSD and control groups (318 ). The elevation in the cortisol concentrations attenuated more rapidly in the
PTSD group than in the control group.

In contrast to the findings in PTSD, ACTH secretion was higher in subjects with PD who developed panic attacks in response to CCK-4
than in those who did not, although even the latter subjects showed brief, less pronounced increases in ACTH concentrations (319 ).
Neither PD subgroup showed significant changes in the plasma cortisol concentration after CCK-4 administration. The elevation of
ACTH concentrations suggested that CRH secretion increases in CCK-4–induced panic in PD (consistent with preclinical evidence
regarding the role of CRH in stress and anxiety and the interaction of CRH and CCK in modulating anxiety) (221 ).

The CCK receptors are classified into CCK-A and CCK-B subtypes. Kennedy et al. reported a significant association between PD and a
single nucleotide polymorphism found in the coding region of the CCK-B–receptor gene (321 ). In contrast, genetic polymorphisms for
the CCK-A–receptor gene and the CCK-pre-pro hormone genes showed no association with PD (321 ). If confirmed by replication,
these data would suggest that a CCK-B–receptor gene variation may be involved in the pathogenesis of PD.

Pande et al. assessed the efficacy of the selective CCK-B–receptor antagonist, CI-988, for preventing panic attacks in PD (322 ). No
differences in the rate of panic attacks were seen between the active drug and placebo treatment groups. Nevertheless, because of
the limited bioavailability of oral CI-988, studies involving this drug may not have sufficiently tested the hypothesis that CCK-B–
receptor antagonism produces antipanic effects in PD.

Other Neuropeptides

Opioid Peptides
Acute, uncontrollable shock increases secretion of opiate peptides and decreases μ-opiate–receptor density (323 ,324 ). The
elevation of opioid peptide secretion may contribute to the analgesia observed after uncontrollable stress and exposure to fear-
conditioned stimuli (325 ). This analgesic effect shows evidence of sensitization, because subsequent exposure to less intense shock
in rats previously exposed to uncontrollable shock also results in analgesia (326 ).

Potentially consistent with these data, Pitman et al. found that patients with PTSD showed reduced pain sensitivity compared with
veterans without PTSD after exposure to a combat film (327 ), an effect that was reversed by the opiate antagonist naloxone (a
finding suggesting mediation by endogenous opiate release during symptom provocation). In the baseline state, the CSF β-endorphin
levels were abnormally elevated in PTSD relative to control samples (328 ). However, Hoffman et al. found lower morning and
evening plasma β-endorphin levels in a PTSD group versus healthy control samples (329 ). Another study found no differences in
plasma methionine-enkephalin concentrations between PTSD subjects and control subjects, although this compound's degradation
half-life was higher in the PTSD group (330 ).

During opiate administration, Bremner et al. reported that some patients with combat-related PTSD experience an attenuation of
their hyperarousal symptoms (331 ). Because preclinical studies in experimental animals have shown that opiates potently suppress
central and peripheral noradrenergic activity, these data appear compatible with the hypothesis that some PTSD symptoms are
mediated by noradrenergic hyperactivity (discussed earlier). Conversely, during opiate withdrawal noradrenergic activity increases,
and it
 P.920

has been noted that some symptoms of PTSD resemble those of opiate withdrawal (170 ).

Neuropeptide Y
NPY administered in low doses intraventricularly attenuates experimentally induced anxiety in a variety of animal models (332 ).
Consistent with these data, transgenic rats that overexpress hippocampal NPY show behavioral insensitivity to restraint stress and
absent fear suppression of behavior in a punished drinking task (333 ). In healthy humans subjected to uncontrollable stress during
military training exercises, plasma NPY levels increased to a greater extent in persons rated as having greater stress resilience (334 ).
During stress exposure, the NPY plasma levels were positively correlated with plasma cortisol concentrations and behavioral
performance, and they were negatively correlated with dissociative symptoms (334 ).

In humans with PD, plasma NPY concentrations were abnormally elevated, and this finding, given NPY's putative anxiolytic effects,
may reflect an adaptive response to anxiety symptoms (335 ). In contrast, patients with combat-related PTSD had lower plasma NPY
concentrations both at baseline and in response to yohimbine challenge than healthy controls (336 ). In the PTSD group, the baseline
NPY levels were inversely correlated with PTSD and panic symptoms and with yohimbine-induced increases in MHPG and systolic
blood pressure (336 ). If this finding proves reproducible, it suggests that a deficit in endogenous NPY secretion may be involved in
the generation of anxiety and sympathetic autonomic symptoms in PTSD.

Thyrotropin-Releasing Hormone and the Thyroid Axis

In the early twentieth century, Graves described cases in which thyroid hormone hypersecretion was associated with anxiety,
palpitations, breathing difficulties, and rapid heart rate in persons recently exposed to traumatic stress. Nevertheless, systematic
epidemiologic studies of the relationship between stress and thyroid disease have not been conducted. Although few studies have
looked at thyroid function in anxiety disorders, Mason et al. found elevated levels of triiodothyronine in patients with combat-
related PTSD (337 ) (Table 63.3 ), a finding consistent with evidence that stress results in long-lasting elevations of thyroid hormone
secretion (338 ).

Respiratory System Dysfunction in Panic Disorder

Associations between respiratory perturbation and acute anxiety have been demonstrated in PD, in which various forms of
respiratory stimulation consistently produce panic anxiety and alterations in parameters of respiratory physiology (339 ,340 ,341
and 342 ). The most straightforward forms of respiratory stimulation that produce panic anxiety produce elevations of carbon
dioxide pressure (hypercapnia). Thus, panic attacks can be consistently induced in patients with PD by rebreathing air, inhaling 5%
to 7% carbon dioxide in air (343 ,344 ), or inhaling a single deep breath of 35% carbon dioxide (345 ,346 ). Other panicogenic
chemical challenges have also been hypothesized to induce anxiogenic effects
 P.921

through respiratory stimulation (340 ,341 ,347 ). Although the panicogenic mechanism of intravenous administration of sodium
lactate remains unclear, it may also involve respiratory stimulation (339 ,340 ).

The evidence that respiratory parameters index risk for panic anxiety includes data showing the following: (a) asymptomatic adult
relatives of patients with PD have abnormally increased sensitivity to respiratory stimulation by carbon dioxide inhalation; (b) among
PD samples, stronger family loading for PD is found among persons with evidence of respiratory dysregulation; and (c) the
respiratory indices associated with PD are heritable, a finding suggesting a shared genetic vulnerability for panic attacks and
respiratory dysregulation (reviewed in Chapter 61 ). Nevertheless, these data partly depend on subjective ratings of dyspnea during
stress or respiratory stimulation, and the mechanisms underlying this sensitivity remain unclear. One possibility is that this
hypersensitivity reflects an overall sensitivity to somatic sensations, because high degrees of anxiety sensitivity are linked to future
panic attacks (348 ).

The associations between respiratory perturbation and acute anxiety are not specific to PD. Exaggerated sensitivity to respiratory
perturbation has also been reported in anxiety-disordered patients with some simple phobias, limited symptom panic attacks,
childhood separation anxiety disorder, or limited-symptom anxiety attacks and in nonpsychiatrically ill subjects with high ratings on
anxiety sensitivity scales. (See Chapter 61 ) For example, children with separation anxiety disorder exhibit greater changes in
somatic symptoms during carbon dioxide inhalation that positively correlate with increases in respiratory rate, tidal volume, minute
ventilation, end-tidal carbon dioxide pressure, and irregularity in respiratory rate during room-air breathing (349 ).

CONCLUDING REMARKS
Part of "63 - Neurobiological Basis of Anxiety Disorders "

The inconsistency in the results of biological investigations of anxiety disorders highlights the importance of addressing the
neurobiological heterogeneity inherent within criteria-based, psychiatric diagnoses. Understanding this heterogeneity will be
facilitated by the continued development and application of genetic, neuroimaging, and neurochemical approaches that can refine
anxiety disorder phenotypes and can elucidate the genotypes associated with these disorders. Application of these experimental
approaches will also facilitate research aimed at elucidating the mechanisms of antianxiety therapies.

The knowledge reviewed herein regarding the neurobiology of fear and anxiety already suggests themes along which the
development of new therapeutic approaches can be organized. In general, anxiolytic treatments appear to inhibit neuronal activity
in the structures mediating fear expression and behavioral sensitization and facilitate endogenous mechanisms for modulating the
neural transmission of information about aversive stimuli and responses to such stimuli. Novel treatments being developed to exploit
the former type of mechanisms include pharmacologic agents that selectively target subcortical and brainstem pathways supporting
specific components of emotional expression (e.g., CRH-receptor antagonists). In contrast, nonpharmacologic treatments for anxiety
may augment the brain's systems for modulating anxiety responses, by facilitating the extinction of putative fear-conditioned
responses or directing the reinterpretation of anxiety-related thoughts and somatic sensations (so they produce less subjective
distress). Informed by increasingly detailed knowledge about the pathophysiology of specific anxiety disorders and the neural
pathways involved in anxiety and fear processing, the development of therapeutic strategies that combine both types of approaches
may ultimately provide the optimal means for reducing the morbidity of anxiety disorders.

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64
Neural Circuitry of Anxiety and Stress Disorders

Michael Davis
Michael Davis: Department of Psychiatry, Emory University School of Medicine, Atlanta, Georgia.

• BRAIN SYSTEMS IN THE GENERATION OF FEAR AND ANXIETY

• BRAIN SYSTEMS IN THE INHIBITION OR SUPPRESSION OF FEAR AND ANXIETY

BRAIN SYSTEMS IN THE GENERATION OF FEAR AND ANXIETY

Part of "64 - Neural Circuitry of Anxiety and Stress Disorders "

Role of the Amygdala

Many data now indicate that the amygdala, along with its many efferent connections, is critically involved in emotion. Although the amygdala complex is generally defined by several distinct groups
of cells, including the lateral, basal, accessory basal central, medial, and cortical nuclei, new data indicate that it is more useful to think of the amygdala as the basolateral amygdala (Bla) and to
think of its several target areas as parts of a broader network that subserve more specialized functions (Fig. 64.1 ). The Bla receives sensory information from the thalamus, cortex (169 ), and
ventral hippocampus (54 ) and then activates or modulates synaptic transmission in target areas appropriate for the reinforcement signal with which the sensory information has been associated.
This involves both positive and negative associations. However, because most of the literature on the amygdala has analyzed the role of the Bla and its adjacent target, the central nucleus of the
amygdala (CeA), in aversive conditioning, this work serves as the main focus of this chapter. Brief summaries of the role of Bla outputs to other targets shown in Fig. 64.1 follow. Because the
periaqueductal gray (PAG) has received considerable attention in the study of defensive behavior and the hippocampus in the study of contextual fear conditioning, these data are reviewed next.
Finally, brain systems and neurotransmitters involved in the inhibition of fear are reviewed, given the clinical significance of this information.

FIGURE 64.1. Schematic diagram of the outputs of the basolateral amygdala to various target areas and how these connections may be involved in fear and anxiety.

Basolateral Nucleus of the Amygdala to CeA or BNST Pathway as It Relates to Conditioned

and Unconditioned Fear
Figure 64.1 shows that the Bla projects directly to the CeA, as well as to a related structure, the lateral division of the bed nucleus of the stria terminalis (BNST), to form part of the
lateral extended amygdala (6 ). Figure 64.2 summarizes work done in many different laboratories indicating that the CeA and BNST have direct projections to various anatomic areas
that may be expected to be involved in many of the symptoms of fear or anxiety (65 ). The CeA and BNST have been grouped together because fibers from the Bla that project to the
BNST pass through the CeA and cells in the lateral division of the CeA project to the BNST. Thus, many effects previously attributed to the CeA may really depend on the BNST.

FIGURE 64.2. Schematic diagram of the outputs of the central nucleus of the amygdala and the lateral division of the bed nucleus of the stria terminalis to various target areas and how
these connections may be related to specific aspects of fear and anxiety. BNST, bed nucleus of the stria terminalis; CER, conditioned emotional response; EEG, electroencephlographic; N,
nucleus.

Autonomic and Hormonal Measures

Anatomically, the CeA and the BNST are well situated to mediate the various components of the fear response. Both structures send prominent projections to areas such as the lateral hypothalamus,
which is involved in activation of the sympathetic autonomic nervous system seen during fear and anxiety (155 ). Direct projections to the dorsal motor nucleus of the vagus, nucleus of the solitary
tract, and ventrolateral medulla may be involved in lateral extended amygdala modulation of heart rate and blood pressure, which are known to be regulated by these brainstem nuclei (222 ).
Projections to the parabrachial nucleus may be involved in respiratory (as well as cardiovascular changes) during fear, because electrical stimulation and lesions of this nucleus are known to alter
various measures of respiration. Indirect projections of the CeA to the paraventricular nucleus through the BNST and preoptic area may mediate the prominent neuroendocrine responses to fearful
or stressful stimuli.

Attention and Vigilance

Projections from the lateral extended amygdala to the ventral tegmental area may mediate stress-induced increases in dopamine metabolites in the prefrontal cortex (101 ). Direct projections to
the dendritic field of the locus ceruleus or indirect projections through the paragigantocellularis nucleus may mediate the increase in firing rates of cells in the locus ceruleus in the presence of a
fearful stimulus. Direct
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projections to the lateral dorsal tegmental nucleus and parabrachial nuclei, which have cholinergic neurons that project to the
thalamus, may mediate increases in synaptic transmission in thalamic sensory relay neurons during states of fear. This cholinergic
activation, along with increases in thalamic transmission accompanying activation of the locus ceruleus, may thus lead to increased
vigilance and superior signal detection in a state of fear or anxiety.

As emphasized by Kapp et al. (141 ), in addition to its direct connections to the hypothalamus and brainstem, the CeA has the
potential for indirect widespread effects on the cortex through its projections to cholinergic neurons that project to the cortex. The
rapid development of conditioned bradycardia during pavlovian aversive conditioning, critically dependent on the amygdala, may
reflect a general increase in attention.

Motor Behavior
Release of norepinephrine onto motor neurons by lateral extended amygdala activation of the locus ceruleus, or through projections
to serotonin containing raphe neurons, could lead to enhanced motor performance during a state of fear, because both
norepinephrine and serotonin facilitate excitation of motor neurons. Direct projections to the nucleus reticularis pontis caudalis, as
well as indirect projections to this nucleus through the central gray, probably are involved in fear potentiation of the startle reflex.
Direct projections to the lateral tegmental field, including parts of the trigeminal and facial motor nuclei, may mediate some of the
facial expressions of fear as well as potentiation of the eyeblink reflex. The lateral extended amygdala also projects to regions of
the central gray that appear to be a critical part of a general defense system and that have been implicated in conditioned fear in
certain behavioral tests including freezing, sonic and ultrasonic vocalization, and stress-induced hypalgesia
(20 ,33 ,78 ,103 ,121 ,155 ).

Elicitation of Fear Responses by Electrical or Chemical Stimulation of the Extended

Amygdala
Electrical stimulation or abnormal electrical activation of the amygdala (i.e., by temporal lobe seizures) can produce a complex
pattern of behavioral and autonomic changes that, taken together, highly resembles a state of fear.

Autonomic and Hormonal Measures

As outlined by Gloor: “The most common affect produced by temporal lobe epileptic discharge is fear…. It arises ‘out
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of the blue.’ Ictal fear may range from mild anxiety to intense terror. It is frequently, but not invariably, associated with a rising
epigastric sensation, palpitation, mydriasis, and pallor and may be associated with a fearful hallucination, a frightful memory
flashback, or both” (98 ). In humans, electrical stimulation of the amygdala elicits feelings of fear or anxiety as well as autonomic
reactions indicative of fear (57 ,99 ). Although other emotional reactions occasionally are produced, the major reaction is one of
fear or apprehension.

Electrical stimulation of the CeA or chemical activation by the cholinergic agonist carbachol or the neurotransmitter glutamate
produces prominent cardiovascular effects that depend on the species, site of stimulation, and state of the animal. CeA stimulation
can also produce gastric ulceration and can increase gastric acid, and these features can be associated with chronic fear or anxiety.
It can also alter respiration, a prominent symptom of fear, especially in panic disorder.

Using very small infusion cannulas and very low doses, Sanders and Shekhar found increases in blood pressure and heart rate when
the γ-aminobutyric acidA (GABAA) antagonist bicuculline was infused into the Bla but not the CeA (215 ). Local infusion of N-methyl-
D-aspartate (NMDA) or AMPA into the basolateral nucleus also increased blood pressure and heart rate (230 ). Repeated infusion of
initially subthreshold doses of bicuculline into the anterior basolateral nucleus led to a “priming” effect in which increases in heart
rate and blood pressure were observed after three to five infusions (216 ). This change in threshold lasted at least 6 weeks and could
not be ascribed to mechanical damage or generalized seizure activity based on EEG measurements. Similar changes in excitability
were produced by repetitive infusion of very low doses of corticotropin-releasing hormone (CRH) or the related peptide, urocortin
(210 ). Once primed, these animals exhibited behavioral and cardiovascular responses to intravenous sodium lactate, a panic-
inducing treatment in certain types of psychiatric patients.

In general, electrical stimulation of the amygdala causes an increase in plasma levels of corticosterone. The effect of electrical
stimulation appears to depend on both norepinephrine and serotonin in the paraventricular nucleus. Depletion of these transmitters
through local infusions of 6-hydroxydopamine or 5,7-DHT, or local infusion of the norepinephrine or serotonin antagonists prazosin or
ketanserin, into the paraventricular nucleus attenuated the effects of electrical stimulation (80 ).

