Presentation on experimental evaluation of memory enhancers

DR AKHIL AGRAWAL
DEPT OF
PHARMACOLOGY
SKNMC PUNE

It is divided into 4 parts:
•INTRODUCTION
oMemory(basic physiology and anatomy)
oMemory enhancers(basics)
•CLASSIFICATION
•EXPERIMENTAL EVALUATION
oIn vitro (Animals)
oIn vivo
oIn humans
•APPLICATION IN MODERN ERA

MEMORY
•It is defined as the process by which
information is encoded, stored and
retrieved
•“Memory” is a slippery concept
because it is still not clear about
complete, consensual notion about
the physical nature of its trace.
• The only sure way to grab at such
phenomenon is by measuring
behaviors and their modifications i.e.
quantifying it in an indirect fashion.
• Such approach is called
“phenomenological”, and opposes
itself to the so-called “mechanistic”
vision, that departs from previously
existent knowledge about the
intrinsic machinery operating behind
the phenomenon

TYPES OF MEMORY
Based on physiological points:
Explicit(recognition)
o Concerned with facts
o Episodic(events)
o Semantic(words,rules,language)
o Area of imp-hippocampus
Implicit (reflexive)
oIncludes skill, habits and
conditioned reflexes
o Priming(facilitate
recognition)
oNon-associative
learning(single stimulus)
oAssociative
learning(relation of
stimulus)
oArea of imp-thalamus

TYPES BASED ON DURATION
 IT IS DIVIDED INTO THREE TYPES:
 Short(seconds-minutes)
o Mechanism-(circuit of reverberating neurons)
 Intermediate(days-weeks)
o Mechanism-(chemical changes in presynaptic and postsynaptic
membrane)
 Long(years-lifetime)
o Mechanism-(structural changes in synapse)

MEMORY PHASES
•The Memory Consolidation Theory, proposed by Müller and Pilzecker is a
fundamental paradigm in the psychobiology and neuropharmacology of
memory
• One consequence of the Consolidation Paradigm is that we can plan our
experimental intervention to take place in two or three different moments
around memory i.e Formation and Recall.
•Since the formation is not an instantaneous process, we may distinguish
two formation phases, Acquisition, better known as learning, and
Consolidation, the labile phase during which the memory trace will be
physically stored
• Recall, also known as memory retrieval, takes place during the reexposition
to the learning context, with or without previously delivered stimuli
Acquisition Consolidation Recall
(1 or N-trials) 0 min hours / days

MEMORY ENHANCERS/NOOTROPICS
 The substance used to enhance cognitive function.
 It can increase memory,motivation,mood or anything
related to cognition and thought.
 They are referred to as “smart” drugs
HISTORY
 The term ‘nootropic’ was coined by DR CORNELEU
GUERGEA in 1972.It is a Greek word with combination of
‘nous’-mind and ‘trepein’- to bend.
 In 1964,he was the first person to synthesize the first
nootropic named PIRACETAM.

COGNITIVE ENHANCERS V/S NOOTROPICS
COGNTIVE ENHANCERS NOOTROPICS
 TEMPORARY
 TOLERANCE PRESENT
 GENERALISED STIMULANT
EFFECT
 CAN CAUSE COGNITIVE
DAMAGE
 MOSTLY NATURAL
 EG-CAFFEINE
 PERMANENT
 NO TOLERANCE
 ABSENT
 NO
 MOSTLY ARTIFICIAL
 EG-PIRACETAM

CRITERIA FOR A NOOTROPIC TO FULFILL IN ORDER TO
QUALIFY FOR CLASSIFICATION(DR GIURGEA’S CRITERIA)
 They should boost memory and learning ability.
 They should also strengthen the ability of learned forms of
behavior as well as memories to withstand forces such as
hypoxiation and electroshock treatment.
 They should afford protection to the brain from a variety of
injuries, either chemical or physical (for example, due to the
ingestion of scopolamine or barbiturates).
 They should make the controlling mechanisms of the cortex
more effective.
 Unlike other kinds of psychotropic drugs, they should have
no sedative or stimulant effects, as well as possessing negligible
side effects.
 They should also have virtually no detectable toxicity.

SKONDIA’S CRITERIA
 Dr Skondia was a second researcher to attempt to classify nootropics.
His criteria was based on a nootropics metabolic approach more than
Dr Giurega’s. Specifically
1. The substance possesses no direct vasoactivity (vasodilatation or
vasoconstriction).
2. The substance shouldn’t change basic EEG rhythm.
3. The substance must cross the blood brain barrier.
4. The substance must possess metabolic activity in the human brain.
5. The substance must have little/no side effects.
6. The substance must undergo clinical trials which reveal metabolic
cerebral improvement.

HOW IT WORKS
 NEUROPLASTICITY
 This term describes neural tissue that can easily grow new structures like
neurons and synapses. Over time, the ability of our brains to remain
flexible and adaptable is key to retaining memory function and staving off
the degenerative disorders associated with aging.
 The brain needs a steady supply of acetylcholine to remain in a state of high
neuroplasticity. This key neurotransmitter is related to all of our major
cognitive functions, including mental focus and memory formation
 MENTAL ENERGY
 It is due to the action of catecholamine hormones dopamine, epinephrine, and nor
epinephrine. The body naturally produces these hormones to create mental energy
in times of “fight or flight”
 They cause vasodilatation indirectly i.e. increase in blood supply to brain.
 They also show signs of neuro preservation and neuro protection.
 They produce their therapeutic effects by augmenting excitatory synaptic
transmission in cortical circuits, primarily through positive modulation of α-amino-
3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs).