Attention and Vigilance

Studies in several species indicate that electrical stimulation of the CeA increases attention or processes associated with increased
attention. For example, stimulation of sites in the CeA that produce bradycardia (142 ) also produce low-voltage fast EEG activity
(140 ). In fact, an attention or orienting reflex was the most common response elicited by electrical stimulation of the amygdala
(16 ,241 ). These and other observations led Kapp et al. to hypothesize that the “central nucleus and its associated structures
function, at least in part, in the acquisition of an increased state of nonspecific attention or arousal manifested in a variety of CRs
which function to enhance sensory processing. This mechanism is rapidly acquired, perhaps via an inherent plasticity within the
nucleus and associated structures in situations of uncertainty but of potential import; for example, when a neutral stimulus (CS)
precedes either a positive or negative reinforcing, unexpected event (US)” (141 ). Electrical stimulation of the amygdala can also
activate cholinergic cells that are involved in arousal-like effects depending on the state of sleep and perhaps the species.

Motor Behavior
Electrical or chemical stimulation of the CeA produces a reduction of prepotent, ongoing behavior, a critical component in several
animal models such as freezing, the operant conflict test, the conditioned emotional response, and the social interaction test.
Electrical stimulation of the amygdala also elicits jaw movements and activation of facial motoneurons, which may mimic
components of the facial expressions seen during the fear reaction. These motor effects may be indicative of a more general effect
of amygdala stimulation, namely, that of modulating brainstem reflexes such as the massenteric, baroreceptor nictitating membrane,
eyeblink, and the startle reflex.

Summary of the Effects of Stimulation of the Amygdala

Viewed in this way, the pattern of behaviors seen during fear may result from activation of a single area of the brain (the extended
amygdala), which then projects to various target areas that themselves are critical for each of the specific symptoms of fear (the
expression of fear), as well as the experience of fear. Moreover, it must be assumed that all these connections are already formed in
an adult organism, because electrical stimulation produces these effects in the absence of prior explicit fear conditioning. Thus,
much of the complex behavioral pattern seen during a state of “conditioned fear” has already been “hard wired” during evolution.

For a formerly neutral stimulus to produce the constellation of behavioral effects used to define a state of fear or anxiety, it is only
necessary for that stimulus to activate the amygdala, which, in turn, will produce the complex pattern of behavioral changes by
virtue of its innate connections to different brain target sites. Viewed in this way, plasticity during fear conditioning probably
results from a change in synaptic inputs before or in the Bla (173 ,192 ,204 ), rather than from a change in its efferent target areas.
The ability to produce long-term potentiation (LTP) in the Bla (55 ,56 ,58 ,91 ,129 ,226 ) that can lead to an increase in
responsiveness to a physiologic stimulus (203 ) and the finding
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that local infusion of NMDA antagonists into the amygdala block the acquisition of fear conditioning (65 ) are consistent with this
hypothesis.

Effects of Lesions of the Amygdala on Conditioned and Unconditioned Fear in Rodents and
Other Species
Many studies in rodents and other species indicate that lesions of the Bla or CeA block many different measures of conditioned fear,
as well as unconditioned fear. Table 64.1 and Table 64.2 show selected examples of such studies in animals, which have been
extensively reviewed elsewhere (64 ,65 ). More recent studies in humans also point to the amygdala in fear and anxiety.

TABLE 64.1. EFFECTS OF LESIONS OF THE AMYGDALA ON CONDITIONED FEAR

TABLE 64.2. EFFECTS OF LESIONS OF THE AMYGDALA ON UNCONDITIONED FEAR

OR STRESS

Effects of Lesions of the Amygdala in Humans and Nonhuman Primates

In nonhuman primates (160 ,184 ,205 ) and in humans (9 ,117 ), cells have been found in the amygdala that respond selectively to
faces or direction of gaze (42 ). In humans, removal of the amygdala has been associated with an impairment of memory for faces
(4 ,138 ,240 ,257 ) and deficits in recognition of emotion in people's faces and interpretation of gaze angle (41 ,50 ,257 ). In a very
rare case involving bilateral calcification confined to the amygdala (Urbach-Wiethe disease), patient SM046 could not identify the
emotion of fear in pictures of human faces and could not draw a fearful face, even though other emotions such as happy, sad, angry,
and disgusted were identified and drawn within the normal range. The deficit in recognizing facial expressions of fear
 P.936

only seemed to occur after bilateral amygdala damage (3 ). This patient and two others also tended to view even the most
threatening faces as trustworthy and approachable (2 ). A more detailed evaluation of patient SM046 showed that she correctly
identified valence (e.g., pleasant versus unpleasant) in faces displaying happy, surprised, afraid, angry, disgusted, or sad emotion,
but she was highly abnormal in rating the level of arousal to the afraid, angry, disgusted, and sad faces (1 ). Another patient (SP)
with extensive bilateral amygdala damage also showed a major deficit in her ability to rate levels of fear in human faces, yet was
perfectly normal in generating a fearful facial expression in comparison with neurologically normal subjects, based on the ratings of
three judges (13 ).

Patients with unilateral (153 ) or bilateral (26 ) lesions of the amygdala also have been reported to have deficits in classic fear
conditioning using the galvanic skin response as a measure of fear. In monkeys, removal of the amygdala decreases reactivity to
sensory stimuli measured with the galvanic skin response (17 ,18 ).

Effects of Local Infusion of Drugs into the Amygdala on Measures of Fear and Anxiety
If the amygdala is critically involved in fear and anxiety, then drugs that reduce fear or anxiety clinically may well act within the
amygdala. It is also probable that certain neurotransmitters within the amygdala may be involved in fear and anxiety. In fact, many
studies indicate that local infusions of GABA or GABA agonists, benzodiazepines, CRH antagonists, opiate agonists, neuropeptide Y,
dopamine antagonists, or glutamate antagonists decrease measures of fear and anxiety in several animal species. Table 64.3 gives
selected examples of some of these studies, which have been extensively reviewed (65 ). Conversely, local infusions of GABA
antagonists, CRH or CRH analogues, vasopressin, thyrotropin-releasing hormone, opiate antagonists, cholecystokinin (CCK) or CCK
analogues tend to have anxiogenic effects. Table 64.4 shows selected examples of such studies that also have been reviewed (65 ).

TABLE 64.3. EFFECTS OF NEUROTRANSMITTER AGONISTS INFUSED INTO THE

AMYGDALA ON FEAR AND ANXIETY
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TABLE 64.4. EFFECTS OF NEUROTRANSMITTER ANTAGONISTS INFUSED INTO THE

AMYGDALA ON FEAR AND ANXIETY
In summary, connections between the Bla and the CeA or BNST are critically involved in various autonomic and motor responses seen
during a state of fear or anxiety. However, it is also the case that connections between the basolateral nucleus and other target
areas are involved in emotional behavior.

Basolateral Nucleus of the Amygdala to Dorsal Striatum Pathway as It Relates to Avoidance

or Escape from Aversive Events
As emphasized by McGaugh, Packard, and others, the amygdala modulates memory in various tasks such as inhibitory avoidance and
motor or spatial learning (49 ,170 ,171 ,185 ,186 ). For example, posttraining intracaudate injections of amphetamine enhanced
memory in a visible platform water maze task but had no effect in the hidden platform, spatially guided task (185 ,186 ). Conversely,
posttraining intrahippocampal infusion of amphetamine enhanced memory in the hidden platform water maze task but not in the
visible platform task. However, posttraining intraamygdala injections of amphetamine enhanced memory in both water maze tasks
(185 ,186 ).
Moreover, preretention intrahippocampal lidocaine injections blocked expression of the memory-enhancing effects of posttraining
intrahippocampal amphetamine injections in the hidden platform task, and preretention intracaudate lidocaine injections blocked
expression of the memory-enhancing effects of posttraining intracaudate amphetamine injections in the visible platform task.
However, preretention intraamygdala lidocaine injections did not block the memory-enhancing effect of posttraining intraamygdala
amphetamine injections on either task. Finally, in the hidden platform task, posttraining intrahippocampal, but not intracaudate,
lidocaine injections blocked the memory-enhancing effects of posttraining intraamygdala the visible platform task, posttraining
intracaudate, but not intrahippocampal, lidocaine injections blocked the memory-enhancing effects of posttraining intraamygdala
amphetamine. The findings indicate a double dissociation between the roles of the hippocampus and caudate-putamen in memory
and suggest that the amygdala exerts a modulatory influence on both the hippocampal and caudate-putamen memory systems.
Perhaps similarly, lesions of the CeA block freezing but not escape to a tone previously paired with shock, whereas lesions of the
basal nucleus of the Bla have just the opposite effect (10 ). However, lesions of the lateral nucleus, which receive sensory
information required by both measures, block both freezing and escape. Lesions of the Bla, but not the CeA, also block avoidance of
a bar associated with shock (146 ). It is possible that basolateral outputs to the dorsal or the ventral striatum mediate the escape
behavior, given the importance of the striatum in several measures of escape or avoidance learning. However, combined, unilateral
lesions of each structure on opposite sides of the brain would be required to evaluate whether this results from serial transmission
from the basolateral nucleus to the striatum.

Basolateral Nucleus of the Amygdala to Hippocampus Pathway as It Relates to Avoidance or

Escape from Aversive Events
As mentioned earlier, posttraining intrahippocampal as well as intraamygdala injections of amphetamine selectively enhance
memory in a hidden platform water maze task (185 ,186 ). Posttraining infusion of norepinephrine into the basolateral nucleus
enhanced retention in the hidden platform water maze task, whereas posttraining infusion of propranolol had the opposite effect
(113 ). These results suggest that
P.938
 P.939

the amygdala exerts a modulatory influence on hippocampal-dependent memory systems, presumably by direct projections from the
basolateral nucleus of the amygdala, perhaps by modulation of LTP in the hippocampus. Lesions (131 ), NMDA antagonists (132 ), or
local anesthetics (134 ) infused into the Bla decrease LTP in the dentate gyrus of the hippocampus. Conversely, high-frequency
stimulation of the amygdala facilitates induction of LTP in the dentate gyrus (130 ,133 ). However, combined, unilateral lesions of
each structure on opposite sides of the brain would be required to evaluate whether this results from serial transmission from the
basolateral nucleus to the hippocampus.

Basolateral Nucleus of the Amygdala to Frontal Cortex Pathway as It Relates to Emotion

Importance of the Bla in US Representation

After pavlovian conditioning, presentation of a conditioned stimulus (CS) elicits some neural representation of the unconditioned
stimulus (US) with which it was paired. For example, the sound of a refrigerator door opening or an electric can opener may bring
the family cat into the kitchen in expectation of dinner. Several studies suggest that the Bla, perhaps by connections with cortical
areas such as the perirhinal cortex (93 ), is critical for these US representations based on studies using a procedure called US
devaluation. In these experiments, a neutral stimulus (e.g., a light) is first paired with food so a conditioned response can be
measured. Some animals then have the food paired with something that makes them sick (US devaluation). After such treatment,
these animals show a reduction in the conditioned response to the light compared with animals that did not experience US
devaluation. This result suggests that, after conditioning, animals have a representation of the value of a reinforcement that is
elicited by the cue paired with that US. When that representation is changed, then the behavior elicited by the cue also is changed
in the same direction. Lesions of the basolateral, but not the CeA, block US devaluation (112 ). In a related paradigm, rats are
trained to be fearful of a weak shock in the presence of a tone. When this is followed by presentation of a stronger shock, without
further tone-shock pairing, more freezing occurs to the tone. Temporary inactivation of the Bla during this inflation procedure
blocks this effect when testing subsequently occurs with a normal, unlesioned, amygdala (5 ).

Second-order conditioning also depends on a US representation elicited by a CS. In this procedure, cue 1 is paired with a particular
US (e.g., shock or food), and cue 2 is paired with cue 1. After such training, cue 2 elicits a similar behavior as that elicited by cue 1,
depending on the US with which cue 1 was paired. Thus, it may elicit approach behavior if cue 1 was formerly paired with food and
avoidance if cue 1 was paired with shock. This indicates that cue 1 elicits a representation of the US that then becomes associated
with cue 2. Lesions of the Bla, but not the CeA, block second-order conditioning (72 ,73 ,112 ), as do local infusions of NMDA
antagonists into the Bla (92 ).

Importance of the Bla Projection to the Frontal Cortex in Using US Representations to

Guide Behavior
Converging evidence now suggests that the connection between the Bla and the prefrontal cortex is critically involved in the way in
which a US representation (e.g., very good, pretty good, very bad, pretty bad) guides approach or avoidance behavior. Patients with
late- or early-onset lesions of the orbital regions of the prefrontal cortex fail to use important information to guide their actions and
decision making (14 ,25 ,63 ). For example, on a gambling task, they choose high, immediate reward associated with long-term loss
rather than low, immediate reward associated with positive long-term gains. They also show severe deficits in social behavior and
make poor life decisions.

Studies using single-unit recording techniques in rats indicate that cells in both the Bla and the orbitofrontal cortex fire
differentially to an odor, depending on whether the odor predicts a positive (e.g., sucrose) or negative (e.g., quinine) US. These
differential responses emerge before the development of consistent approach or avoidance behavior elicited by that odor (220 ).
Many cells in the Bla reverse their firing pattern during reversal training (i.e., the cue that used to predict sucrose now predicts
quinine and vice versa) (221 ), although this has not always been observed (217 ). In contrast, many fewer cells in the orbitofrontal
cortex showed selectivity before the behavioral criterion was reached, and many fewer reversed their selectivity during reversal
training (221 ). These investigators suggest that cells in the Bla encode the associative significance of cues, whereas cells in the
orbitofrontal cortex are active when that information, relayed from the Bla, is required to guide choice behavior.

Taken together, these data suggest that the connection between the Bla and the frontal cortex may be involved in determining
choice behavior based on how an expected US is represented in memory. The necessity for communication between the amygdala
and the frontal cortex was shown in monkeys using a “disconnection approach” in which the amygdala on one side of the brain and
the frontal cortex on the other side were lesioned together (22 ). Because the reciprocal connections between the two structures
are ipsilateral, this procedure completely eliminated activity of the network connections while preserving partial function of each
structure. Using this approach in rhesus monkeys, Baxter et al. found a decrease in US devaluation after unilateral neurotoxic lesions
of the basolateral nucleus in combination with unilateral aspiration of orbital prefrontal cortex (22 ). These monkeys continued to
approach a food on which they had recently been satiated, whereas control monkeys consistently switched to the other food.
 P.940

Neuroimaging Studies of the Amygdala in Humans

As reviewed by Whalen (244 ), neuroimaging studies in normal human subjects have shown activation of the amygdala by
presentation of biologically relevant sensory stimuli that probably induce strong negative emotional states. For example, the
functional magnetic resonance imaging (fMRI) signal intensity within the amygdala is greater when subjects view graphic
photographs of negative material (e.g., mutilated human bodies) compared with when they view neutral pictures (135 ). Positron
emission tomography metabolic activity within the amygdala increased to negative material presented by film clips (199 ), and the
amount of amygdala activity during film clips predicted later recall (47 ). In addition, fMRI signal intensities in humans during classic
fear conditioning increased in response to stimuli that predicted an aversive event (45 ,150 ,179 ).

Amygdala activation also seems to be greater during presentations of fearful faces compared with neutral facial expressions
(40 ,180 ), happy facial expressions (180 ,246 ), or when subjects looked at a fixation point on an otherwise blank screen (246 ).
Whalen et al. used a backward masking technique in which very brief presentations of fearful and happy facial expressions (33
milliseconds) were followed immediately by presentations (167 milliseconds) of neutral faces (245 ). Most study subjects reported
seeing neutral “expressionless” faces, but not any afraid or smiling faces. Nonetheless, the amygdala demonstrated greater fMRI
signal intensity to masked fearful faces compared with masked happy faces. In addition, subjects reported that these masked stimuli
did not induce any noticeable changes in their state of emotional arousal. As suggested by Whalen (244 ), “this study offers
preliminary support for the notion that the amygdala constantly monitors the environment for such signals. More than functioning
primarily for the production of strong emotional states, the amygdala would be poised to modulate the moment-to-moment
vigilance level of an organism.”

Role of the Periaqueductal (Central) Gray

Outputs from the CeA to the ventral central gray appear to mediate several components of the fear response including freezing,
conditioned analgesia, and fear-related vocalizations, but, surprisingly, maybe not cessation of operant behavior (11 ). More dorsal
regions of the PAG play a role in active defensive responses (33 ), depending on whether the threat is distal or proximal (31 ,71 ).
Fanselow (79 ), for example, showed that dorsal, but not ventral, PAG lesions eliminate activity bursts elicited by foot shock,
whereas ventral, but not dorsal, PAG lesions diminish freezing responses elicited by cues previously paired with foot shock. Fanselow
suggested that these stimuli (i.e., foot shock versus stimuli predicting foot shock) access different points on a “predatory
imminence” continuum in which proximal threats activate the dorsal PAG to generate active defensive behaviors. More distal threats
activate the ventral PAG and generate passive or preparatory defensive behaviors such as freezing and analgesia. From a similar
perspective, Deakin and Graeff et al. proposed that moderately threatening stimuli inhibit the dorsal PAG (66 ,104 ), but this
inhibition is overcome with more extreme danger, thus allowing active defense or panic behaviors to emerge.

Results from stimulation studies have suggested an anatomic division of function within PAG. In particular, Depaulis and colleagues
showed that chemical or electrical stimulation of PAG regions lateral to the aqueduct produces active behaviors such as forward
avoidance, defensive aggression, and cardiovascular activation (67 ,68 ), whereas stimulation of more ventral regions of the PAG
elicits passive responses such as behavioral arrest and decreased cardiovascular output (21 ,69 ). Electrical stimulation of the dorsal
PAG in humans produces a pattern of cardiovascular effects that resemble those seen during a natural panic attack, and patients
often experience fear, anxiety, and the desire to terminate stimulation (162 ). Exposure of rats to a cat or high-frequency
vocalizations of conspecifics that often signal a predator in the environment increases neuronal firing in the dorsal PAG inferred
from an increase in the immediate early gene c-fos (162 ).

Based on these and other data, several investigators have suggested that the dorsal PAG may be involved in panic attacks in humans,
perhaps resulting from a dysregulation of various transmitters systems within this structure (162 ). The dorsal PAG has heavy
innervation of the panicogenic peptide CCK, which has been shown to excite the majority of cells in this region. CCK antagonists
functionally decrease the effects of electrical stimulation of the dorsal PAG, as does elevating serotonin, perhaps relevant to the
use of serotonin reuptake inhibitors in the treatment of panic disorder. Whether these effects depend on connections between the
amygdala and the PAG or whether they represent examples where the PAG can function autonomously remains to be determined.

Role of the Hippocampus in Contextual Fear Conditioning

Rats given cue-shock pairings learn to be afraid of the cue as well as the place where cue-shock pairings occurred (context
conditioning). In 1992, two seminal articles reported that the hippocampus was necessary for context but not explicit cue
conditioning (148 ,190 ). Both studies found that electrolytic lesions of the dorsal hippocampus blocked freezing in the presence of a
fearful context but not in the presence of a cue paired with shock in that context. Kim and Fanselow found such effects when
lesions were made shortly after training (1 or 7 days) but not 28 days later (148 ), whereas Phillips and LeDoux used pretraining
lesions (190 ).
 P.941

Although the role of the hippocampus in contextual fear conditioning had been discovered earlier using a place aversion measure
(223 ), these articles were more influential because they integrated the well-known role of the hippocampus in spatial learning with
a simple, yet powerful measure of classic fear conditioning. Contextual freezing was quickly adopted by investigators interested in
the role of hippocampal LTP in learning because contextual fear conditioning was rapid and long lasting, like LTP, and it was easy to
measure without complex or expensive equipment. The idea was that the hippocampus was required to form a representation of the
context and that this representation was then associated with shock, perhaps in the amygdala. The hippocampus was not needed
when an explicit cue, such as a tone, was used because this could be relayed directly to the amygdala without having to be
processed by the hippocampus.

As attractive as this hypothesis is, there are problems with concluding that the hippocampus is involved in all forms of contextual
conditioning (96 ,97 ,202 ). Hippocampal lesions often produce substantial behavioral activation, which may interact with the
expression of freezing and lead to a disruption of the freezing response itself, rather than of contextual fear. In fact, hippocampal
lesions disrupt not only conditioned freezing responses, but also unconditioned freezing responses, such as the response elicited by a
rat when confronted by a cat (32 ,34 ,147 ). The finding that hippocampus lesions did not block freezing to an explicit cue makes
this competing response interpretation more difficult to accept, but some studies have found that hippocampal lesions disrupt
freezing to an explicit cue (167 ). However, increases in activity cannot account for disruption of contextual freezing by
hippocampal lesions in all instances. In an elegantly designed study, Anagnostaras et al. showed that hippocampal lesions disrupted
freezing to a context that had been paired with shock shortly before surgery (12 ). In the same subjects, however, freezing to a
second context, that had been paired with shock 28 days preoperatively, was not impaired. Thus, the freezing deficit to the recently
conditioned context could not have resulted from an inability to freeze.

Although pretraining electrolytic lesions of the dorsal hippocampus (167 ) block contextual fear conditioning, neurotoxic lesions fail
to do so (97 ,167 ,202 ), as does local infusion of muscimol (128 ). To explain this difference, Maren et al. suggested that rats with
damage to cells in the hippocampus pick out salient explicit cues in the context and use these as elemental cues for fear
conditioning (167 ). However, these investigators suggested that rats with electrolytic lesions do not do this because the lesion
disrupts fibers that connect the ventral subiculum to the nucleus accumbens, which decreases exploration and thus sampling of the
context to pick out salient explicit cues to associate with shock. In fact, experiments found a blockade of the acquisition but not the
expression of contextual fear conditioning measured by freezing using infusion into the nucleus accumbens of a local anesthetic
(108 ). This effect did not occur using tone-shock pairings, even using a weaker trace conditioning procedure that produced
relatively low levels of freezing to the tone.

Another possibility is that fibers from the dorsal to the ventral hippocampus are important in these anterograde amnestic effects of
electrolytic lesions of the dorsal hippocampus because neurotoxic lesions of either the entire hippocampus (102 ,202 ) or just the
ventral hippocampus blocked contextual freezing, whereas neurotoxic lesions of the dorsal hippocampus again failed to block
contextual conditioning (202 ). However, in contrast to the hypothesis that contextual fear conditioning involves processes similar to
spatial learning, lesions of the ventral hippocampus did not block but instead actually facilitated spatial learning in a water maze
task (202 ). As these investigators concluded, these data directly contradict the “widely held notion that spatial and contextual
forms of learning are essentially different manifestations of the same basic underlying process” (202 ). Because these lesions also
impaired freezing to a tone, these authors suggested that the ventral lesions disrupted freezing by increasing activity.

Because all these studies have relied on freezing as the measure of conditioned fear, it is important to assess the effects of
hippocampal lesions on other behavioral or autonomic responses associated with fear. If hippocampal lesions disrupted multiple
measures of contextual fear, it would provide further support for the hippocampal theory of context conditioning. However,
posttraining hippocampal lesions were found not to disrupt context-specific potentiated startle, even though context-elicited
freezing was disrupted in the same animals (174 ). This could not be explained by an excitatory effect of hippocampal lesions on
startle amplitude itself (96 ). In contrast, lesions of the CeA completely blocked both freezing and startle.

In another experimental design (95 ), lesions of the dorsal hippocampus failed to block a phenomenon called contextual blocking,
whereby prior contextual fear conditioning retards subsequent cue conditioning. However, as in other studies, freezing to the
fearful context was blocked by hippocampal lesions. These data, along with several other reports in the literature (96 ,97 ,202 ),
severely limit the general impression that the hippocampus is required for contextual fear conditioning. However, it does seem to be
involved in certain situations, so further work is needed to predict those occasions in which it is and is not involved.

BRAIN SYSTEMS IN THE INHIBITION OR SUPPRESSION OF FEAR AND

ANXIETY
Part of "64 - Neural Circuitry of Anxiety and Stress Disorders "

Extinction
The inability to suppress unwanted fear memories or irrational worry is a major problem in many psychiatric disorders, yet very
little is known about brain systems involved
 P.942

in the inhibition of fear. One way to study this important problem is to analyze brain systems involved in extinction, defined as a
reduction in conditioned fear when the CS is presented many times in the absence of the US. Although such a procedure can
decrease the conditioned response, this does not result from an erasure of the original fear memory. Instead, something new is
learned that overcomes or competes with the original fear memory. For example, an extinguished conditioned response can return
with the passage of time (spontaneous recovery, 187 ), after a subsequent stressor (reinstatement, 201 ), or when testing occurs in a
different context (renewal, 36 ). Such results indicate that extinction (but see discussion in ref. 74 ) may involve a form of active
inhibition that is fragile compared with conditioned fear itself.

Conditioned Inhibition
In a conditioned inhibition procedure, cue 1 predicts food or shock, and a compound stimulus (cue 1 plus cue 2) predicts the absence
of these USs. There is general agreement that conditioned inhibition, closely related to extinction, does involve active inhibition. In
fact, it has been argued that extinction is a special case of conditioned inhibition (38 ). The summation test is the basic method for
observing conditioned inhibition (200 ). In this procedure, the putative conditioned inhibitor (e.g., a light) is presented in compound
with an excitatory CS (e.g., a tone). If the combination produces a decrease in the conditioned response below the level observed
when the CS is presented alone, then that stimulus is said to act as a conditioned inhibitor. When the conditioned inhibitor is
removed, excitation returns to its original level. Various control procedures indicate that a stimulus trained in this way is in fact
acting by inhibition.

Because psychotherapy often involves procedures to rid patients of unwanted fear memories, a behavioral analysis of extinction or
conditioned inhibition has certain clinical implications, as suggested by Bouton and Swartzentruber (39 ). As they pointed out,
“performance after extinction is inherently unstable” (39 ). Phenomena such as spontaneous recovery and reinstatement may
explain why conditioned fears and phobias in humans sometimes seem to return spontaneously without any obvious cause. The
renewal effect may explain why fears reduced successfully in the therapist's office reappear when the patient returns home or to
work. If a drug is used as an adjunct to therapy, renewal of fear could occur when the fearful stimulus is encountered in the absence
of the drug. In fact, animal experiments show that when benzodiazepines are given during extinction, fear of the CS returns when
testing occurs in the absence of the drug (37 ).

Brain Areas in Extinction or Conditioned Inhibition

Sensory Cortex
Assuming that extinction results from active inhibition (see earlier), one could expect that lesions of various brain areas would
disrupt either the development or expression of extinction. LeDoux, Romanski, and Xagoraris reported that rats given ablations of
visual cortex before light–foot shock pairings failed to show extinction of lick suppression relative to sham controls over days (156 ).
In a similar study employing heart rate conditioning in the rabbit, Teich et al. showed that although bilateral lesions of either
auditory or visual cortex did not disrupt acquisition of fear conditioning to a tone CS, auditory cortex lesions, but not visual cortex
lesions, blocked extinction of conditioned heart rate responses to the tone (237 ). Based on known anatomic connections between
sensory cortex and thalamic structures, the authors of both experiments concluded that, during extinction, sensory cortices exert a
modality specific inhibition of the thalamic structures important for the performance of conditioned responses.

However, my colleague and I found no effect of complete visual cortex lesions on extinction of fear-potentiated startle using a
visual CS when the lesions were made either before light-shock pairings or after light-shock pairings and extinction (77 ). Although
there were procedural differences between these reports, the conclusion that sensory cortex is universally involved in extinction of
conditioned fear is not supported.

Frontal Cortex
Rats with lesions of the ventral medial prefrontal cortex made before fear conditioning required more days to reach an extinction
criterion using an auditory CS and freezing as the measure of fear (178 ). However, in these same animals, extinction of conditioned
fear to contextual cues was not impaired. In an extensive series of experiments, my colleagues and I found normal rates of
extinction to both explicit and contextual cues after total removal of the ventral medial prefrontal cortex using both freezing and
fear-potentiated startle as measures of conditioned fear (94 ). Because the lesions in the study by Morgan et al. were performed
before fear conditioning (178 ), the apparent blockade of extinction after ventral medial prefrontal cortex lesions may have resulted
from an increase in the strength of original fear conditioning, rather than from interfering with the process of extinction. Although
the lesions and shams groups did not differ significantly in their level of freezing before the extinction sessions, freezing to explicit
cues often becomes maximal after a very few training trials, so “ceiling effects” may well have been operating. Because extinction
rate can be a more sensitive index of the strength of original
 P.943

conditioning than the terminal level of performance before the initiation of extinction (15 ), the slower rate of extinction in the
lesioned animals may have reflected a stronger degree of original learning. Although these authors do not believe their effects can
be explained in this way (177 ), the finding that the lesions had no effect on the rate of extinction of context conditioning, which
clearly was not at the ceiling of the freezing scale, is consistent with this interpretation. Similarly, we did not find any effect of
pretraining ventral prefrontal cortex lesions on extinction of contextual fear-potentiated startle or freezing (94 ). In addition, we
did not find any effect of ventral medial prefrontal cortex lesions on extinction when lesions were made after fear conditioning but
before extinction (94 ). Morgan and LeDoux also found no effect on the rate of extinction when ventral prefrontal cortex lesions
were made after fear conditioning, but before extinction (176 ). If the frontal cortex is required for the development of extinction
or for the inhibition of fear after extinction, one would expect lesions to block the development of extinction, irrespective of
whether the lesions were made before or after the initial phase of fear conditioning.

Similarly complex effects on extinction have been reported regarding depletion of dopamine in the prefrontal cortex (181 ).
Preconditioning lesions of dopamine terminals in the medial prefrontal cortex retarded the rate of extinction when a 0.8-mA shock
was used but not when a 0.4-mA shock was used. Inspection of the results strongly suggests that the 0.8-mA group was at the ceiling
of the measurement scale at the beginning of the extinction session, whereas the 0.4-mA group was not. Conversely, 6-
hydroxydopamine lesions of the frontal cortex substantially retarded extinction after 0.8-mA tone-shock pairings, even when the
lesions were made after training (181 ). Thus, it is possible that dopamine levels in the prefrontal cortex are important for
extinction when conditioning has produced high, but not more moderate, levels of fear, although further studies using posttraining
lesions are required to verify this.

Quirk et al. found that pretraining lesions of the ventral medial prefrontal cortex did not block the development of conditioned
freezing or the rate of within session extinction (191 ). However, the lesioned rats showed much more spontaneous recovery
measured 24 hours later. Similar results were found in rats given systemic administration of an NMDA antagonist (193 ). In contrast,
we found no change in the rate or final level of extinction, measured with fear-potentiated startle and freezing, when extinction
was assessed over 18 daily sessions using a small number of CS presentations each day (94 ). There also were no differences in the
degree of spontaneous recovery measured 5 days later or in shock-induced reinstatement measured 24 hours after a single foot
shock. Hence, the findings of Quirk et al. may depend critically on the use of a relatively small amount extinction training
(191 ,193 ). Moreover, their lesions were generally more ventral than ours, and this may have contributed to the differences. Clearly,
more work needs to be done to examine the limits of the role of the prefrontal cortex in extinction of conditioned fear, given the
clinical importance of these data.

Hippocampus
Although a complete review of the hippocampal literature on extinction is beyond the scope of this chapter, this brain area has
received a great deal of experimental attention and was once widely believed to be involved in extinction. Theories of extinction
confront the problem of designing a mechanism capable of discriminating occasions of reinforcement from nonreinforcement.
Douglas suggested that the hippocampus is a nonreinforcement detector providing the organism with the means to “tune out”
information that is of no motivational consequence (70 ). It is possible that the hippocampus recognizes that the CS is no longer
followed by the US and inhibits relevant sensory or conditioned response production centers.

Various conditioning paradigms have been used to assess the role of the hippocampus in extinction, including the rabbit nictitating
membrane response (29 ,219 ,228 ), conditioned heart rate (44 ), and conditioned suppression (152 ). Although some of these
experiments have found that hippocampal lesions attenuate extinction (219 ), others have found no effect (228 ), and still another
has shown facilitated extinction (152 ).

Because extinction is context specific (see earlier), one could expect that lesions of the hippocampus would disrupt this contextual
control of extinction. However, direct tests of this hypothesis have not found a disruption of context specific extinction using
pretraining lesions. Hence, neither fimbria-fornix lesions (252 ) nor excitotoxic lesions of the entire hippocampus (87 ) had any
effect on the rate of extinction or on renewal of conditioned fear, although both types of lesions disrupted reinstatement. In
contrast, large hippocampal lesions were not found to disrupt reinstatement of appetitively conditioned behavior (84 ). Overall,
therefore, the role of the hippocampus in extinction remains uncertain.

Neurotransmitters in Extinction

Role of NMDA Receptors in Extinction

Local infusion of NMDA antagonists into the amygdala blocked the development of extinction measured with fear-potentiated startle
(76 ). Intraamygdala infusion of AP5 also blocked extinction using an auditory CS and freezing as a measure of conditioned fear
(158 ). Perhaps similarly, systemic administration of the NMDA antagonist MK-801 fully blocked extinction of conditioned analgesia, a
reliable
 P.944

measure of conditioned fear (62 ), and this effect could not be explained by state-dependent context extinction. A similar blockade
of extinction was reported using a lick-suppression paradigm (19 ), as well as extinction of the rabbit nictitating membrane
preparation (143 ). Taken together, these data indicate that NMDA antagonists can block the development of extinction measured on
subsequent test sessions. This may even occur under conditions in which the antagonist does not block the development of short-
term extinction. Thus, systemic injection of the NMDA antagonist CPP before extinction blocked the expression of conditioned
freezing by about 40% but did not block the development of extinction. However, the CPP group showed substantial recovery of
conditioned freezing measured 24 hours later, a finding suggesting that CPP blocked the long-term development of extinction. (193 ).
Interestingly, these investigators found a similar effect with preconditioning lesions of the ventral prefrontal cortex (191 ), although
the connection between these two sets of data remains to be made.

Role of GABA in Extinction

Several studies have suggested that GABA agonists given before nonreinforced CS presentations interfere with the development of
extinction (37 ,74 ). However, many of these effects may be attributable to state-dependent learning rather than to a blockade of
learning during nonreinforced CS exposure. For example, Bouton, using lick suppression as a measure of fear, showed a blockade of
extinction when rats were given chordiazepoxide during nonreinforced CS presentations and were then tested in the absence of the
drug (37 ). However, when chordiazepoxide also was given before testing, extinction was still evident. This finding suggests that the
benzodiazepine did not actually block the learning that was occurring during extinction, but, instead, the change in drug state
between extinction and testing was the factor that produced renewal (36 ).

Interestingly, a series of experiments by Harris and Westbrook found similar effects on excitatory conditioning. For example, rats
given fear conditioning after injection with benzodiazepines showed an impairment in conditioned freezing measured 24 hours later
in the same context compared with rats trained under the drug but tested in a different context (111 ) or rats given a stressor
before testing (109 ). Thus, the benzodiazepines did not actually prevent original learning, but instead produced a state during
conditioning that interfered with retrieval during testing.

Hence, it would seem that GABA agonists do not directly interfere with either excitatory or inhibitory learning, but, instead, act on
processes that are important for retrieval of prior learning. However, if extinction is a form of active inhibition, it is possible that
GABA may be one of the neurotransmitters necessary for the expression of extinction. In fact, in an elegant set of experiments,
Harris and Westbrook provided evidence that extinction is mediated by GABA release (110 ). Pretraining or pretesting administration
of the inverse agonist FG 7142, which decreases GABA transmission, blocked both development and expression of extinction to an
auditory CS paired with foot shock, using freezing as a measure. This effect could not be ascribed to state dependency or to a
ceiling effect. Pretest administration of FG 7142 reinstated freezing when assessed in the context where extinction took place, but
not in a novel context, which itself reinstated freezing, and the two effects were not additive statistically. However, the disruption
of extinction by FG 7142 was not complete, a finding leaving open the possibility that other mechanisms and neurotransmitters also
may be involved.