Trends in use of memory
enhancers
•Nature, an online website
ran its own informal survey.
1,400 people from 60
countries responded to the
online poll.
•Usage of 2 drugs-
methylphenidate,modafinil
were considered for the poll
•Among the users, students
formed the major chunk
followed by office goers.

Classification of nootropics
 RACETAM(piracetam,oxiacetam) –piracetam is first nootropic agent discovered
 Choline (Acetylcholine Precursors)-(lecithin)
 Acetyl cholinesterase Inhibitors (Over-The-Counter)
 a) Huperzine A
 Ampakines(aniracetam)
 Herbal
 Bacopa Monneri
 Vinpocetine
 Gingko biloba
 Rhodiola rosea
 Mucuna pruriens – precursor to dopamine
 Gotu Kola
 Other Nootropics
 Sulbutiamine(vitamin b1 derivative)
 Pyritinol
 Citicholine
 L-theanine
 Picamilon
 Phenylethylamine (PEA)
 noopept(peptide)
 Dihydroergotoxine
 Piribedil

CLASSFICATION OF MEMORY ENHANCERS
1) DRUGS WITH SOME CLINICAL BENEFIT
a)Drugs increasing central cholinergic activity(for treatment of AD)-
(tacrine,galantamine and donezepil)
b) Nootropics
c)NMDA receptor(glutamate receptor) antagonist-(memantine)
2) Clinical benefit is uncertain-
a)cerebral vasodilators(pyritinol)
b)dopamine agonist(piribedil)
c)others(ginkgo biloba)
DRAWBACKS OF AVAILABLE DRUGS-
• Mechanism of action of these drugs are not clear
• Therapeutic benefit is also limited
• Possible cerebral steal phenomenon
• Prolonged use may cause serious side-effects
• Diversity in different classes and so poorly classified

Safety of nootropics
Common Side Effects Include:
 Headache
 Jitteriness
 Insomnia
 Gastrointestinal Problems
 There are many other side-effects
depending upon individual drug.
 Roughly half of users reported
unpleasant side effects, and some
discontinued use because of them.
 As the frequency drug decreases, the
frequency of side-effects decrease.

Limitations
•Observing an effect in one
behavioral task of a specific drug
applied into certain brain structure
does not warrant that other
behavioral tasks would be equally
affected
•A more diffused, wide-embracing
systemic treatment drug is harder to
interpret. Since this treatment may
simultaneously target very different
CNS Structures.
•All scientific conclusions obtained
from experimental animals cannot
be extrapolated to human clinical
cases.

IN VIVO METHODS
 PASSIVE AVOIDANCE
 ACTIVE AVOIDANCE
 DISCRIMINATION LEARNING
 CONDITIONED RESPONSE
 STUDY IN MONKEYS
 ELECTROPHYSIOLOGICAL METHODS
 ANIMAL WITH MEMORY DEFICIT

PASSIVE AVOIDANCE(aversive task)
 It is usually employed to describe experiments in which animal learns
to avoid a noxious event by suppressing a particular behavior
 STEP DOWN
o Purpose and rationale-
An animal(mouse or rat) spends most of the time close to walls
and in corners. When placed on an elevated platform in the center of
rectangular compartment, it steps down immediately to the floor to
explore the enclosure and to approach the wall

oPROCEDURE-
 Mice/rat of either sex used. A rectangular box (50x50cm) with
electrifiable grid floor and 35 cm fits over the block. The grid floor is
connected to a shock device which delivers scrambled foot shocks.
A typical paradigm consists of 3 phases:
Familiarization(animal is placed on platform, released after raising the
cylinder and latency is measured. After 10 s of exploration, it is returned to
home cage)
Learning(immediately after animal descends from platform an
unavoidable foot shock applied-50hz,1.5ma,1s and animal returned to home
cage)
Retention test (24 h after the learning trial the animal is again placed on
the platform and the step-down latency is measured. The test is finished
when the animal steps down or remains on the platform (cut-off time: 60 s).

EVALUATION
The time of descent during the learning phase and the time
during the retention test is measured. A prolongation of the step-
down latency is defined as learning.
MODIFICATION
•Most common modification is to induce amnesia in animals.
•There are different methods to do so including
(i) electroconvulsive shock, (ii) scopolamine, (iii) alcohol, (iv)
CO2
• Ricceri studied the effect of nerve growth factor on passive
avoidance learning and retention

CRITICAL ASSESSMENT
•There are some critical parts in the experimental procedure:
(i) Placing the animal on the platform, since the tendency of the animal
to escape the human hand may shorten the step-down latencies.
(ii) Another important point is the timing of the electric shock. It must
not be applied at the first contact of the animal with the floor, since
the light touch with the forelimbs does not cause the required shock
intensity.
(iii) It is also necessary to keep the room sound-proof, and this can be
done by using a white noise generator (60–70 dB).
LIMITATION
The variability of this method is relative high, therefore, it is necessary to
test large groups of animals (minimum 10 animals per group).