Role of Adrenocorticotropic Hormone and Vasopressin in Extinction

Work by DeWied, Van Wiersima, Izquierdo, and Richardson and their co-workers indicated that administration of various peptides
such as adrenocorticotropic hormone or vasopressin either before or after extinction training attenuates subsequent extinction
performance (for review see ref. 74 ).

Neural Systems in Conditioned Inhibition

Using fluorodeoxyglucose autoradiography to measure neural activity, Mcintosh and Gonzalez-Lima compared region-specific activity
in parallel auditory pathways in two groups presented with a tone-light compound (172 ). In both groups, the tone was a fear-
eliciting CS. The groups differed with respect to the significance of the light, which had been trained as a conditioned inhibitor in
one group and as a neutral stimulus in the other. Thus, the experiment allowed for an analysis of whether a conditioned inhibitor
modulates activity in sensory areas normally activated by an auditory fear-eliciting CS. Interestingly, in only one area, the ventral
medial geniculate nucleus, was there a significant difference in activation between the two groups. This structure showed less
activation in the conditioned inhibition group, a finding suggesting that a conditioned inhibitor may act at this locus in the auditory
pathway to inhibit conditioned fear normally produced by an auditory CS.

We found normal conditioned inhibition of fear-potentiated startle, using a visual excitatory stimulus and an auditory conditioned
inhibitor after lesions of either the medial prefrontal cortex (94 ) or the nucleus accumbens (75 ). In addition, local infusion into the
nucleus accumbens of either amphetamine or glutamate antagonists did not alter the magnitude of conditioned inhibition, as they
alter responding to conditioned reinforcers trained in an operant situation (46 ,236 ).

In an appetitive learning situation, Holland et al. reported that lesions of the hippocampus appeared to block
 P.945

feature negative conditional discrimination (127 ), a phenomenon closely related to conditioned inhibition.
Several studies suggested that the lateral septum may play an important role in conditioned inhibition. Using pavlovian
discriminative fear conditioning, single-unit firing rates in the dorsal lateral septal nucleus increased in the presence of a
conditioned inhibitor and decreased in the presence of a conditioned excitor (238 ,254 ). This finding was not seen when recordings
were made in the medial septal nucleus (253 ). More recently, Yadin and Thomas reported that stimulation of the same area of
dorsolateral septal nucleus inhibited restraint stress-induced ulcers (255 ). Using c-fos mRNA as a measured of neuronal activation,
we found a unique increase in c-fos in a ventral part of the lateral septum, the so-called septohypothalamic nucleus, when a
conditioned inhibitor of fear was presented (53 ). Curiously, however, lesions of the lateral septal nucleus did not block the
expression of conditioned inhibition in preliminary pilot studies, although further work certainly is required to evaluate the role of
the lateral septum, perhaps using acute inactivation techniques rather than lesions.
One study suggests that the dorsal central gray may play an important role in conditioned inhibition of fear. Fendt reported that
posttest infusions of 5 ng of picrotoxin (a GABA chloride channel blocker) into the dorsal central gray, but not the lateral or
ventrolateral central gray, reduced the expression of conditioned inhibition without affecting the expression of conditioned fear
(81 ). Although this result is complicated by the finding that neither 2.5-ng doses nor 10-ng doses affected conditioned inhibition, it
raises the intriguing possibility that a conditioned inhibitor of fear releases GABA into the dorsal central gray. Alternatively, because
low doses of picrotoxin would be expected to activate the dorsal central gray by removing tonic inhibition, these results could be
interpreted as indicating that the dorsal central gray is involved in inhibiting an unknown brain structure mediating conditioned
inhibition (81 ). Given the prominent role of the central gray in the expression of fear (162 ), more work is needed to investigate the
role of the dorsal central gray in conditioned inhibition of fear.

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65
Structural and Functional Imaging of Anxiety and Stress Disorders

Scott L. Rauch

Lisa M. Shin

Scott L. Rauch: Department of Psychiatry, Harvard Medical School; Departments of Psychiatry and Radiology, Massachusetts
General Hospital, Boston, Massachusetts.

Lisa M. Shin: Department of Psychiatry, Harvard Medical School; Department of Psychiatry, Massachusetts General Hospital, Boston,
Massachusetts; Department of Psychology, Tufts University, Medford, Massachusetts.

 GENERAL PRINCIPLES
 RELEVANT NEUROIMAGING FINDINGS IN HEALTHY SUBJECTS
 POSTTRAUMATIC STRESS DISORDER
 OBSESSIVE-COMPULSIVE DISORDER
 SOCIAL AND SPECIFIC PHOBIAS
 PANIC DISORDER
 CONCLUSIONS AND FUTURE DIRECTIONS

GENERAL PRINCIPLES
Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "

Neuroimaging research has emerged as a powerful force in shaping neurobiological models of psychiatric disorders. In this chapter,
neuroimaging findings pertaining to anxiety and stress disorders are reviewed. This review necessarily extends previous ones that we
have written, together with our colleagues, on this same and related topics (97 ,98 ,103 ).

Anxiety and Stress Disorders

Whereas anxiety disorders comprise a discrete category within the current version of the Diagnostic and Statistical Manual (2 ), the
concept of stress disorders is less well defined. Although growing evidence suggests that stress may play a role across a variety of
psychiatric disorders (such as major depression), posttraumatic stress disorder (PTSD) and acute stress disorder remain the only
conditions for which exposure to traumatic stress is explicitly acknowledged as an etiologic factor and a criterion for diagnosis. In
this chapter, we survey the imaging data pertinent to models of PTSD and other anxiety disorders, including obsessive-compulsive
disorder (OCD), specific phobias (SpP), social phobia (SoP; also called social anxiety disorder) and panic disorder (PD).

In general, patients with anxiety disorders suffer either exaggerated fear responses to relatively innocuous stimuli (e.g., phobias) or
spontaneous fear responses in the absence of true threat (e.g., PD). Thus, it is important to consider the mediating neuroanatomy of
normal threat assessment and the fear response. In fact, contemporary models focus on these systems as candidate neural
substrates for the anxiety disorders.

Relevant Neuroanatomy
The limbic system plays an important role in mediating human emotional states, including anxiety. Anterior paralimbic cortex (i.e.,
posterior medial orbitofrontal, anterior temporal, anterior cingulate, and insular cortex) links cortical regions subserving higher
level cognition and sensory processing with deep limbic structures, such as the amygdala and the hippocampus (81 ).

Modern models of threat assessment and the normal fear response have focused on the role of the amygdala (1 ,75 ). The amygdala
is positioned to receive input regarding the environment both directly and, thus, rapidly from the thalamus as well as from sensory
cortex. The amygdala appears to serve several related functions, including preliminary threat assessment; facilitation of fight-or-
flight responses; facilitation of additional information acquisition; and enhancement of arousal and plasticity, so the organism can
learn from the current experience to guide responses optimally in future similar situations.

Conversely, several brain areas provide important feedback to the amygdala (1 ,75 ): medial frontal cortex (e.g., anterior cingulate
and orbitofrontal cortex) may provide critical “top-down” governance over the amygdala, thus enabling attenuation of the fear
response once danger has passed or when the meaning of a potentially threatening stimulus has changed; the hippocampus provides
information about the context of a situation (based on information retrieved from explicit memory stores); and
corticostriatothalamic circuits mediate “gating” at the level of the thalamus and thereby regulate the flow of incoming information
that reaches the amygdala.

Finally, neuromodulators influence the activity within each of these various brain areas, as well as the interactions
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among the nodes of the entire system outlined earlier. Ascending projections from the raphe nuclei (serotonin) and the locus
ceruleus (norepinephrine), as well as widespread local γ-aminobutyric acid–ergic (GABAergic) neurons, are perhaps most relevant to
the physiology of anxiety (30 ,65 ,114 ). These transmitter systems likely serve as the principal substrates for contemporary
anxiolytic medications, including serotonergic reuptake inhibitors, monoamine oxidase inhibitors, other antidepressant medications,
and benzodiazepines.

Neuroimaging Methods
Morphometric magnetic resonance imaging (mMRI) methods can be used to test hypotheses regarding abnormalities in the size or
shape of particular brain structures. Functional imaging methods include positron emission tomography (PET) with tracers that
measure blood flow (e.g., oxygen-15–labeled carbon dioxide) or glucose metabolism (i.e., fluorine-18–labeled fluorodeoxyglucose
[FDG]), single photon emission tomography (SPECT) with tracers that measure correlates of blood flow (e.g., technetium-99– labeled
hexamethylpropylene amine oxime [TcHMPAO]), and functional MRI (fMRI) to measure blood oxygenation level–dependent (BOLD)
signal changes. Each of these techniques yields maps that reflect regional brain activity.

Functional imaging methods can be applied in the context of various experimental paradigms. In neutral state paradigms, subjects
are studied during a nominal “resting” state or while they perform a nonspecific continuous task. Thus, between-group comparisons
are made to test hypotheses regarding group differences in regional brain activity, without particular attention to state variables. In
treatment paradigms, subjects are scanned in the context of a treatment protocol. In pre/posttreatment studies, subjects are
scanned both before and after a trial. Then, within-group comparisons are made to test hypotheses regarding changes in brain
activity profiles associated with symptomatic improvement. Alternatively, correlational analyses can be performed to identify
pretreatment brain activity characteristics that predict good or poor treatment response. In symptom provocation paradigms,
subjects are scanned during a symptomatic state (after having their symptoms intentionally induced) as well as during control
conditions. Within-group comparisons can be made to test hypotheses regarding the mediating anatomy of the symptomatic state;
group-by-condition interactions can be sought to distinguish responses in patient versus control groups. Behavioral and
pharmacologic challenges can be used to induce symptoms. In some cases, when symptomatic states occur spontaneously,
experiments are designed to capture these events without the need for provocation or induction per se. In cognitive activation
paradigms, subjects are studied while they perform specially designed cognitive-behavioral tasks. This approach is intended to
increase sensitivity by employing tasks that specifically activate brain systems of interest. Again, group-by-condition interactions are
sought to test the functional responsivity or integrity of specific brain systems in patients versus control subjects.

Imaging studies of neurochemistry have employed PET and SPECT methods in conjunction with radiolabeled high-affinity ligands. In
this way, regional receptor number and or affinity can be characterized in vivo (i.e., receptor-characterization studies). Other
approaches include the use of magnetic resonance spectroscopy (MRS) to measure the regional relative concentration of select
“MRS-visible” compounds. For instance, MRS can be used to measure the compound N-acetylaspartate (NAA), which is a purported
marker of healthy neuronal density.

These various neuroimaging techniques should be viewed as complementary. Convergent findings across paradigms and laboratories
yield the most cohesive and compelling models of pathophysiology.

RELEVANT NEUROIMAGING FINDINGS IN HEALTHY SUBJECTS

Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "

Anxiety and Other Negative Emotional States

Behaviorally Induced Fear and Anxiety

Fischer and colleagues used PET to study regional cerebral blood flow (rCBF) in bank officials while they viewed security camera
videotape of a robbery that they had experienced previously (46 ). Watching the robbery video was associated with rCBF increases in
orbitofrontal cortex, visual cortex, and posterior cingulate gyrus; rCBF decreases occurred in Broca's area, left angular gyrus,
operculum, and secondary somatosensory cortex. Paradiso et al. studied rCBF in healthy elderly subjects who viewed emotionally
evocative film clips; a fear/disgust versus neutral comparison revealed activation in orbitofrontal cortex, among other areas (93 ).
Similarly, Kimbrell et al. studied induced emotional states in healthy adults (67 ); relative to a neutral condition, an anxiety
condition was associated with rCBF increases in left anterior cingulate and left temporal pole, whereas rCBF decreases were found
in nonparalimbic frontal cortical regions. Liotti et al. used PET and autobiographic memory scripts to examine the neural correlates
of anxiety and sadness in healthy women (77 ); relative to a neutral condition, an anxiety condition was associated with rCBF
increases in insular, orbitofrontal, and anterior temporal cortex and rCBF decreases in parahippocampal gyri.

Pharmacologically Induced Fear and Anxiety

Benkelfat et al. examined rCBF changes associated with the administration of cholecystokinin tetrapeptide versus saline in healthy
persons (10 ). Administration of cholecystokinin
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tetrapeptide was accompanied by increased heart rate and panic symptoms and rCBF increases in right cerebellar vermis, left
anterior cingulate gyrus, bilateral claustrum-insula-amygdala, and bilateral temporal poles. However, further analyses suggested
that the apparent temporal pole activations were attributable to extracranial artifacts of jaw muscle contraction. Ketter et al. used
PET to study rCBF changes during procaine versus saline administration in healthy persons (66 ). In the procaine versus saline
contrast, rCBF increases occurred in amygdala and anterior paralimbic structures, including anterior cingulate gyrus, insular cortex,
and orbitofrontal cortex. Blood flow in left amygdala was positively correlated with fear intensity and was negatively correlated
with euphoria intensity. Similar results were reported by Servan-Schreiber et al. (126 ).

Other Negative Emotions

Several studies examining rCBF associated with behaviorally induced sadness in healthy subjects have implicated both anterior
paralimbic and nonparalimbic frontal cortical areas (4 ,54 ,55 ,77 ,94 ). Some studies of behaviorally induced sadness have also
found rCBF changes within the amygdala (71 ,120 ,121 ). Of note, studies of other behaviorally induced negative emotions, including
anger (39 ,67 ) and guilt (127 ), have likewise found activation of anterior paralimbic cortical territories.

Processing Unpleasant, Arousing, or Threat-Related Stimuli

In functional imaging studies of responses to unpleasant pictures (73 ), several studies have found amygdala activation when
contrasted with a neutral (63 ,72 ) or pleasant picture (92 ) comparison condition. In a separate study, Lane et al. reported that
anterior paralimbic regions were activated when study subjects attended to the emotions evoked by the pictures rather than when
subjects attended to physical attributes of the depicted scenes (70 ). Functional imaging studies have also demonstrated a
correlation between amygdala activity during encoding of emotionally arousing pictures or film clips and subjects' subsequent
memory of them (29 ,59 ).

Several functional imaging studies have shown greater amygdala responses to overtly presented fearful human facial expressions in
comparison with neutral or happy faces (14 ,86 ). Whalen et al. used fMRI and a technique called “backward masking” to study
amygdala responses to emotional faces in the absence of explicit knowledge (142 ). Although subjects were unaware of seeing the
“masked” emotional faces, a comparison of the masked fear and masked happy conditions yielded activation in the amygdala
bilaterally.

Habituation
The term habituation refers to a decrement in responses over repeated presentations of a stimulus. Measures of habituation can be
obtained peripherally (e.g., skin conductance) or more centrally (e.g., rCBF or fMRI BOLD signal). For example, declining fMRI BOLD
signal within the amygdala has been observed in response to repeated presentations of fearful faces, regardless of whether subjects
are aware the stimuli are present (14 ,142 ).

A few studies have directly examined the neural correlates of habituation. Fischer et al. used PET to study rCBF changes over
repeated presentation of videotaped scenes in healthy women (45 ). In separate scanning conditions, subjects watched two
repeated videotaped presentations of neutral park scenes and snakes. From the first to the second presentation of the videotapes,
rCBF decreased in bilateral secondary visual cortex and right medial temporal cortex, including amygdala and hippocampus. In an
fMRI study, Fischer et al. also found response decrements over repeated presentations of human face stimuli in amygdala and
hippocampus, as well as thalamus, and prefrontal, inferior temporal, and posterior cingulate cortex (47 ). Similar results have also
been reported by Wright et al. (146 ).

Conditioning and Extinction

Fear conditioning involves the presentation of a neutral stimulus (i.e., a conditioned stimulus [CS]), such as a tone, that is
temporally paired with an aversive stimulus (i.e., the unconditioned stimulus [US]), such as a shock. After repeated presentations of
the CS and US, the CS alone begins to elicit fear-related autonomic changes, such as skin conductance responses. Subsequently, over
repeated presentations of the CS without the US, fear responses decline, and this process is referred to as extinction. Existing
research suggests that the amygdala plays a critical role in fear conditioning (27 ,35 ,74 ,75 ,141 ), and the medial prefrontal cortex
may play a critical role in the process of extinction (56 ,83 ,84 ).

Fredrikson and Furmark and their colleagues used PET to study healthy subjects who viewed a videotape of snakes (CS) both before
and after the video was paired with shock (US) (49 ,52 ). The findings revealed a significant correlation between rCBF changes in
right amygdala and electrodermal activity changes. Hugdahl et al. used PET to compare patterns of blood flow before and after
classic conditioning in healthy male study subjects, by employing a paradigm in which a tone (CS) was paired with brief shock (US)
(62 ). Extinction was associated with widespread activations in right prefrontal, including orbitofrontal, cortex, as well as left
occipital and superior frontal cortex.

In a different conditioning paradigm, Morris et al. showed study subjects pictures of faces that were previously paired with an
aversive burst of white noise (CS+) and faces that were never paired with the noise (CS-) (85 ). A comparison of the CS+ versus CS-
conditions yielded activation in right thalamus, orbitofrontal cortex, and superior frontal gyrus. There was a positive correlation
between
 P.956

activation in thalamus and in right amygdala, orbitofrontal cortex, and basal forebrain. Morris and colleagues subsequently used PET
and backward masking techniques to study rCBF responses to conditioned face stimuli with and without awareness in healthy male
subjects (87 ). When all CS+ conditions were compared with all CS- conditions, bilateral activation in amygdala was observed. Right
amygdala activation was found in the condition in which subjects were aware; left amygdala activation was found in the condition in
which subjects were unaware of the emotionally expressive face stimuli.

In a single-trial fMRI study, LaBar et al. examined amygdala activation during both acquisition and extinction in a mixed-gender
cohort (69 ). In the acquisition condition, a colored shape (CS+) was paired with a shock (US), whereas a different colored shape (CS-)
was never paired with shock. No shocks were delivered during the extinction condition. Comparing CS+ with CS- trials revealed
activation in periamygdaloid cortex and amygdala during early acquisition and early extinction trials, respectively. Activation in both
these regions declined over time. Büchel and colleagues also used a single-trial fMRI to study the neural correlates of fear
conditioning in healthy subjects (26 ). Study subjects were scanned during an acquisition phase in which two neutral faces (CS+)
were presented with a loud tone (US) and two other neutral faces were presented alone (CS-). To disambiguate the effects of the
CS+ and US, the US was not presented on half of the CS+ trials (i.e., CS+unpaired). The critical comparison, CS+unpaired versus CS-, revealed
activation in anterior cingulate, bilateral insular, parietal, supplementary motor, and premotor cortex. A time by event type
interaction revealed that fMRI signal in amygdala decreased over time in the CS+unpaired condition relative to the CS- condition. Similar
results were reported by Büchel et al. in a trace conditioning study, in which a temporal gap occurs between the offset of the CS
and onset of the US (28 ). These researchers also found conditioning-related hippocampal activation that declined over time.

Summary
Taken together, functional imaging studies in healthy human subjects extend findings from animal research. Normal anxiety and fear
reactions are associated with increased activity in limbic and paralimbic regions, whereas other territories of heteromodal
association cortex exhibit decreased activity. However, similar patterns of limbic and paralimbic activation may be observed in
association with other emotional states, and hence this general profile should not be taken as specific to anxiety or fear. Exposures
to unpleasant, arousing, or threat-related stimuli often produce detectable amygdala responses, which can be associated with
enhanced memory. Additional paralimbic recruitment may be related to a person's attention to his or her emotional state.
Habituation can be observed in widely distributed brain regions, including limbic, paralimbic, and sensory areas. Consistent with
animal data, human imaging results point to a role for the amygdala in fear conditioning and for the frontal cortex in extinction. The
accessory role of the hippocampus in these processes remains less well defined.

POSTTRAUMATIC STRESS DISORDER

Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "

Amygdalocentric Neurocircuitry Model

We previously presented a neurocircuitry model of PTSD that emphasizes the central role of the amygdala and its interactions with
the hippocampus, medial prefrontal cortex, and other heteromodal cortical areas purported to mediate higher cognitive functions
(103 ). Briefly, this model hypothesizes hyperresponsivity within the amygdala to threat-related stimuli, with inadequate top-down
governance over the amygdala by medial prefrontal cortex, specifically, the affective division of anterior cingulate cortex (142 ),
and the hippocampus. Amygdala hyperresponsivity mediates symptoms of hyperarousal and explains the indelible quality of the
emotional memory for the traumatic event; inadequate influence by the anterior cingulate cortex underlies deficits of habituation,
and decreased hippocampal function underlies deficits in identifying safe contexts, as well as accompanying explicit memory
difficulties (21 ). Further, we propose that in threatening situations, patients with PTSD exhibit an exaggerated reallocation of
resources to regions that mediate fight-or-flight responses and away from widespread heteromodal cortical areas, as a neural
substrate for dissociation.

Structural Imaging Findings

mMRI studies have reported smaller hippocampal volumes in veterans with PTSD than in comparison subjects. Bremner and
colleagues (21 ) found that right hippocampal volumes were 8% smaller in 26 veterans with PTSD than in 22 civilians without PTSD. In
addition, the PTSD group exhibited poorer performance on a standard measure of verbal memory, and their percent retention scores
on this test were directly correlated with right hippocampal volume (i.e., lower scores were associated with smaller right
hippocampal volumes). Gurvits and colleagues (58 ) used mMRI to study seven Vietnam combat veterans with PTSD, seven Vietnam
combat veterans without PTSD, and eight nonveterans without PTSD. These investigators found significantly smaller hippocampal
volumes bilaterally for the PTSD group in comparison with both control groups. Across the 14 veterans, hippocampal volume was
inversely correlated with extent of combat exposure and PTSD symptom severity.

Similar hippocampal volumetric differences also have been reported in mMRI studies of PTSD resulting from childhood abuse.
Bremner and colleagues (22 ) found 12%
 P.957

smaller left hippocampal volumes in 17 adult survivors of childhood abuse with PTSD than in 21 nonabused comparison subjects.
Stein and colleagues (134 ) found 5% smaller left hippocampal volumes in 21 adult survivors of childhood sexual abuse (most of
whom had PTSD) than in 21 nonabused comparison subjects. Furthermore, total hippocampal volume was smaller in abused subjects
with high PTSD symptom severity than in those with low PTSD symptom severity. In contrast to these results, DeBellis et al. (36 )
failed to find decreased hippocampal volumes in 44 maltreated children and adolescents with PTSD, compared with 61 nonabused
healthy subjects. However, the PTSD group had smaller intracranial and cerebral volumes than did the comparison group.

Taken together, the results of structural neuroimaging studies of adult samples suggest that PTSD is associated with reduced
hippocampal volume, which, in turn, may be associated with cognitive deficits and PTSD symptom severity. Although the extent of
traumatic exposure may be correlated with hippocampal volume, it appears that differences between PTSD and control groups
cannot be explained by traumatic exposure alone (58 ). The results of DeBellis et al. (36 ) suggest that hippocampal volumetric
differences between groups may not be evident in samples of children and adolescents or in samples of persons with relatively
recent traumatic exposures.

Stress, Glucocorticoids, and the Hippocampus

In this review about neuroimaging of anxiety and stress disorders, it is worth elaborating on the potential relationship between
stress and hippocampal findings in PTSD. Animal research has provided evidence that stress is associated with damage to the
hippocampus (16 ). For example, sustained, fatal social stress in vervet monkeys was associated with degeneration of neurons in the
CA3 region of the hippocampus (139 ); chronic restraint stress in rats was associated with atrophy of apical dendrites of hippocampal
CA3 pyramidal neurons (140 ); and exposure to cold water immersion stress in rats was related to structural damage to hippocampus
(CA3 and CA2 fields) and deceased local CBF in hippocampus (42 ).

The effect of stress on hippocampus appears to be mediated by glucocorticoid hormones. Exposure to glucocorticoids is associated
with hippocampal damage in both rats and primates. For example, Sapolsky et al. reported that chronic exposure to corticosterone
in rats led to a loss of neurons in the CA3 region of the hippocampus (115 ). Woolley and colleagues found that daily corticosterone
injections decreased dendritic branching and length in the CA3 region of the rat hippocampus (145 ). In a study of primates,
Sapolsky et al. reported that chronic exposure to cortisol (through steroid-secreting pellets stereotactically implanted in
hippocampus) was related to neuronal shrinkage and dendritic atrophy in the CA3 region (117 ). Moreover, chronic stress during
development is capable of inhibiting normal cellular proliferation within the hippocampus, a process mediated by glucocorticoids
and glutamatergic transmission by an N-methyl-D-aspartate receptor–dependent excitatory pathway (57 ).

Clinical research has also revealed decreased hippocampal volumes in humans with elevated cortisol levels resulting from Cushing's
syndrome (130 ); furthermore, in these patients, a treatment-related reduction of cortisol levels is associated with increased
hippocampal volumes (131 ). High cortisol levels and decreased hippocampal volumes have also been found in patients with major
depressive disorder (25 ). The hippocampus is also involved in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis, and
lesions to hippocampus appear to increase the release of glucocorticoids during stress (43 ,60 ); this, in turn, may further damage
the hippocampus (116 ).

Although these findings may have great relevance to anxiety and stress disorders, the picture is complicated by the finding that
cortisol levels are characteristically reduced, rather than elevated, in PTSD (147 ). One parsimonious theory suggests that patients
with PTSD suffer hypersensitivity to glucocorticoids, resulting in both reduced levels of cortisol (because of accentuated feedback
inhibition) and reduced hippocampal volume (147 ).

Functional Imaging Findings

Semple and colleagues used PET to study six patients with combat-related PTSD and comorbid substance abuse versus seven normal
control subjects (125 ). rCBF was measured in three conditions: resting state, an auditory continuous performance task, and a word
generation task. Compared with the control group, the PTSD group exhibited greater rCBF during both task conditions within
orbitofrontal cortex.

Rauch and colleagues studied a mixed-gender cohort of eight subjects with PTSD, using PET and a script-driven imagery method for
inducing symptoms (104 ). In the provoked versus control condition, patients exhibited increased rCBF within anterior cingulate
cortex, right orbitofrontal, insular, anterior temporal and visual cortex, and right amygdala. rCBF decreases occurred within left
inferior frontal (Broca's area) and left middle temporal cortex. Interpretations of this initial study, with regard to the
pathophysiology of PTSD, were limited by the absence of a comparison group.

Using a similar paradigm and a comparison group, Shin and colleagues studied eight women with childhood sexual abuse–related
PTSD and eight matched trauma-exposed control subjects without PTSD (129 ). In the traumatic versus neutral comparison, both
groups exhibited anterior paralimbic activation. However, a group-by-condition interaction revealed that the control group
manifested a
 P.958

significantly greater rCBF increase within anterior cingulate cortex than did the PTSD group, whereas the PTSD group showed
significantly greater rCBF increases within anterior temporal and orbitofrontal cortex. Bremner and colleagues (20 ) also used script-
driven imagery and PET to study rCBF in ten female survivors of childhood sexual abuse with PTSD and 12 without PTSD. Consistent
with the findings of Shin et al. (129 ), Bremner and colleagues (20 ) reported relatively attenuated recruitment of anterior cingulate
cortex in the PTSD group.

Bremner and colleagues (23 ) studied rCBF responses to trauma-related pictures and sounds in ten Vietnam veterans with PTSD and
in ten veterans without PTSD. In the combat versus neutral comparison, the PTSD group exhibited rCBF decreases in medial
prefrontal cortex (subcallosal gyrus) and anterior cingulate cortex. Liberzon and colleagues (76 ) used SPECT to study rCBF in 14
Vietnam veterans with PTSD, 11 veteran control subjects, and 14 healthy nonveterans. In separate scanning sessions, subjects
listened to combat sounds and white noise. In the combat sounds versus white noise comparison, all three groups showed activation
in anterior cingulate/medial prefrontal cortex, but only the PTSD group exhibited activation in the left amygdaloid region.

Bremner et al. (17 ) used PET to examine the effect of yohimbine challenge on glucose metabolic rates in ten combat veterans with
PTSD and in ten nonveteran subjects without PTSD. Yohimbine administration was associated with increased anxiety and panic
symptoms, as well as widespread decreases in cerebral glucose metabolism in the PTSD group.

Shin and colleagues studied seven patients with combat-related PTSD and seven matched trauma-exposed control subjects without
PTSD in the context of a PET cognitive activation paradigm (128 ). Subjects were required to make judgments about pictures from
three categories: neutral, general negative, and combat-related. Subjects performed two types of tasks: one involved responding
while actually seeing the pictures (perception), and another involved responding while recalling the pictures (imagery). In the
combat imagery versus control conditions, the PTSD group exhibited rCBF increases in right amygdala and ventral anterior cingulate
gyrus and rCBF decreases in left inferior frontal gyrus (Broca's area).

Using another cognitive activation paradigm, Rauch et al. studied the functional integrity of the amygdala in eight combat veterans
with PTSD and eight healthy combat veterans (108 ). During fMRI, subjects viewed fearful and happy faces temporally masked by
neutral faces. Healthy persons are typically aware of seeing only the neutral faces, although they show amygdala activation to the
masked fearful faces (142 ). Rauch and colleagues found greater amygdala activation to masked fearful faces in persons with PTSD
than in control subjects (108 ). Furthermore, the magnitude of amygdala activation was correlated with PTSD severity. These results
suggest that PTSD may be characterized by amygdala hyperresponsivity to general threat-related stimuli, consistent with our
neurocircuitry model of PTSD.

Imaging Studies of Neurochemistry

Schuff and colleagues used mMRI and MRS to study seven veterans with PTSD and seven nonveteran control subjects (123 ). Although
these investigators found a nonsignificantly smaller (6%) right hippocampus in the PTSD group by mMRI, they found an 18% reduction
in right hippocampal NAA by MRS, a finding suggestive of reduced density or viability of neurons in this region.

DeBellis and colleagues used MRS to study NAA/creatine ratios in 11 maltreated children and adolescents with PTSD and 11 healthy
comparison subjects without histories of maltreatment (37 ). The PTSD group had lower NAA/creatine ratios in pregenual anterior
cingulate gyrus. This result is consistent with those of symptom provocation PET studies (20 ,23 ,129 ), which have reported failure
to activate anterior cingulate in PTSD.

Bremner et al. (18 ) used SPECT and [123I]iomazenil to study benzodiazepine-receptor binding in 13 veterans with PTSD and 13
nonveterans without PTSD. These investigators found decreased benzodiazepine-receptor binding in prefrontal cortex in the PTSD
group, relative to the control group.

Summary
Taken together, data from neuroimaging studies are consistent with the current neurocircuitry model of PTSD that emphasizes the
functional relationship among the amygdala, hippocampus, and medial prefrontal cortex. Hippocampal volumes and NAA levels
appear to be decreased in persons with PTSD. These findings dovetail with animal research that points to a relationship among stress,
HPA axis function, and cell viability within the hippocampus. Functionally, in comparison with control subjects, patients with PTSD
exhibit the following: (a) greater activation within orbitofrontal cortex, anterior temporal poles, and the amygdala; (b) diminished
activation in anterior cingulate and medial prefrontal cortex, as well as reduced NAA/creatine ratios in pregenual anterior cingulate;
and (c) decreased activation within widespread areas that are associated with higher cognitive functions, such as Broca's area and
dorsolateral prefrontal cortex.

OBSESSIVE-COMPULSIVE DISORDER
Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "

Corticostriatal Model
One current neuroanatomic model of OCD focuses on corticostriatothalamocortical circuitry (98 ,106 ). According to
 P.959

this model, the primary disorder lies within the striatum (specifically, the caudate nucleus). This leads to inefficient gating at the
level of the thalamus, which results in hyperactivity within orbitofrontal cortex (corresponding to the intrusive thoughts) and
hyperactivity within anterior cingulate cortex (corresponding to anxiety, in a nonspecific manner). Compulsions are conceptualized
as repetitive behaviors that are performed to recruit the inefficient striatum ultimately to achieve thalamic gating and hence to
neutralize the unwanted thoughts and anxiety.

Structural Imaging Findings

The results of several mMRI investigations of OCD have suggested volumetric abnormalities involving the caudate nucleus, although
the nature of the observed abnormalities has been somewhat inconsistent. Scarone et al. (119 ) studied a mixed-gender cohort of 20
patients with OCD versus 16 matched controls and found increased right caudate volume in the OCD group. Robinson et al. (111 )
studied a mixed-gender cohort of 26 patients with OCD versus 26 matched controls and found bilaterally decreased caudate volumes
in the OCD group. Jenike et al. (64 ) studied an all-female cohort of ten patients with OCD versus matched controls and found trends
toward a rightward shift in caudate volume (p = .06) as well as overall reduced caudate volume (p = .10) in the OCD group. Aylward
et al. (3 ) studied a mixed-gender cohort of 24 patients with OCD versus 21 matched controls and found no significant differences in
striatal volumes. Rosenberg et al. (112 ) studied 19 treatment-naive pediatric subjects with OCD and 19 case-matched
psychiatrically healthy comparison subjects. These investigators found reduced striatal volumes in the OCD group and an inverse
correlation between striatal volume and OCD symptom severity.

Functional Imaging Findings

Neutral state paradigms employing PET and SPECT have most consistently indicated that patients with OCD exhibit increased
regional brain activity within orbitofrontal and anterior cingulate cortex, in comparison with neurologically normal control subjects
(6 ,7 ,78 ,89 ,113 ,136 ). Observed differences in regional activity within the caudate nucleus have been less consistent (6 ,113 ).

Pre/posttreatment studies have reported treatment-related attenuation of abnormal regional brain activity within orbitofrontal
cortex, anterior cingulate cortex, and caudate nucleus (8 ,9 ,61 ,95 ,124 ,137 ). In addition, both pharmacologic and behavioral
therapies appear to be associated with similar brain activity changes (8 ,124 ). Some treatment studies have also reported that
lower pretreatment glucose metabolic rates in orbitofrontal cortex predict a better response to serotonergic reuptake inhibitors
(24 ,118 ,136 ).

Symptom provocation studies employing PET (80 ,99 ) as well as functional MRI (15 ) have also most consistently shown increased
brain activity within anterior-lateral orbitofrontal cortex, anterior cingulate cortex, and caudate nucleus during the OCD
symptomatic state.

Cognitive activation studies using PET and fMRI have probed the functional integrity of the striatum in OCD (102 ,105 ). In these
studies, patients with OCD perform an implicit (i.e., nonconscious) learning paradigm shown reliably to recruit striatum in healthy
individuals (101 ,107 ). In both studies, patients with OCD failed to recruit striatum normally and instead activated medial temporal
regions typically associated with conscious information processing.

Imaging Studies of Neurochemistry

Several MRS studies have been performed to measure NAA concentrations in patients with OCD versus healthy comparison subjects.
Ebert and colleagues (41 ) found reduced relative NAA levels in right striatum and anterior cingulate cortex in 12 patients with OCD
in comparison with six healthy control subjects. Bartha and colleagues (5 ) found lower left striatal NAA concentrations in 13
patients with OCD than in 13 matched control subjects.

MRS has also been used to demonstrate elevated glutamatergic concentrations within the striatum of a child with OCD (82 ).
Glutamate is the principal transmitter mediating frontostriatal communication. Interestingly, elevated striatal glutamate levels
were attenuated toward normal after successful pharmacotherapy. These findings suggest that orbitofrontal hyperactivity in OCD
may be mirrored by elevated glutamate at the site of orbitofrontal ramifications in striatum, and treatment-related attenuation of
orbitofrontal activity may be accompanied by decreased glutamate concentration within the striatum.