STEP THROUGH
PURPOSE AND RATIONALE
•This test uses normal behavior of mice and rats.
• These animals avoid bright light and prefer dim
illumination.
• When placed into a brightly illuminated space
connected to a dark enclosure, they rapidly enter
the dark compartment and remain there.
PROCEDURE
• The test apparatus consists of a small chamber
connected to a larger dark chamber via a guillotine
door.
•In the acquisition trial the animal is placed in the
illuminated compartment at a maximal distance
from the guillotine door, and the latency to enter
the dark compartment is measured.
• Immediately after the animal enters the dark
compartment, the door is shut automatically and
an unavoidable foot shock is delivered.

EVALUATION
•The time to step-through during the learning phase is measured and the time
during the retention test is measured.
• In this test a prolongation of the step-through latencies is specific to the
experimental situation. An increase of the step-through latency is defined as
learning.
MODIFICATIONS OF THE TEST
•This test procedure is employed in different modifications.To test drugs usually
several test-groups can be tested.
• (i) CXC-group: vehicle – without amnesia – vehicle; (ii) DXD-group: drug –
without amnesia – drug; (iii) CAC-group: vehicle – amnesia – vehicle; (iv)
DADgroup: drug – amnesia – drug; (v) CAD-group: vehicle – amnesia – drug; (vi)
DAC-group: drug – amnesia – vehicle.
•Itokazu showed that l-dopa caused memory deficit in mice by this method and
hence recommended as a model in human dementia
•LIMITATION AND CRITICAL ASSESMENT
Same as previous experiment

TWO COMPARTMENT TEST
•A rodent in an open field tends to enter any recesses in the walls and to hide
there.
• When placed into a large box, connected through a narrow opening with a
small dark compartment, the animal rapidly finds the entrance into the small
chamber, enters it and spends most of its time there. The times spent in the
large and small compartments are measured.
•The latency of the first entrance into the dark chamber and the number of
crossings from one compartment into the other can be used as auxiliary
criteria.
PROCEDURE
•Same as in step down experiment
CRITICAL ASSESSMENT AND LIMITATION
Same as in step down experiment

UPHILL AVOIDANCE
 Many animal species exhibit a negative geotaxis, i.e. the tendency to
orient and move towards the top when placed on a slanted surface.
When placed on a tilted platform with head facing down-hill, rats and
mice invariably turn around and move rapidly up the incline
PROCEDURE
 The experimental apparatus is a 50 × 50 cm box with 35 cm high
opaque plastic walls.
 The animal is first fitted with the tail-electrode to give shock and then
placed onto the grid with its nose facing down. During baseline trials
the animal’s latency to make a 180° turn and initiate the first climbing
response is measured.
 During the experimental trials the latencies are measured and
additionally a tail-shock (1.5 or 2 mA) was administered contingent on
the first climbing response after the 180° turn.

EVALUATION
The latencies are measured
CRITICAL ASSESSMENT OF THE METHOD
Its most obvious advantage is that it can be administered to animals debilitated
in sensory-motor coordination by pharmacological or surgical treatments that
would preclude use of other inhibitory (passive) avoidance tasks.
LIMITATION
Though it is a useful to the existing arsenal of inhibitory avoidance methods, it is
still highly variable and hence large number of animals are required

Trial-to-criteria inhibitory
avoidance
 PURPOSE AND RATIONALE
 As animals experience different sensitivity to the foot shock punishment
applied in the dark area, immediately after the first trial the animal is returned
to the lighted area to evaluate if the task has been acquired.
 A criteria is established to determine the learning of the test, usually requiring
the animal to remain in the lighted area for a period of 30–60 s.
 PROCEDURE
 The animals are trained in the same way as in the step-through version. They
are placed in the lighted compartment and after they entered with the four
paws into the dark area, the door is closed and a mild foot shock is delivered.
 The number of trials to attain criteria are counted as an indication of the speed
of acquisition.

EVALUATION
•The animals are placed in the lighted area, the door opened and the latency to
step with the four paws into the dark area is recorded.
• A cut-off latency of 180 or 300 s is usually imposed.
CRITICAL ASSESSMENT OF THE TEST
•This modification of the classical inhibitory avoidance procedure is useful to
determine the effect of drugs on acquisition as amnesic drugs significantly
increase the number of trials to reach the criterion.(eg-diazepam,scopolmine)
•Once the animals have been trained to a predetermined criteria any effect of
the drug on the retention trial can be associated to drug effects on
consolidation rather on the acquisition process (any nootropic-piracetam)
This test can also be performed as a continuous trial to criterion. In this
procedure the door is not closed after the animal enters the dark area and
upon delivery of the shock the animal can escape to the lighted area.
LIMITATION
Same as in uphill avoidance method

Scopolamine-induced amnesia in mice
 The administration of the antimuscarinic agent scopolamine to young
human volunteers produces transient memory deficits
 Analogously, scopolamine has been shown to impair memory retention
when given to mice shortly before training in a dark avoidance task
PROCEDURE
 Five min after i.p. administration of scopolamine hydrobromide, each
mouse is individually placed in the bright part of a two-chambered
apparatus for training.
 After a brief orientation period, the mouse enters the second, darker
chamber. Once inside the second chamber, the door is closed which
prevents the mouse from escaping, and a foot shock is applied through
the grid floor.
 The latency in entering the second darker chamber in untreated
control animals is 250s, whereas treatment with scopolamine reduces
latency to 50 s.
 A prolonged latency indicates that the animal remembers that it has
been punished and, therefore, does avoid the darker chamber.