Summary
Taken together, these neuroimaging findings are consistent with disorders in corticostriatothalamocortical circuitry. Consistent with
the hypothesis of a primary abnormality in the striatum, MRI and MRS studies of OCD have shown reduced striatal volumes and
reduced striatal NAA, respectively. PET studies have revealed hyperactivity within orbitofrontal cortex, and the magnitude of this
hyperactivity predicts response to treatment. In addition, in neurologically normal persons, the performance of repetitive motor
routines does facilitate striatal recruitment in the service of thalamic gating, whereas this pattern is not readily demonstrated in
patients with OCD. These imaging data further support the working model of striatal pathology and striatothalamic inefficiency,
together with orbitofrontal hyperactivity.

SOCIAL AND SPECIFIC PHOBIAS

Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "
 P.960

Neuroanatomic Models
Currently, there are no cohesive neuroanatomically based models for the phobias (53 ,132 ). One possibility is that phobias are
learned and hence reflect another example of fear conditioning to specific stimuli or situations. Alternatively, phobias may
represent the product of dysregulated systems for detecting potentially threatening stimuli or situations. For instance, if humans
have evolved a neural network specifically designed to assess social cues for threatening content, and another to assess threat from
small animals, these may represent the neural substrates for the pathophysiology underlying phobias.

Structural Imaging Findings

Given the high prevalence of phobias and the relative ease with which medication-free phobic subjects without significant
comorbidities can be recruited, it is striking how few imaging studies have been conducted in this arena. Potts et al. used mMRI to
examine volumetric measures of total cerebrum, caudate, putamen, and thalamus in 22 patients with SoP and in 22 matched
healthy control subjects (96 ). The groups did not significantly differ on any of these measures.

Functional Imaging Findings

Studies of SpP to date have principally employed PET symptom provocation paradigms and have reported somewhat inconsistent
results. Mountz and colleagues found that persons with small-animal phobia exhibited increased heart rates, respiratory rates, and
subjective reports of anxiety during exposure to phobic stimuli; however, no changes in rCBF measurements were observed (88 ).

Wik and colleagues studied six patients with snake phobias during exposure to videotapes of neutral, generally aversive, and snake-
related scenes (143 ). During the phobic condition, they found significant rCBF increases in secondary visual cortex and rCBF
decreases in prefrontal cortex, posterior cingulate cortex, anterior temporopolar cortex, and hippocampus. These findings were
similar to those of two other studies of phobia from the same laboratory (50 ,51 ).

Using in vivo exposure and PET, Rauch and colleagues studied rCBF in seven persons with a variety of small animal phobias (100 ). In
the provoked versus control condition, patients with phobias exhibited rCBF increases within multiple anterior paralimbic territories
(i.e., right anterior cingulate, right anterior temporal pole, left posterior orbitofrontal cortex, and left insular cortex), left
somatosensory cortex, and left thalamus.

Whereas one neutral-state SPECT study of patients with SoP and healthy control subjects found no significant between-group
differences in rCBF (133 ), more recent cognitive activation studies performed in conjunction with fMRI have yielded more
informative results. Birbaumer et al. (11 ) used fMRI to study seven patients with SoP and five healthy control subjects during
exposure to slides of neutral human faces or aversive odors. In comparison with the control group, the SoP group exhibited
hyperresponsivity within the amygdala that was specific to the human face stimuli. In a follow-up study, Schneider et al. used fMRI
to study 12 patients with SoP and 12 healthy control subjects (122 ). The researchers used a classic conditioning paradigm in which
neutral face stimuli were the conditioned stimuli and odors (negative odor and odorless air) served as the unconditioned stimuli. In
response to conditioned stimuli associated with the negative odor, the SoP group displayed signal increases within amygdala and
hippocampus, whereas healthy comparison subjects displayed signal decreases in these regions.

Imaging Studies of Neurochemistry

Davidson et al. (34 ) used MRS to study NAA in 20 patients with SoP and in 20 healthy control subjects. Relative to the control group,
the SoP group exhibited decreased NAA in white matter and cortical and subcortical gray matter (e.g., caudate and thalamus).

Tiihonen et al. used SPECT and I-123–labeled β-CIT to measure the density of dopamine reuptake sites in 11 patients with SoP and in
28 healthy comparison subjects (138 ). They found significantly reduced striatal dopamine reuptake binding site density in the SoP
versus control group.

Summary
Although relatively few neuroimaging studies of SpP have been conducted, findings from existing research suggest activation of
anterior paralimbic regions and sensory cortex corresponding to stimulus inflow associated with a symptomatic state. Although such
results are consistent with a hypersensitive system for assessment of or response to specific threat-related cues, they do not provide
clear anatomic substrates for the pathophysiology of SpP. Cognitive activation neuroimaging studies of SoP reveal exaggerated
responsivity of medial temporal lobe structures to human face stimuli; this hyperresponsivity may reflect a neural substrate for
social anxiety in SoP.

PANIC DISORDER
Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "

Neuroanatomic Models
Neurobiological models of PD have emphasized a wide range of disparate elements (31 ). Satisfactory models of PD must account for
spontaneous panic attacks, which are a
 P.961

defining feature of PD. It is possible that spontaneous panic corresponds to a normal physiologic anxiety response that is mediated
by intact fear-anxiety circuits but, owing to homeostatic deficits, occurs in inappropriate, threat-free situations. This is consistent
with theories of hypersensitivity to carbon dioxide at the level of the brainstem (i.e., the suffocation false alarm model), as well as
theories regarding fundamental monoaminergic dysregulation. Another possibility is that panic attacks emerge in the context of
what should be minor anxiety episodes because of failures in the systems responsible for limiting such normal responses;
hippocampal deficits may underlie such a failure to inhibit anxiety responses. Finally, panic episodes described as spontaneous (i.e.,
without identifiable precipitants) could reflect anxiety responses to stimuli that are not processed at the conscious (i.e., explicit)
level, but instead, recruit anxiety circuitry without awareness (i.e., implicitly). There is strong evidence that the amygdala can be
recruited into action in the absence of awareness that a threat-related stimulus has been presented (142 ). By this model, PD may
be characterized by fundamental amygdala hyperresponsivity to subtle environmental cues, triggering full-scale threat-related
responses in the absence of conscious awareness.

Structural Imaging Findings

Fontaine et al. (48 ) published a qualitative MRI study involving 31 patients with PD and 20 matched healthy control subjects. The
frequency of gross structural abnormalities was higher in the PD group (40%) than in the control group (10%); the most striking focal
findings in the PD group involved abnormal signal or asymmetric atrophy of the right temporal lobe.

Functional Imaging Findings

In an initial PET neutral-state study, Reiman and colleagues studied 16 patients with PD and 25 normal control subjects (109 ). In the
subset of patients who were vulnerable to lactate-induced panic (n = 8), the investigators found abnormally low left/right ratios of
parahippocampal blood flow. DeCristofaro et al. used SPECT to measure rCBF at rest in seven treatment-naive patients with PD and
in five age-matched healthy control subjects (38 ). Relative to the control group, the PD group exhibited elevated rCBF in left
occipital cortex and reduced rCBF in the hippocampal area bilaterally. Nordahl et al. used PET and FDG to measure regional cerebral
metabolic rate glucose (rCMRglc) in 12 patients with PD and 30 normal control subjects during an auditory continuous performance
task (90 ). The investigators found that the PD group exhibited a lower left/right hippocampal ratio. In a follow-up experiment (91 ),
these investigators used PET-FDG methods to study imipramine-treated subjects with PD and found a rightward shift in symmetry of
rCMRglc within hippocampus and posterior inferior frontal cortex. In comparison with the untreated group, the imipramine-treated
group exhibited rCMRglc decreases in posterior orbital frontal cortex. Bisaga et al. (12 ) used PET and FDG to study a cohort of six
women with PD and six matched control subjects. In contrast to previous studies, the PD subjects displayed elevated rCMRglc in the
left hippocampus and parahippocampal area.

The literature contains three symptom provocation studies of PD, all of which have employed pharmacologic challenges. Stewart et
al. used SPECT and the xenon inhalation method to measure CBF during lactate infusion in ten patients with PD and in five healthy
control subjects (135 ). The patients with PD who experienced lactate-induced panic attacks (n = 6) displayed global cortical CBF
decreases. Woods et al. used SPECT and yohimbine infusions to study six patients with PD and six normal control subjects (144 ). In
the PD group, yohimbine administration increased anxiety and decreased rCBF in bilateral frontal cortex. In a PET study, Reiman et
al. measured rCBF during lactate infusions in 17 patients with PD and in 15 normal control subjects (110 ). The eight patients who
suffered lactate-induced panic episodes exhibited rCBF increases in bilateral temporopolar cortex and bilateral insular
cortex/claustrum/putamen. Healthy control subjects and patients with PD who did not experience lactate-induced panic attacks did
not exhibit such rCBF changes. Of note, the temporopolar findings were subsequently questioned as possibly reflecting extracranial
artifacts from muscular contractions (40 ,10 ). In a symptom capture case report, Fischer and colleagues (44 ) found that a
spontaneous panic attack was associated with rCBF decreases in right orbitofrontal, prelimbic (area 25), anterior cingulate, and
anterior temporal cortex.

Imaging Studies of Neurochemistry

Dager and colleagues used MRS to measure brain lactate levels during hyperventilation in seven treatment-responsive patients with
PD and in seven healthy comparison subjects (32 ). The PD group showed a significantly greater rise in brain lactate in response to
the same level of hyperventilation. Dager et al. also used MRS to measure brain lactate levels during lactate infusions in 15 patients
with PD and in ten healthy comparison subjects (33 ). The PD group exhibited a significantly greater brain lactate level during
lactate infusion, a finding consistent with the interpretation of reduced clearance, rather than higher production, of lactate in PD.

Three studies have used SPECT and [123I]iomazenil to measure benzodiazepine-receptor binding in PD. Kuikka et al. (68 ) studied 17
subjects with PD and 17 matched healthy comparison subjects and found that the PD group exhibited a greater left/right ratio in
benzodiazepine-receptor uptake that was most prominent in prefrontal cortex. Brandt et al. (13 ) studied 12 medication-naive
patients with PD and nine
 P.962

matched healthy control subjects and found that the PD group exhibited significantly elevated benzodiazepine-receptor binding
within right supraorbital frontal cortex, as well as a trend toward elevated binding in the right temporal cortex. Bremner et al. (19 )
included 13 patients with PD and 16 healthy comparison subjects and found that the PD group showed decreased benzodiazepine-
receptor binding in left hippocampus and precuneus.

Using PET and carbon-11–labeled flumazenil, Malizia et al. studied seven patients with PD and eight healthy comparison subjects
(79 ). These investigators found that the PD group exhibited a global reduction in benzodiazepine binding that was most pronounced
in right orbitofrontal and right insular cortex.

Summary
Resting state neuroimaging studies have suggested abnormal hippocampal activity in PD. Symptom provocation studies have revealed
reduced activity in widespread cortical regions, including prefrontal cortex, during symptomatic states. MRS studies have reported
greater brain lactate levels in response to hyperventilation and lactate infusions. Finally, receptor-binding studies of PD suggest
widespread abnormalities in the GABAergic/benzodiazepine system. Consistent with prevailing neurobiological models of PD, it is
possible that fundamental abnormalities in monoaminergic neurotransmitter systems, originating in the brainstem, underlie the
abnormalities of metabolism, hemodynamics, and chemistry found in widespread territories of cortex. Further, regional
abnormalities within the medial temporal lobes provide some support for theories regarding hippocampal or amygdala dysfunction in
PD.

CONCLUSIONS AND FUTURE DIRECTIONS

Part of "65 - Structural and Functional Imaging of Anxiety and Stress Disorders "

Neuroimaging research is helping to advance neurobiological models of anxiety and stress disorders. At the current early stage of
this scientific enterprise, there are hints of commonalities across anxiety disorders as well as leads regarding disorder-specific
features. Beyond the need for a general expansion of the existing database, it will be critical to explore the specificity of initial
findings by conducting studies with psychiatric comparison groups in addition to healthy control subjects. This is of particular
relevance to the concept of stress disorders, in which common etiologic factors or vulnerability factors may have corresponding
pathophysiologic profiles that are independent of our current diagnostic scheme. For instance, the relationship between early or
chronic life stress and hippocampal structure and function may well span anxiety, mood, and even psychotic disorders. In this light,
longitudinal and developmental studies may be of particular importance in elucidating the neural correlates and consequences of
stress. Similarly, genetic studies in animals and humans will benefit from neuroimaging methods that can illuminate the
bidirectional link from behavior to brain structure, function, and chemistry. For instance, research regarding the heritability of
anxious temperament may be enhanced by using extended phenotypes of conditionability or distributed brain function within
amygdala, hippocampus, and medial frontal cortex. In fact, the gamut of existing animal and human experimental paradigms with
relevance to anxiety and stress disorders provides a promising context for advancing integrated models across scales and
neuroscientific modes of inquiry. As such integrated models evolve, targets for new and improved neuropsychopharmacotherapies
are destined to emerge. Indeed, neuroimaging is likely to play a role not only in conceptually motivating but also in discovering and
testing such new therapies as part of the next generation of progress in this domain.

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66
Current and Emerging Therapeutics of Anxiety and Stress Disorders

Jack M. Gorman

Justine M. Kent

Jeremy D. Coplan

Jack M. Gorman, Justine M. Kent, and Jeremy D. Coplan: Columbia University, New York State Psychiatric Institute, New York,
New York.

During the 1960s and 1970s the concept of “pharmacologic dissection” became popular as a putative method for differentiating
among different categories of psychiatric illness. At the time, it was widely held that anxiety disorders, but not depression, respond
to benzodiazepines, whereas depression, but not anxiety disorders, responds to antidepressants. Panic disorder was held to be the
one exception, responding only to antidepressants. Alprazolam, selective serotonin reuptake inhibitors (SSRIs), and buspirone had
not yet been tested. On the basis of these observations, it was asserted that anxiety and depression are clearly distinct categories of
illness.

Thirty years later we find that the situation has changed dramatically. The first inconsistency with the notion of pharmacologic
dissection was the clear finding that panic disorder does indeed respond to benzodiazepines. For a while, alprazolam and then
clonazepam were regarded by some authorities and clinicians as first-line therapies for panic disorder, replacing tricyclics and
monoamine oxidase inhibitors.

An even more potent challenge, however, has come from the evidence not only that anxiety disorders respond to antidepressants
but also that antidepressants work better than benzodiazepines for most of them. As this chapter discusses, antidepressants are now
considered the appropriate pharmacologic intervention for panic disorder, social anxiety disorder, posttraumatic stress disorder
(PTSD), and generalized anxiety disorder (GAD). The latter case is particularly interesting. Once considered the sole domain of
benzodiazepines, GAD was then shown to be responsive to a drug in an entirely new category, with no relationship whatsoever to
the benzodiazepine receptor or γ-aminobutyric acid (GABA)—buspirone. At about the same time evidence began to accumulate from
just a few studies that GAD might also respond to antidepressants. This evidence was largely ignored, and pharmaceutical companies
were advised to stay away from GAD, a condition supposedly so placebo-responsive that no drug would ever be shown effective in
large clinical trials. To the contrary, venlafaxine is now approved by the Food and Drug Administration (FDA) for the treatment of
GAD, and there is also evidence for the efficacy of paroxetine.

At this point, rather than “dissecting” among the anxiety disorders or between anxiety disorders and depression, pharmacologic
grounds might lead one to assume that these conditions are variants of each other. This also would probably be an oversimplification.
Anxiety and depression are different and we can make distinctions among the anxiety disorders. Nevertheless, the finding that
antidepressants are so ubiquitously effective across categories raises interesting questions and challenges. We review here the
evidence for responsiveness of four anxiety disorders to medication.

 PANIC DISORDER
 GENERALIZED ANXIETY DISORDER
 SOCIAL PHOBIA
 POSTTRAUMATIC STRESS DISORDER
 CONCLUSION
 ACKNOWLEDGMENTS

PANIC DISORDER
Part of "66 - Current and Emerging Therapeutics of Anxiety and Stress Disorders "
Panic disorder (PD) has a reported lifetime prevalence of between 1.5% and 3.5% (1 ,2 ), is highly comorbid with major depression,
and is associated in its own right with significant impairment in psychosocial functioning independent of depressive symptomatology.
In the Epidemiologic Catchment Area study, subjective reports of patients with PD indicate that approximately one-third experience
poor physical and emotional health, rates comparable with major depression (2 ).
Historical Notes
Recognized as a distinct disorder that could be distinguished from the general diagnosis of “anxious neurosis,” in part
 P.968

through the pharmacologic dissection work of Klein and Fink (3 ,4 ) in the 1960s, PD was first categorized as a discrete diagnostic
entity in the Diagnostic and Statistical Manual of Mental Disorders, third edition (DSM-III), in 1980. Despite some minor changes in
diagnostic criteria in the third edition revised (DSM-III-R) and the fourth edition (DSM-IV), primarily involving the number and
frequency of attacks required, the major criteria remain essentially the same. The key triad of symptoms are (a) the occurrence of
spontaneous panic attacks; (b) the presence of anticipatory anxiety; and (c) the presence of phobic avoidance, resulting in some
degree of functional impairment. The pharmacologic treatment of PD has evolved dramatically since the heterocyclic
antidepressants were first established as possessing powerful antipanic properties in the early 1960s (4 ). Throughout the 1970s and
1980s, the heterocyclic antidepressants continued to be the mainstay of pharmacologic treatment of PD, with the monoamine
oxidase inhibitors (MAOIs) used primarily in patients who failed trials of heterocyclic antidepressants. The high potency
benzodiazepines were increasingly prescribed as both primary and adjunct treatments throughout this same time period. With the
introduction of the SSRIs in the United States in the late 1980s and early 1990s for the treatment of depression, this class of drug
began being used in the treatment of PD with promising results. In the late 1990s, several large-scale, controlled trials established
the SSRIs to be effective and safe treatments for PD, thus supplanting the heterocyclic antidepressants and benzodiazepines as first-
line treatment. Although the serotonin, norepinephrine, and GABA systems remain the traditional targets for the majority of
antipanic medications, widely different classes of drugs targeting an array of neurochemical systems are now being explored as
potential treatments for PD.

Heterocyclic Antidepressants
Numerous controlled trials have confirmed the efficacy of the heterocyclic antidepressants, since the initial observations of Klein
(4 ), in both the acute and long-term treatment of PD. In general, heterocyclics with greatest serotoninergic reuptake inhibition
effect, such as imipramine and clomipramine, appear to be most effective in the treatment of PD (5 ,6 and 7 ). By far the best
studied of this class of antidepressants is imipramine, which due to its well-established efficacy has been generally accepted as the
gold standard of PD treatment (8 ). In the Cross-National Collaborative Panic Study, more than 1,000 patients in 14 countries were
randomized into a study comparing imipramine, alprazolam, and placebo (9 ). At the study's end, imipramine and alprazolam were
found to have comparable efficacy, and both were significantly superior to placebo on most outcome measures. A positive dose–
response relationship between imipramine levels and clinical improvement has been reported, with plasma levels of 140 mg/mL
associated with the greatest improvement in panic symptoms (10 ). Although several other of the heterocyclic antidepressants have
been used in the treatment of PD (amitryptyline, desipramine, nortriptyline, clomipramine), far less controlled data are available
supporting their efficacy (11 ,12 ). Although effective, side effects often limit the use of this class of medication in the treatment of
PD. This is particularly true in the case of clomipramine, which due to its anticholinergic and antihistaminergic effects can be
difficult for patients to tolerate (13 ). The use of the heterocyclic antidepressants is often limited by the presence of comorbid
medical conditions such as cardiac disease and glaucoma. Lethality in overdose is another concern given the reported high rates of
suicide in this population when depression is comorbid (14 ,15 and 16 ). Although the SSRIs are often touted as offering a more
tolerable side-effect profile than the heterocyclics, the side-effect burden of imipramine has recently been shown to be comparable
to, although different in nature from, that of the SSRIs, with most side effects (with the exception of dry mouth, sweating, and
constipation) not persisting beyond the first few weeks of treatment (17 ).

Monoamine Oxidase Inhibitors

Like the heterocyclic antidepressants, the MAOIs, are clearly established to be effective in the treatment of PD, yet have yielded to
newer antidepressants with similar antipanic efficacies but less drug–drug and dietary interactions. Among the MAOIs, phenelzine is
the best studied in PD, and its efficacy in the acute treatment of PD is supported by several studies (18 ,19 and 20 ). Dietary
restrictions, lethality in overdose, and drug interaction concerns limit the widespread use of the traditional MAOIs. Stemming from
these concerns, the reversible inhibitors of MAOI (RIMAs) were developed and have demonstrably fewer drug–drug and dietary
interactions. These include moclobemide and brofaromine, neither of which is currently marketed in the United States, but are used
extensively throughout Europe and other parts of the world. The efficacy of moclobemide has been shown in the treatment of PD in
placebo-controlled studies (21 ), and moclobemide has been found to be comparable in efficacy to clomipramine (21 ) and
fluoxetine (22 ). Moclobemide has also been shown to be as effective as fluoxetine in maintenance treatment of PD (23 ).
Brofaromine was shown to be comparable to fluvoxamine (24 ) and clomipramine (25 ) in small randomized, double-blind studies
lacking a placebo. In a placebo-controlled study of the efficacy of brofaromine, 30 patients with PD (DSM-III-R definition) were
treated for 12 weeks. Although there was no significant reduction in the number of panic attacks for those patients treated with
brofaromine, patients demonstrated clinical improvement on several other measures, including agoraphobic avoidance (26 ).
 P.969

Benzodiazepines
The high-potency benzodiazepines, another mainstay of treatment for PD, have been shown to be effective, well tolerated, and safe. In the absence
of comorbid substance abuse, concerns about abuse potential have proven largely unfounded in this population (27 ,28 ,29 and 30 ). Among the
benzodiazepines, alprazolam and clonazepam are labeled for the treatment of panic disorder and have been shown in numerous, controlled trials to
be effective treatments (31 ,32 ,33 ,34 ,35 and 36 ). Clonazepam, with a long half-life of 20 to 50 hours, allows fewer doses per day than the short-
acting alprazolam, and may reduce the likelihood of rebound symptoms between doses. The benzodiazepines have repeatedly been shown to offer an
early advantage in the treatment of anxiety by providing almost immediate relief of anxiety-related somatic symptoms such as muscle tension and
insomnia (27 ,37 ,38 ). However, in the long term, antidepressants may offer the advantage of better targeting and relief of psychic symptoms of
anxiety (37 ), and provide the added benefit of treating associated depressive symptoms. Discontinuing long-term pharmacotherapy with
benzodiazepines can be difficult, with as many as a third of patients with PD being unable to discontinue use due to dependence/withdrawal (27 ).
Thus, despite their efficacy and safety, many clinicians remain concerned about the risk of dependence (39 ).

Selective Serotonin Reuptake Inhibitors (SSRIs)

Among the antidepressants currently used in the treatment of PD, the SSRIs have become first-line treatments (40 ,41 ). Following in the wake of
numerous promising open and controlled trials, several large, multicenter, placebo-controlled studies involving hundreds of subjects each have
demonstrated the efficacy of the SSRIs in the treatment of PD (Table 66.1 ) (42 ,43 ,44 ,45 ,46 ,47 ,48 ,49 and 50 ). Both sertraline and paroxetine
are labeled in the United States for the treatment of PD, and citalopram is approved in several European countries for this indication. Although
fluvoxamine has not been studied in clinical trials on the scale of the other SSRIs, it has been shown to be efficacious in smaller (<100 subjects),
randomized, placebo-controlled studies (25 ,44 ). Despite their established efficacy in the treatment of PD, there are certain problems inherent in
prescribing the SSRIs in patients with PD. Of primary concern on the initiation of treatment is the commonly observed anxiogenic effect, which,
despite being dose-dependent, can nonetheless make the initial several days of treatment a challenge for patients. Initiating treatment with a dose
of one-fourth to one-half that of the normal starting dose may greatly reduce the patient's feelings of restlessness and increased anxiety. The
delayed onset of anxiolytic action contributes to making the period of SSRI initiation difficult for patients with PD. Other effects such as sexual
dysfunction and weight gain with long-term use can make these drugs as problematic as earlier classes for some patients. Overall, though, the side-
effect burden associated with SSRI treatment has been shown to be more tolerable for most patients than the heterocyclics and benzodiazepines
(51 ). Because the SSRIs have been associated with a discontinuation syndrome characterized by anxiety, tremor, dizziness, paresthesias, nausea,
and other symptoms when abruptly stopped, these medications should be tapered over a few weeks, if possible, to minimize discontinuation
symptoms (38 ).

TABLE 66.1. EFFICACY OF THE SSRIs

IN THE ACUTE TREATMENT OF PANIC
DISORDER BASED ON LARGE-SCALE,
PLACEBO-CONTROLLED STUDIES

Newer Antidepressants
Among the newer antidepressants, several have demonstrated promise in PD. Venlafaxine, a serotonin-norepinephrine reuptake inhibitor, has shown
efficacy (on some measures) in a small, placebo-controlled study (52 ). Nefazodone, a weak serotonin-norepinephrine reuptake inhibitor with
serotonin receptor subtype 2C (5-HT2C) antagonist properties, has been shown to reduce anxiety in depressed patients with comorbid PD (53 ).
Mirtazapine enhances both noradrenergic and serotoninergic neurotransmission without reuptake inhibition. Results of an open study involving ten
patients suggested that mirtazapine might be effective in the treatment of PD (54 ). More recently, in a double-blind randomized trial comparing
mirtazapine and fluoxetine in the treatment of PD, both drugs showed comparable efficacy on the primary outcome measures and on most secondary
outcome measures (55 ). Adverse events differed between treatments, with weight gain occurring more frequently in those patients receiving
mirtazapine, whereas nausea and paresthesias occurred more often in those receiving fluoxetine.

Anticonvulsants
Among the anticonvulsants being used in the treatment of PD are valproate and carbamazepine, and the newest anticonvulsants gabapentin,
lamotrigine, pregabalin, and vigabatrin. Valproate has shown promise in several open trials (56 ,57 and 58 ), and one small placebo-controlled study
(59 ). It may be particularly effective when mood instability is comorbid (60 ). There is far less support for the use of carbamazepine in the
treatment of PD, with uncontrolled studies in patients with PD with EEG abnormalities demonstrating some benefit from carbamazepine treatment
(58 ). However, the only controlled trial of carbamazepine in a small number of PD patients (N = 14) did not report a significant difference for
carbamazepine versus placebo in reducing panic attack frequency (61 ). Gabapentin has shown promise (62 ) and is recognized as having a benign
side-effect profile. Lamotrigine, pregabalin, and vigabatrin are currently under investigation.
P.970
 P.971

Beta-Blockers
Although the beta-blockers are more commonly used in the treatment of performance anxiety and as adjunctive treatment in PTSD,
a small number of open studies suggest they may be effective in the treatment of PD (63 ), although they are not considered a first-
line treatment.

Future Directions
Several classes of drugs, although initially viewed as promising, have shown limited efficacy in the treatment of PD. These include
buspirone, bupropion, ondansetron, and the cholecystokinin (CCK) antagonists (64 ,65 ,66 and 67 ). A number of new classes of
drugs are being studied, including the benzodiazepine partial agonists such as abecarnil and pagaclone, and the corticotropin-
releasing hormone (CRH) inhibitors. Experimental agents showing promise in panic-like models in rodents include the group II
metabotropic glutamate receptor agonist LY354740 (68 ) and drugs acting at the neuropeptide receptors, including neuropeptide-Y
agonists and neurokinin substance P antagonists (69 ).

In summary, the expansion of the antipanic armamentarium suggests that, as with many of the psychiatric disorders, there is no
single effective treatment of PD. Among the most commonly prescribed classes of drugs for the treatment of PD [benzodiazepines,
SSRIs, tricyclic antidepressants (TCAs), and MAOIs], there are probably no major differences in treatment efficacy, with most
reported differences in efficacy between classes probably attributable to differences in study design and samples (27 ). The use of
antidepressants, however, and particularly the SSRIs, has supplanted the long-term use of benzodiazepines for this disorder.
Antidepressant use has two main advantages over the benzodiazepines: (a) it provides antidepressant benefits in a population highly
susceptible to depressive symptomatology and comorbid major depression (70 ), and (b) it eliminates the difficulties associated with
withdrawal symptoms upon benzodiazepine discontinuation. In the case of comparable efficacy, medication choice is based on
factors such as latency to onset of therapeutic action, safety, and the individual side-effect profiles of each medication. In this
regard, the SSRIs are currently considered first-line treatment for PD, demonstrating comparable efficacy and superior tolerability
to other treatment classes. Combination therapies are frequently used for treatment resistance, and an approach of prescribing a
benzodiazepine at the initiation of treatment with an SSRI, and later tapering it, has proved to be popular with clinicians and has
recently been demonstrated to be advantageous in the early stages of treatment over an SSRI alone (71 ).

GENERALIZED ANXIETY DISORDER

Part of "66 - Current and Emerging Therapeutics of Anxiety and Stress Disorders "

The diagnostic criteria for generalized anxiety disorder (GAD) have evolved over the past two decades, undergoing substantial
revision to the current definition emphasizing excessive, unrealistic worry as the cardinal feature of this disorder. Defined in 1980 in
DSM-III as a disorder of continuous or persistent worry symptoms of at least 1 month's duration, the diagnosis of GAD was reframed in
DSM-III-R to require symptoms extended for 6 months or longer, and an emphasis on unrealistic worry was stressed. With DSM-IV,
GAD was defined as excessive and persistent
 P.972

worry, accompanied by three or more physical or psychological symptoms of anxiety, persisting 6 months or longer. Because of the
shift in diagnostic criteria and required duration of symptoms over the years, comparison of pharmacologic treatment studies
performed prior to the introduction of DSM-III-R is difficult.

In comparison with panic disorder, PTSD, and obsessive-compulsive disorder (OCD), there are fewer publications devoted to GAD
overall, and only a limited number of published controlled clinical medication trials. Several reasons have been proposed for this
deficiency, including underrepresentation in clinical settings and a view of GAD as a “mild” disorder (72 ). In reality, GAD is one of
the most commonly diagnosed anxiety disorders, with a lifetime prevalence of 4.1% to 6.6% (73 ,74 ), which is often chronic (75 ),
and associated with significant compromise in functioning (76 ,77 ). There remains substantial controversy surrounding the validity
of GAD as a primary disorder, as opposed to a comorbid condition or a prodromal/residual phase of another disorder (78 ,79 ).
Findings from epidemiologic studies of GAD suggest that current comorbidity with other disorders is as high as 58% to 65% (73 ,76 ),
and lifetime comorbidity rates are between 80% and 90% (76 ,80 ). Non-comorbid, “pure” GAD lifetime prevalence was found to be
only 0.5% in the National Comorbidity Survey (76 ). Overall, the sum of studies examining quality of life issues support the idea that
non-comorbid GAD is relatively rare, but is associated with significant impairment in its own right (81 ,82 ).

Historical Notes
Prior to the introduction of the benzodiazepines, the main agents available for the treatment of anxiety were the tricyclic
antidepressants (doxepin, imipramine, amitryptyline), antihistamines (diphenhydramine, hydroxyzine), barbiturates (mephobarbital),
and the sedative antianxiety agent meprobamate (Milltown). The development of the benzodiazepines in the mid-1950s led to the
introduction of chlordiazepoxide (Librium) in 1959, and ushered in an era of benzodiazepine use in the treatment of a wide range of
anxiety symptoms related to anxiety and mood disorders, psychosis, and alcohol withdrawal. Greater tolerability and the superior
safety profiles of the benzodiazepines resulted in a sharp decline in the use of barbiturates for anxiety disorders (83 ). The
benzodiazepines have remained a common treatment choice for GAD throughout the past two decades. However, concerns about
dependence and withdrawal, short-term memory impairment, interactions with alcohol, and psychomotor impairment have resulted
in increased interest in alternative medications. The introduction of drugs such as buspirone (1986), SSRIs (from 1980 on), and the
serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine (1994) have broadened the available treatment armamentarium
for GAD significantly.

Tricyclic Antidepressants
Tricyclic antidepressants (TCAs) have been in use in the treatment of GAD for many decades; however, data supporting their
efficacy from controlled clinical trials are extremely limited. Imipramine is the only TCA shown to be effective in placebo-controlled
trials of GAD patients without comorbid depression (84 ,85 ). In comparator trials, imipramine has been shown to have clinical
efficacy comparable to the benzodiazepines (84 ,86 ,87 ).

Benzodiazepines
Five benzodiazepines (alprazolam, chlordiazepoxide, clorazepate, diazepam, and lorazepam) are currently labeled as treatments for
anxiety (GAD) (88 ). Clonazepam and alprazolam are labeled specifically for the treatment of PD. There are a limited number of
clinical trials demonstrating the efficacy of benzodiazepines in the treatment of GAD in its current (DSM-IV) definition (89 ,90 );
however, benzodiazepines have been shown effective in controlled studies using DSM-III criteria for GAD (91 ).

Buspirone
Buspirone is a serotonin receptor subtype 1A (5-HT1A) partial agonist with anxiolytic properties. In a metaanalysis of placebo-
controlled comparator trials with benzodiazepines, buspirone showed comparable efficacy to the benzodiazepines in eight trials in
735 patients meeting DSM-III criteria (1 month's duration of illness) for GAD (92 ). In a metaanalysis of eight placebo-controlled
studies in over 500 GAD patients with coexisting depressive symptoms, buspirone demonstrated significant superiority to placebo
(93 ). Prior recent treatment with a benzodiazepine (<1 month) has been shown to predict poor response to subsequent buspirone
treatment in GAD (92 ). This may be due to a combination of factors including the presence of benzodiazepine withdrawal,
exacerbation of benzodiazepine discontinuation symptoms by buspirone and its metabolite a-(2-pyridinyl)-piperazine via
enhancement of noradrenergic activity (94 ), and psychological factors such as patient expectations.

SSRIs
The first published study of a medication with significant serotonin reuptake inhibition properties in GAD involved a small, open-
label trial of clomipramine (95 ). The suggestion of efficacy in this study, along with the success of clomipramine in treating other
anxiety disorders, raised interest in pharmacologic agents for GAD that target the serotoninergic system. Following several years
later, the first comparison trial of an SSRI in the treatment of GAD was published by Rocca and colleagues (89 ). Treatment efficacy
of paroxetine was compared with the tricyclic imipramine and the
 P.973

benzodiazepine 2′-chlordesmethyldiazepam in 81 subjects with GAD. Of the 63 patients who completed the randomized, 8-week
study, 68% of the paroxetine group, 72% of the imipramine group, and 55% of the 2′-chlordesmethyldiazepam group were judged to
be responders as measured by a 50% or more decrease in Hamilton Anxiety Rating Scale (HAM-A) scores. The greatest improvement
during the first 2 weeks occurred in the group receiving the benzodiazepine, as expected by the early relief of physical anxiety
symptoms and insomnia provided by this class of medication. However, from the fourth week forward, the paroxetine and
imipramine groups demonstrated superior benefits, particularly in the area of psychic symptoms of anxiety. More recently, the
efficacy of paroxetine was demonstrated in a large, fixed-dose study of more than 500 patients with a DSM-IV diagnosis of GAD
without major depression (96 ). Patients were randomized to receive paroxetine 20 mg/day, paroxetine 40 mg/day, or placebo for 8
weeks. Patients receiving both doses of paroxetine demonstrated significant differences in the primary outcome measure, reduction
in HAM-A score, versus placebo, with 68% on 20 mg paroxetine and 81% on 40 mg paroxetine rated as responders based on a Clinical
Global Impression (CGI-I) score of 1 or 2, versus 52% on placebo.