•In spite of the fact that the pathogenesis of primary degenerative
dementia (Alzheimer’s disease) in man has been only partially
elucidated, the scopolamine amnesia test is widely used as primary
screening test for so-called anti-Alzheimer drugs.(eg-Rivastigmine)
MODIFICATIONS OF THE METHOD
•Lenègre investigated the effects of piracetam on amnesias induced by
scopolamine, diazepam and electroconvulsive shock.
•Sparks and schreurs reported that trace amounts of copper in water
induced B-amyloid plaques and learning deficit in rabbit model for
Alzheimer's disease
LIMITATION
The neuropathology of Alzheimer's disease is not confined to only
cholinergic system

Cognitive deficits on chronic low dose
MPTP-treated monkeys
 Röltgen and Schneider described the
effect of chronic dose of (MPTP) exposure
on cognitive functions in monkeys.
 These animals develop cognitive deficits
and difficulties in performing previously
learned tasks, such as delayed response,
delayed alternation and object retrieval,
as well as dopamine depletions in several
brain areas without severe Parkinsonian
signs as found after high MPTP doses
(methyl phenyl tetrahydropyridine)

PROCEDURE
•Adult monkeys are trained to perform a delay response task while seated
in a restraining chair placed inside a sound attenuating modified
Wisconsin General Test apparatus.
• The monkey sits behind an opaque screen that when raised allows access
to a sliding tray that contains recessed food wells.
• Monkeys are trained to retrieve a raisin from one of the food wells after
observing the experimenter baits the well.
•Animals are trained until performance with a 5s delay is 90% correct or
better for at least 5 consecutive days
•Once the animals are performing at criterion level,MPTP is administered
intravenously in doses ranging from 0.05 mg/kg at the start of the study to
0.20 mg/kg.
•Animals receive cumulative doses up to 60 mg over periods up to one year.
Pharmacological data are obtained after animals consistently show at least
a 15% performance deficit on delayed response

EVALUATION
•Delayed response performance after drug administration is compared with
matched control performance obtained on the same day prior to drug
administration The total number of correct responses as well as the number of
mistakes and ‘no response’ errors are tabulated for each session.
•Date are then expressed as mean (± standard deviation) performance. All
animals serve as their own controls. Statistical analysis consists of analysis of
variance, repeated measures design, with post hoc comparisons (Bonferroni t-
test).

ACTIVE AVOIDANCE
 Active avoidance learning is a fundamental behavioral
phenomenon .
 As in other instrumental conditioning paradigms the
animal learns to control the administration of the
unconditioned stimulus by appropriate reactions to the
conditioned stimulus preceding the noxious stimulus.
 The first stage of avoidance learning is usually escape,
whereby a reaction terminates the unconditioned stimulus

Run away aviodance
 A straightforward avoidance situation features a fixed aversive gradient
which can be traversed by the animal.
 The shock can be avoided when the safe area is reached within the time
allocated
PROCEDURE
 The same box as used in the step-through model can be used in this
experiment.
 A loudspeaker mounted above the starbox serves for presenting the
acoustic conditioned stimulus
 The animal is allowed to explore the whole apparatus for 5 min. The
guillotine door is then closed and the animal is placed into the light
starting area. After 10 s the acoustic CS is applied and the door is
simultaneously opened.
 Shock is turned on after 5 s.The training is continued until the animal
attains the criterion of 9 avoidances in 10 consecutive trials.

EVALUATION
•The time the animal needs to reach the safe area on is recorded.
•Also, the number of errors committed is recorded
CRITICAL ASSESSMENT AND LIMITATION
Same as in step down method

Shuttle box avoidance (two-way shuttle box)
 The animal learns that a random
stimulus (a tone, the CS) is a reliable
predictor for a coming aversive
experience , and can prompt an
evasive action in order to avoid it,
i.e., it moves to the other side of the
shuttle box (the CR) when the
stimuli predict aversive events.
 Since there is the possibility to learn
how to escape, this task may be
classified as an operant
conditioning, i.e., the animal must
learn the relation between CS and
US in order to anticipate US with a
CR (escape) and avoid it.

PROCEDURE
•The apparatus used consists of a rectangular box with high metal walls,
and an electrifiable grid floor.
•Simple programming equipment provides for automatic delivery of the
conditioned stimulus (CS) and the unconditioned stimulus (US). The
animal is allowed to explore the apparatus for 5 min with the connecting
door open and the compartment lights switched off.
•The guillotine door is then closed. After 20 s the light is switched on in the
compartment containing the animal, and the door is opened.
• A tone (CS) is presented and 5 s later the floor shock is applied in the
illuminated compartment and continued until the animal escapes to the
dark side of the compartment
EVALUATION
The time the animal needs to reach the safe area on both days is measured.
In addition, the number of errors (not reaching the safe area) are recorded

•The task is rather difficult due to the lack of a permanent safe area
•Lack of a simple instrumental response
•Presence of a variable aversive gradient
• Increased weight of emotional factor.
Salmi described a computer-assisted two way avoidance conditioning
equipment for rats.
LIMITATION
•Compared to runway avoidance, shuttle box avoidance is a more difficult
task.
• Since the animal is not handled between trials, the shuttle box can be
easily automated

Jumping avoidance (one-way shuttle box)
 It is a simplified one-way avoidance, allowing for the
spontaneous or forced return of the animal to the
start.
 In order to enhance the start-goal distinction a
vertical gradient is introduced which requires the
animal to perform a discrete response of an all-or-none
character, such as the jump, which clearly differs from
the more continuous translational movements
required in the usual avoidance tasks

PROCEDURE
•The apparatus used consists of a rectangular box with high
metal walls, electrifiable grid floor and a Plexiglas ceiling.
• Flush with the horizontal surface of the pedestal moves a
vertical barrier, which can either be retracted to the rear wall
of the apparatus to expose the goal area or pushed forward to
block access to the goal completely.
•The animal is placed into the apparatus for 5 min with the
goal area exposed.The barrier is then moved forwards and the
goal is blocked for 2 s.
• The first trial starts by exposing the goal area and applying
an acoustic . Electric shocks – US are applied 5 s later , and
continued together with the CS until the animal jumps onto
the platform .
•After 30 s the barrier pushes the animal off the platform onto
the grid floor.

EVALUATION
The time the animal needs to reach the safe area on both days is
measured. In addition, the number of error (not reaching the safe area)
is recorded.
In automated procedures extinction is more rapid, especially when short
inter test intervals are used
LIMITATION
A high degree of automation and minimum handling is required

DISCRIMINATION LEARNING
 In the experiments described above the animals have no
choice between the conditioned stimuli. They have only
one conditioned stimulus.
 The experiments can be classified either as simultaneous or
successive discrimination paradigms.

Spatial habituation learning
PURPOSE AND RATIONALE(classical non-aversive
and non-associative task.)
 The open-field test utilizes the natural
tendency of rodents to explore novel
environments in order to open up new
nutrition, reproduction and lodging
resources .
 The rate of exploratory behaviors exhibited
in an unfamiliar environment is limited
through the inherent necessity to avoid
potential dangers.
 Spatial habituation learning is defined as a
decrement in reactivity to a novel
environment after repeated exposure to that
now familiar environment.
 The test can be used to examine short-term
spatial memory and/or long-term spatial
memory

PROCEDURE
•The open-field apparatus is a rectangular chamber made of painted wood or
grey PVC.
•The rodent is placed on the center or in a corner of the open-field for 5–10
minute sessions
EVALUATION
The exploratory behaviors registered are:
(1) Rearing or vertical activity: the number of times an animal was standing
on its hind legs with forelegs in the air o against the wall.
(2) The duration of single rearing a a measure of non-selective attention
(3) Locomotion or horizontal activity: the distance in centimeters an animal
moved.

•In order to assess emotionality parameters the aversivity of the open-field can
be varied by increasing the size and/or the illumination density of the
apparatus .
• The emotional behaviors registered are: (1) Corner time: the time spent in the
4 corner squares (2) Wall time: the time an animal spent close to the wall as a
measure for thigmotaxis (scanning the walls of the apparatus with the
vibrissae). (3) Center time: the time spent in the center of the open field . (4)
Defecation: number of boli deposited. (5) Freezing: the time the animal stays
completely immobile except for movements associated with respiratory
activity .
The open-field paradigm is a well validated, simple and time economical test,
which has been widely used to examine the neurobiological foundations sub
serving spatial memory, general activity and emotionality in rodents with
different approaches including: lesions, drugs, electrophysiology,
neuroanatomy, and neurogenetics
LIMITATION
The training session is a quick procedure, this task is not simple to be
automated

Spatial discrimination
 In the simplest case of discrimination learning the animal distinguishes
between two symmetric stimulus response sets, the equal probability of
which has been changes by differential reinforcement events. Position of
the cues with respect to the animal’s body defines the CS+ and CS–.
 Usually right-left discrimination is employed.
PROCEDURE
 The apparatus used is usually a simple T- or Y-maze, with an electrifiable
grid floor. The last 10 cm of each arm are separated from the rest of the
apparatus by a swing-door which prevent the animal from seeing the food
cup or the plastic sheet covering the grid in the goal area.
 In an aversively motivated spatial discrimination learning the animal is
trained to escape and/or to avoid foot shocks by always going to the right.
 Then the animal is placed on the start and after 5 s electric shocks (0.5 s,
50 Hz, 1.0 mA) are applied at 3 s intervals.
 After a 60 min interval the safe goal area is shifted to the other arm of the
maze and the discrimination is reversed

EVALUATION
•Errors are scored. An error means that the animal enters the wrong arm with
all four legs.
• During retention the number of trials until the animal makes correct
choices are counted.
•The ecology of rodents makes these animals specially proficient in spatial
discrimination learning, which is usually mastered in a few trials.
•Barnes and others introduced the radial maze as a modification of spatial
discrimination.
•This method is now well established and widely used to test for any new
cognitive enhancer.
LIMITATION
There are many initial errors due to inability of the animal to remember the
correct solution but rather to the tendency of the animal to explore
alternative pathway

Spatial learning in the radial arm maze
 Olton and co-workers have developed a spatial discrimination task for
rodents that has been extensively used in learning and memory
studies, and that has served as the basic task for one of the most
important theories on the role of the hippocampus .
 The rat uses spatial information provided by the distal cues in the room
to efficiently locate the baited arms. The radial arm-maze allows the
study of spatial reference and working memory processes in the rat.
 In reference memory procedures, information is useful for many
sessions/days and may usually be needed during the entire experiment.
 On the contrary, working memory procedures have a major temporal
component as the information presented in the maze is useful for one
session but not for subsequent ones; the rat has to remember the
information during a delay interval . Correct choices in the radial arm-
maze are rewarded by food.