Venlafaxine
Venlafaxine is an inhibitor of SNRI. Venlafaxine has recently been demonstrated in humans, using peripheral measures, to have
primarily 5-HT reuptake inhibition properties at low doses (75 mg/day), with increasing norepinephrine (NE) reuptake inhibition
properties at higher doses (375 mg/day) (97 ). Shown to be effective in the treatment of anxiety symptoms associated with major
depression (98 ,99 ), the extended release (XR) form of venlafaxine has been shown to be effective in the treatment of GAD (DSM-IV
criteria) in several placebo-controlled studies (100 ,101 ). In a placebo-controlled multicenter comparator trial, 405 patients with
GAD were randomized to receive venlafaxine XR (75 or 150 mg/day), buspirone (30 mg/day), or placebo for 8 weeks. For the 365
patients for whom efficacy measures were obtained, there was no significant difference between groups in improvement on the
primary outcome measure, the HAM-A. However, both doses of venlafaxine were shown to be superior to placebo in improving HAM-
A psychic anxiety and anxious mood scores at the endpoint (week 8), and venlafaxine demonstrated superiority to placebo and
buspirone on the CGI-S at the same time point. More robust efficacy findings for venlafaxine were reported in a recent large,
multicenter trial, involving 377 outpatients with GAD without comorbid depression (101 ). Patients were randomly assigned to
receive either placebo or venlafaxine XR at one of three doses (75, 150, or 225 mg/day) for 8 weeks. Of the 349 patients included in
the efficacy analysis, those receiving 225 mg/day demonstrated significant improvement across seven of the eight outcome
measures, and the 225-mg/day group was the only group to show significant improvement in scores on both of the CGI subscales
(severity, global improvement) versus placebo.

Other Agents (Trazodone, Nefazodone, Anticonvulsants, Partial GABA Agonists)

In a randomized, placebo-controlled comparator trial of trazodone, diazepam, and imipramine in the treatment of 230 patients with
GAD, trazodone was found to be superior to placebo yet somewhat less effective than diazepam and imipramine at the study's
endpoint (84 ). The antidepressant nefazodone, which antagonizes the 5-HT2C receptor and inhibits the reuptake of both serotonin
and NE, has shown promise in the treatment of GAD in an open trial (102 ). Anticonvulsants such as valproate and carbamazepine
have been used in the treatment of GAD; however, evidence of their efficacy is primarily anecdotal, as there are no controlled
clinical trials for either of these medications in the treatment of GAD (103 ). Other agents such as the partial GABA agonist abecarnil
have not demonstrated significant efficacy versus placebo in GAD (101 ).

In summary, although the benzodiazepines have been the mainstay of pharmacotherapy for GAD since their introduction, significant
concerns regarding their long-term use in GAD have fueled the search for other effective treatments. Given the chronic nature of
GAD, medications such as buspirone, and the antidepressants venlafaxine and paroxetine, which have fewer effects on cognitive and
psychomotor function, now represent first-line therapies for GAD.

SOCIAL PHOBIA
Part of "66 - Current and Emerging Therapeutics of Anxiety and Stress Disorders "

Social phobia (SP) (or social anxiety disorder) is reported to be the most common anxiety disorder with a 1-year prevalence of 7% to
8% and a lifetime prevalence of 13% to 14% in patients aged 15 to 54 years. Social anxiety disorder can be classified into two
subtypes—discrete and generalized. Level of disability with SP can be high, and 70% to 80% of patients have comorbid psychiatric
disorders, particularly depression and substance abuse (104 ). Given the high degree of burden of illness in SP, its treatment has
become a major priority.

Historical Notes
Liebowitz et al. (105 ) noted that SP, like atypical depression (106 ), had a specific responsivity to the MAOIs, whereas TCAs,
although effective for PD and “typical” major depression, were not effective for SP (107 ). The efficacy of the MAOIs, which block
reuptake of dopamine in addition to NE and serotonin, prompted speculation about a potential
 P.974

“dopaminergic” component to the neurobiology of SP. Several open clinical studies have attempted to utilize the “dopamine
component” concept in phamacotherapy with some success, e.g., seligiline (108 ) and pergolide (109 ). However, as data
accumulated, other systems were also implicated, and the pharmacologic dissection approach seemed less applicable (see above).
Positive results with the high-potency benzodiazepine clonazepam (110 ) suggested a GABAergic component. However, the
suitability of benzodiazepines for long-term treatment of a chronic condition such as social anxiety disorder has been questioned. In
addition, the benzodiazepines are ineffective against comorbid depression.

RIMAs (Reversible Inhibitor of Monoamine Oxidase A)

Although phenelzine demonstrated efficacy, the need for dietary restrictions severely limited its use. Moclobemide is a RIMA with a
much lower propensity to induce hypertensive crises and has a more favorable side-effect profile. Moclobemide had been reported
to have efficacy in early studies in the treatment of social phobia (111 ). However, conflicting results have subsequently been
reported in placebo-controlled trials. Some studies have shown moclobemide to be more effective than placebo, whereas two
recent, large, randomized placebo-controlled trials conducted in the United States have reported less robust results (112 ,113 ).
Brofaromine, another drug in the RIMA class, may still hold promise. The safety and efficacy of brofaromine were examined in a
multicenter trial of 102 outpatients with SP (114 ). Brofaromine produced a significantly greater change from baseline in Liebowitz
Social Anxiety Scale (LSAS) scores compared with placebo.

SSRIs
Based on clinical evidence, SSRIs are the first-line treatment in social anxiety disorder (115 ). The most extensive database for the
treatment of social anxiety disorder exists for the SSRI paroxetine. Several large, multicenter, placebo-controlled trials have been
completed on three different continents (116 ,117 ,118 and 119 ). In all cited studies, a significantly greater proportion of patients
responded to paroxetine treatment compared with placebo. Paroxetine is currently the only SSRI licensed for use in this condition in
the United States. The SSRIs are particularly attractive agents due to their favorable tolerance and safety profile, although typical
SSRI side effects may nevertheless be problematic.

Despite promising open studies with fluvoxamine, fluoxetine, and citalopram (120 ,121 ) only fluvoxamine has been tested under
double-blind, randomized, placebo-controlled trial conditions (122 ). Like paroxetine, fluvoxamine yielded efficacy data superior to
placebo. A report on a multicenter sertraline trial was pending at the time of this writing.

As is the case with PD, buspirone does not appear effective for SP as monotherapy in placebo-controlled double-blind studies (123 ).
It may, however, have a role in augmentation of the SSRIs.

Serotonin/Norepinephrine Reuptake Inhibitors

One open label study (124 ) aimed to evaluate the clinical response to venlafaxine in SP in 12 patients who were nonresponders to
SSRIs, and to assess how the response could be influenced by Axis II comorbidity with avoidant personality disorder (APD). The
duration of the study was 15 weeks using an open flexible-dosing regimen in individuals with or without concomitant APD.
Venlafaxine improved SP and/or APD symptomatology, as demonstrated by decreasing LSAS total scores. Similar favorable open-
labeled results have been reported for nefazodone (125 ,126 ). Placebo-controlled studies are warranted.

Anticonvulsants
A randomized, double-blind, placebo-controlled, parallel-group study was conducted to evaluate the efficacy and safety of
gabapentin in relieving the symptoms of social phobia. A significant reduction in the symptoms of social phobia was observed among
patients on gabapentin compared with those on placebo as evaluated by clinician- and patient-rated scales (127 ). Adverse events
were consistent with the known side-effect profile of gabapentin. The efficacy of other novel anticonvulsants remains to be
investigated, although encouraging results have been reported for the gabapentin-like compound, pregabalin (128 ).

POSTTRAUMATIC STRESS DISORDER

Part of "66 - Current and Emerging Therapeutics of Anxiety and Stress Disorders "

Despite the high prevalence, chronicity, and associated comorbidity of PTSD in the community, relatively few placebo-controlled
studies have evaluated the efficacy of pharmacotherapy for this disorder. The symptom overlap between PTSD and other
pharmacotherapy-responsive disorders has suggested that pharmacotherapy might be effective. Nevertheless, in those placebo-
controlled trials investigating the pharmacotherapy of PTSD that have been carried out, statistically significant efficacy for the
treatment being studied has traditionally been inconsistent. One of the key methodologic limitations has been the fact that most
studies have been conducted with war veterans, who are likely to constitute a more treatment-refractory population.

SSRIs
More recently, a total of 187 civilian outpatients with DSM-III-R PTSD (73% were women, and 61.5% experienced
 P.975

physical or sexual assault) were treated with the SSRI sertraline in a placebo-controlled design (129 ). Sertraline effectively
diminished symptoms of PTSD of moderate to marked severity in comparison to placebo. Using a conservative last-observation-
carried-forward analysis, treatment with sertraline resulted in a responder rate of 53% at the study's endpoint compared with 32%
for placebo (p = .008). Sertraline is the first medication approved by the FDA for the treatment of PTSD. Similar positive results have
been reported for the SSRI fluoxetine in civilian populations (130 ,131 ). In a study by van der Kolk et al. (132 ), fluoxetine was
found to be most effective in the nonveteran versus veteran portion of his study sample. Although published placebo-controlled data
for paroxetine are not available, Marshall et al. (133 ) have argued that this particular SSRI may have specific advantages because of
its relatively low activating properties. Direct comparative studies are lacking.

Combat Veteran PTSD

Among combat veterans, PTSD is a highly prevalent and often chronic disorder, persisting in as many as 15% of Vietnam veterans for
at least 20 years (134 ). Treatment response in veterans with combat-related PTSD has been disappointing. Although anxiolytics,
anticonvulsants, antipsychotics, and antidepressants, including SSRIs, have been tried, none has been consistently associated with
improvement in all primary symptom domains (i.e., intrusive recollections, avoidance/numbing, and hyperarousal). In an open study
using nefazodone, at mean daily doses of 430 mg (range, 200 to 600 mg/day), 19 treatment-refractory PTSD patients demonstrated
benefit after 12 weeks (134 ). Double-blind placebo controlled studies would be of interest.

The efficacy of the antidepressant drug bupropion in the treatment of male combat veterans with chronic PTSD was investigated in
an open-label study of 6 weeks’ duration (135 ). Improvement was seen in hyperarousal symptoms but was less significant than the
change in depressive symptoms. Mirtazapine, a novel drug with both noradrenergic and serotoninergic properties, may be effective
in individuals who demonstrate intolerance to side effects of, or a limited response to, SSRIs. Three of six severely refractory PTSD
patients treated with mirtazapine were assessed as responders in a pilot study (136 ). Case reports have suggested benefit for
refractory patients treated with venlafaxine (137 ) and risperidone (138 ). Raskind et al. (139 ) reported that the α1-adrenergic
antagonist prazosin ameliorated combat nightmares in a small sample of veterans with PTSD.

Monoamine Oxidase Inhibitors

Traditional MAOIs have shown efficacy in the treatment of PTSD, but their use is limited by serious drug and food interactions.
Moclobemide, a RIMA, is relatively free of these limitations and is therefore potentially useful in the treatment of PTSD.
Moclobemide was highly effective in an open-labeled design (140 ). However, in a double-blind, randomized, placebo-controlled,
multicenter study, brofaromine, also a RIMA, failed to surpass efficacy levels seen with placebo. Thus, the role for RIMAs remains
unclear at this time.

Anticonvulsants
Despite a long-recognized role for anticonvulsants in the treatment of PTSD (141 ), few placebo-controlled studies have been
reported. An open study of divalproex reported favorable results (142 ). In a placebo-controlled study, patients treated with
lamotrigine showed improvement on reexperiencing and avoidance/numbing symptoms compared to placebo-treated patients (143 ).
The authors concluded that lamotrigine may be effective as a primary psychopharmacologic treatment in both combat and civilian
PTSD and could also be considered as an adjunct to antidepressant therapy used in the treatment of PTSD. Further large-sample,
double-blind, placebo-controlled trials are warranted.

Summary and Future Directions

Current management of PTSD is well summarized by Davidson et al. (144 ). Clearly, there are many challenges associated with the
treatment of PTSD. Different patients with PTSD may not respond to pharmacotherapy in the same manner, and it is unclear
whether this is related to gender, trauma type, or other factors. Antidepressants, particularly the SSRIs, are currently the form of
pharmacotherapy for patients with PTSD with greatest support in the literature. Psychosocial techniques, such as cognitive-
behavioral therapy or stress inoculation training, are effective and may be considered as adjunctive therapy with medication. Larger
placebo-controlled studies for many different classes of medications would be desirable in moving the field forward. In addition,
carefully conducted polypharmacy in which drug interactions are well understood may well be necessary for more difficult cases.

In a review by Shalev and colleagues (145 ), a synthesis of findings in PTSD studies is provided. Most studies explored a single
treatment modality (e.g., pharmacologic, behavioral). The cumulated evidence from these studies suggests that several treatment
protocols reduce PTSD symptoms and improve the patient's quality of life. The magnitude of the results, however, was often limited,
and remission was rarely achieved. Given the shortcoming of unidimensional treatment of PTSD, it was suggested by the authors that
combining biological, psychological, and psychosocial treatment yields the best results.

A host of novel compounds show promise for the treatment of PTSD (146 ). Such classes of compounds include corticotropin-
releasing factor antagonists, neuropeptide-Y
 P.976

enhancers, antiadrenergic compounds, drugs that down-regulate glucocorticoid receptors, more specific serotoninergic agents,
agents that normalize opioid function, substance P antagonists, N-methyl-D-aspartate facilitators, glutamatergic antagonists, and
antikindling/antisensitization anticonvulsants.

CONCLUSION
Part of "66 - Current and Emerging Therapeutics of Anxiety and Stress Disorders "

Antidepressants are the logical first choice for most patients with anxiety disorders, based on their efficacy and tolerability.
Although maintaining a role, the use of benzodiazepines for first-line or long-term therapy is now less likely. Does this mean that
anxiety disorders are a variant of depression? Certainly, anxiety disorders and depression are highly comorbid. Untreated, the
majority of patients with anxiety disorders eventually develop depression, whereas a large fraction of depressed patients suffer
from clinically significant comorbid anxiety, if not an overt syndromal anxiety disorder.

Pharmacologic dissection is clearly perilous, leaving us prone to inferences based on limited knowledge. Most of the antidepressants
that successfully treat anxiety disorders manipulate the reuptake of serotonin, norepinephrine, or both. Altering the brain circuits
through these modulatory neurotransmitters in turn has wide-ranging effects on many other systems in the brain. The release of CRH
from extrahypothalamic sites like the amygdala is only one such system. Hence, there may be a common denominator among
anxiety disorders and between anxiety disorders and depression at one or more points in these complex circuits.

The observation, then, that antidepressants work for the four anxiety disorders discussed in this chapter warrants, in our opinion,
only the following inference: it is highly likely that some substrate of the serotonin and norepinephrine systems is malfunctioning in
several anxiety disorders and depression. This could be the locus of a common genetic or environmental vulnerability to both
categories of illness. Although it will not likely tell us that anxiety and depression are fundamentally the same thing, the search for
such common substrates and vulnerabilities, suggested but not guaranteed by the psychopharmacologic findings, is likely to be very
illuminating.

ACKNOWLEDGMENTS
Part of "66 - Current and Emerging Therapeutics of Anxiety and Stress Disorders "

Dr. Gorman has received research support from Pfizer, Eli Lilly, the National Institute of Mental Health, and NARSAD. In addition, he
has been a consultant and received honoraria from a number of pharmaceutical companies, including Pfizer, Eli Lilly, Bristol-Myers
Squibb, Wyeth-Ayerst, SmithKline Beecham, Astra Zeneca, Janssen, Organon, Forrest, Parke-Davis, Lundbeck, Solvay, Merck, Sanofi-
Synthelabo, and Aventis. Dr. Kent has served as a consultant for Bristol-Myers Squibb and SmithKline Beecham. Dr. Coplan has
received research support from SmithKline Beecham, Eli Lilly, and Janssen. In addition, he has served on a speakers’ bureau and/or
an advisory board for the following companies: SmithKline Beecham, Wyeth-Ayerst, and Bristol-Myers Squibb.

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67
The Economic Burden of Anxiety and Stress Disorders

Ronald C. Kessler

Paul E. Greenberg

Ronald C. Kessler: Department of Health Care Policy, Harvard Medical School, Boston, Massachusetts.

Paul E. Greenberg: Analysis Group, Cambridge, Massachusetts.

No society can afford to guarantee universal health insurance coverage for treatment of all illnesses for all of its citizens. The
number of illnesses is simply too large and the costs of treatment too great for such a guarantee even in the most economically
advantaged societies. Resource allocation rules are consequently needed (1 ). The most widely accepted of these rules emphasizes
cost-effectiveness. According to this rule, medical interventions are appropriate only if their expected benefits clearly exceed the
sum of their direct costs and their expected risks (2 ).

The difficulty in implementing this decision rule is that no obvious comparability exists between the single metric in which the costs
of treatment are usually defined (i.e., dollars) and the many different metrics in which the benefits of treatment can be defined
(e.g., physical pain, discomfort, psychological distress, and role impairment). To create transformations across these different
metrics to allow for comparisons of costs and benefits on a single metric, a number of strategies have been developed, such as
assessments of willingness to pay, time trade-off, standard gamble, and other utility or quasi-utility measures (3 ). In addition, a
special interest has evolved in the indirect economic costs of illness and the benefits of treatment in terms of sickness absence and
disability from work. The costs of these role impairments can be more easily assessed than the costs of other adverse effects of
illness and represent the cost-benefit trade-off to purchasers of employer-sponsored health insurance plans (4 ).

The most ambitious effort to date to evaluate the costs of illness in terms of role impairments and disabilities is the World Health
Organization (WHO) Global Burden of Disease (GBD) Study, an initiative de