PROCEDURE
•The apparatus is a wooden elevated eight-arm radial maze with the arms
extending from a central platform 26 cm in diameter.
• Food pellets (reward) are placed at the end of the arms. During the test,
rats are fed once a day and their body weights maintained at 85% of their
free feeding weight to motivate the rat to run the maze.
•The session is terminated after 8 choices and the rat has to obtain the
maximum number of rewards with a minimum number of errors.
EVALUATION
The number of errors (entries to non-baited arms) are counted during the
session

Depending on the hypothesis being tested in the radial arm-maze
(1) animals can be trained extensively and then receive specific brain lesions
(hippocampus) after recovery from the surgery the rats are re-trained to
determine the cognitive ability or the speed of recovery as these lesions severely
disrupt processing of spatial information
(2) In working memory studies animals are forced to obtain reward in specific arms ,
and after a time delay they have to either return to the same arms or to avoid these
arms and obtain the food in the rest of the arms .
(3) Animals are trained to find food in only one arm, and after a delay they are
required to return to the same arm.

•The radial arm-maze has been widely used to determine the neurobiological
mechanisms underlying spatial learning in rodents and to evaluate the effect of
drugs.
•The deleterious effects of scopolamine, benzodiazepines, haloperidol, ketamine,
PCP, as well as the facilitator effects of nicotine, pramiracetam, picrotoxin,
naloxone have been reported in the literature
LIMITATION
• One of the disadvantages of the test is that hypothalamic lesions or the anorectic
effect of certain drugs (amphetamine) affect the appetitive nature of the maze
and animals do not master the maze for this reason.

Visual discrimination
 Vision is better than any other sensory system for the analysis
of spatial relationships in the environment of the animal. The
organization of the visual system ensures processing of visual
information according to simple principles, i.e. by fitting the
distribution of light over the receptive surface to elementary
geometrical concepts and by comparing these patterns with
images stored in the memory.
 Visual pattern recognition is one of the most challenging
problems of contemporary neurophysiology and experimental
psychology, with significant implications for mathematical
and technical modeling of perceptual phenomena

PROCEDURE
• The apparatus consists of a square separated by a Plexiglas sliding door
from the choice area, which is connected by swing doors to the goal
compartment.
•The animal is placed into the apparatus with all doors open and allowed
to explore it. Then it is placed in the start and after 5 s release by raising
the Plexiglas door.
• After another 5 s, electric shocks are applied until the animal escapes
through either of the open doors to the safe goal compartment where it is
left for some seconds.
CRITICAL ASSESSMENT AND LIMITATION OF THE METHOD
The method is time consuming and only useful to address a specific
hypothesis

Spatial learning in the water maze
PURPOSE AND RATIONALE(explicit associative memory)
 A task was developed where rats learn to swim in a water tank to
find an escape platform hidden under the water.
 As there are no proximal cues to mark the position of the platform,
the ability to locate it efficiently will depend on the use of a
configuration of the cues outside the tank.
 Learning is reflected on the shorter latencies to escape and the
decrease on the length of the path to find the platform
PROCEDURE
 The apparatus is a circular water tank filled to a depth of 20 cm
with 25 °C water. The tank is divided in four equal quadrants and a
small platform is located in the centre of one of the quadrants.
 The rat is released into the water and allowed 60–90 s to find the
platform.

EVALUATION
The latency to reach the escape
platform is measured during the
training days.
•In the “cue” version of the water
maze, the platform is clearly visible
over the water and allows the
evaluation of the motor and
motivational aspects of the rat under
study, as the animals should easily
find the visible platform in 10–15 s.
• To evaluate working memory
processes in the water maze, the rat
can be trained to find a new
platform position and later its
performance is tested after a short
delay

•Nitta found an impairment of the performance of the water maze task in
rats treated by infusion into the cerebral ventricle for 14 days with β-amyloid
protein which consisted of senile plaques of Alzheimer’s disease.
•It is recommended as a model for alzeimer’s disease
•This test allows the researcher to study working and reference memory
processes, and to dissociate the memory deficits induced by brain lesions or
drug injections from the motor, motivational or sensory deficits.
•This test has been used to study the effect of drugs on memory , the
participation of the hippocampus, amygdala and catecholamine depletions,
as well as recovery of function .
LIMITATION
It has comparitvely less specificity as was once believed

Water maze 8 arm radial maze
Advantages
 No motivational problems – problem
cases are easily tested.
 Olfactory cues are not present.
 Animals learn readily and quickly.
Disadvantages
 Differences in platform finding
latency are the product of learning
and motor performance; it is not
possible to subtract one from another
 Working memory cannot be tested
independently.
 A videotracking system is essential for
evaluating the swim tracks of
animals.
 A fairly large pool has to be installed
in a fairly dedicated room
Advantages
 Distinguishes between motor
impairment and spatial learning
 Different memory types can be
tested: working memory, long-term
(reference) memory, motor
(egocentric) memory.
 A tracking program is not essential.
 Cheap, simple set-up, which can be
dismantled and easily stored after
use
 Animals must be well-handled and
motivated to perform the tasks
Disadvantages
 Appetitive task: the animals must be
mildly food-deprived to be
motivated to look for baited arms
 Training the animals usually takes
much more time than water maze

Olfactory learning
 Odors provide rodents with important information on the environment
and the learning of successive olfactory discrimination problems in rats is
closely related to the acquisition rules of higher primates.
 Odor-reward associations are learned in few trials as odors exert more
discriminative control over other sensory modalities like tones or lights .
 Animals have to learn to discriminate an arbitrary designated positive odor
(i.e., banana) from a negative one (i.e., orange) to receive a reward.
Procedure
 The olfactory apparatus is a rectangular box with a photosensitive cell
mounted on top of the water spout/odor outlet.
 Rats are trained to approach the water spout and to brake the light beam.
Responses to the positive odor are rewarded with water while responses to
the negative odor results in the presentation of a light flash.
 Sessions are terminated when the rat makes 90% correct choices or after
400 trials.

EVALUATION
•The animal is rewarded with 0.05 ml of water when it brakes the beam to
the positive odor or when it does not respond to the negative odor.
•Results are reported as % correct responses or as a logit transformation of
the %correct/incorrect response ratio
•The anatomical connections from the olfactory bulbs to cortical as well as
subcortical areas are fairly well known, and brain lesions that impair
olfactory discrimination learning could be used as models of amnesia.
•Systemic injections of scopolamine disrupts the performance of rats
during long delays with no effect on immediate recall, data that are
consistent with the effect of scopolamine in humans
•The test can also be used to evaluate the cognitive effects of drugs
such as ACTH analogs that facilitate the storage of olfactory
information

•A T-maze with controlled access to the reward compartment was
developed , that allows the test to evaluate working memory processes
after time delays
•Willer examined the effects of a competitive NMDA agonist on the ability
of rats to acquire potentiated aversions to the odor element of a taste-odor
compound
LIMITATION
It requires lot of training for animals

Aversive discrimination in
chickens
 One to 2-days old chicks have been extensively used to
study learning and memory. The chicks are trained to
discriminate between an aversive and a non-aversive
stimulus in a single 10–15 s learning trial.
 Following this learning trial, retention is monitored
over a comprehensive range of learning retention
intervals to chart the course and to differentiate
possible stages of memory formation.

PROCEDURE
• In a single passive avoidance learning trial, chicks are pre-trained to
peck at a 4 mm chromed bead, dipped in water and presented for 10 s.
•In a discrimination paradigm, chicks are trained to avoid a red bead and
are tested on red and blue beads successively and discrimination ratios
registered.
•Twenty different chicks are used for each data point. Retention in both
paradigms is indexed by the proportion of chicks avoiding the aversive
bead, with the provision that in the discrimination paradigm is indicated
by the avoidance of the red bead and pecking on the blue bead.

EVALUATION
Discrimination ratios of treated animals after various time intervals
following administration of drugs are statistically compared with saline
treated controls.
The test has been used with slight modifications successfully by many
authors in several research groups
•Bourne compared the taste and odor avers an methyl anthranilate with
the odorless quinine as averants.
•Clements and Bourne and Stamatakis injected drugs, e.g., GABA agonists
or α2-noradrenergic agonists and antagonists, directly into the
intermediate median hyperstriatum ventral of day-old chicks, prior to
training on a chrome bead dipped in either methyl anthranilate or quinine.

Conditioned response
Conditioned nictitating membrane response in rabbits
 The rabbit’s classically conditioned eye blink response has become a widely
used model system for studying associative learning in mammals and to
find drugs potentially useful in the treatment of age-related memory
disorders (alzeimer’s disease)
Apparatus and general procedure
 A small loop of surgical nylon is sutured into the right nictitating
membrane, and the surrounding hair is removed. One day later, the rabbit is
placed in a Plexiglas restrainer, and two stainless-steel wound clips are
applied to the skin over the parietal region.
 The transducer assembly converts nictitating membrane movements into
electrical signals that are subjected to an analog to digital conversion using a
5-ms sampling rate and a resolution of 0.06 mm actual membrane extension.
 During the course of the experiment, two stimuli are employed as
conditioned stimulus: a) a 1000-ms, 1-kHz, 84-dB tone ; b) a 1000-ms,
intermittently presented light produced by interruption of the house lights
at 10 Hz to yield a change in illumination, measured at the eye level of the
rabbit from 32.11 to 8.01.
 The unconditioned stimulus is a 100ms,3mA,60Hz shock delivered to wound
clips by constant shock generator

DRUGS
Drug solutions or saline are injected subcutaneously into the cervical area of
the rabbit via an infusion pump at a rate of 3 ml/min, 30 min before
behavioral testing
PROCEDURE
•Experimentally naive rabbits are randomly assigned in equal numbers to
each of the treatments (n = 10 per treatment).
• The experiment consists of two phases:
•Phase 1 is an adaptation day followed by 9 days of acquisition training.
•Each acquisition session consists of 30 tone-shock and 30 light-shock trials
presented in a randomized sequence within 10 trial blocks, with the
restriction that no more than three consecutive tone or light trials can occur.
• On each conditioned stimulus –unconditioned stimulus trial, the offset of
the 1 000-ms tone or light conditioned stimulus occurs simultaneously with
the onset of the 100-ms unconditioned stimulus.
• A response is defined as at least a 0.5-mm extension of the nictitating
membrane.

EVALUATION
The data are analyzed by repeated measures analyze of variance and
Tukey tests.
The rabbit nictitating membrane response has been used widely to study
the effects of drugs on learning
•Several authors used a corneal air puff as unconditioned stimulus
•Scavio studied post-training effects of several drugs, such as
amphetamine, chlorpromazine, ketamine, and scopolamine, on the
acquisition and extinction of the rabbit’s conditioned membrane
response.
•Solomon described behavioral paradigm for delay, trace, and long-delay
conditioning. Various drugs influence the nictitating membrane
response in rabbits, such as cocaine , sodium pentobarbital , haloperidol
, MDA (3,4-methylenedioyamphetamine)

Automated learning and memory model
in mice
 Vanover and Barrett developed and validated pharmacologically an
automated, relatively rapid, and reproducible behavioral model of
learning and memory using an autoshaped procedure in mice.
PROCEDURE
 For experimental sessions, mice are placed in enclosures containing
specially designed operant chambers equipped with a recess for
dipper accessibility on one wall of the chamber and two additional
smaller holes on either side of dipper well.
 The dipper can be raised into the dipper well for a sucrose solution
reinforcer presentation. Each recess has a photocell monitoring
nose-poke responses.
 Five to 9 mice per group are tested in a 2-day procedure designed to
measure acquisition and retention of nose-poke response under an
autoshaping schedule of reinforcement.

• During the autoshaping procedure which is used to measure acquisition
and retention of the response reinforcement for nose-poking a tone is
added. This tone sounds on a variable-interval schedule of presentation and
stays for either 6 s or until a nose-poke in the dipper well is made before the
end of the 6 s period, at which time the tone is turned off and a dipper with
sucrose solution is presented
•A dipper well nose-poke response made during the presence of the tone is
counted as reinforced response.
• Each sessions lasts for 2 h or until 20 reinforcers have been earned,
whichever comes first.
•Drugs in various doses or vehicle are administered i.p. immediately before
the session.
•The latency of the response to the 10th reinforcer is considered as a measure
of acquisition and retention, because all mice have been exposed to all
possible intervals(in random order) by the 10th tone.

EVALUATION
•The mean and standard error of the mean are calculated for every group.
Two-tailed Student t-tests are used to compare two independent groups
on any one dependent
variable.
•One way analyses of variance (ANOVA) are conducted across groups for
effects of dose on acquisition (day 1 performance). One way analyses of
covariance (ANCOVA) are conducted across groups for effects of dose for
retention (day 2 of performance) with the performance on day 1 as the
covariate.
• When an ANOVA or ANCOVA shows statistical significance, post-hoc
Duncan’s multiple range tests are conducted with every group compared
with vehicle.
LIMITATION
If a mouse fails to give a nose poke response,still it is given dipper at the
termination of tone.it may lead to false positive results

Studies in aged monkeys
 Nonhuman primates have the closest taxonomic relationship to humans,
sharing many morphologic and physiologic similarities in the central
nervous system.
 They offer additional advantages for neurobehavioral animal models of
aging in that many of the behavioral processes thought to be affected by
aging (e.g. reaction time, learning and memory) can be studied easily
PROCEDURE
 The apparatus developed specifically for the series of studies used to develop
the primate model was the Automated General Experimental Device
(AGED),
 The AGED is a totally automated, computer-controlled testing system,
whose prominent feature consists of a 3 × 3 matrix of stimulus response (SR)
panels. Each SR panel is hinge mounted directly in front of the
reinforcement well so that when a panel is pushed, a red switch is
magnetically activated and a reinforcement well is exposed.
 Both colored and patterned stimuli can be projected onto the SR panels. A
plastic partition with a stimulus window and armholes separates the monkey
from the SR matrix.

EVALUATION
The monkey must remember the stimulus location to get a reinforcement. Number
of correct answers will be counted as well as the time until the monkey answer
correctly.
•This system provides an accurate and objective mean of collecting data under a
number of behavioral paradigms.
•Further, this system provides experimental control over a number of variables that
might confound behavioral measures
Cai and Arnstein described dose-dependent effects of Dopamine D1 receptor
agonists on spatial working memory in aged monkeys.
LIMITATION
•Ethical issues
•Catching procedures are risky
•Mandatory presence of technician is time consuming and costly

Electrophysiological methods
LONG-TERM POTENTIATION IN HIPPOCAMPAL
SLICES
 This procedure is perhaps the most dramatic example of activity-
dependent synaptic plasticity that has yet been identified in the
mammalian brain . A brief tetanus to any one of a number of
monosynaptic excitatory pathways in the hippocampus can
enhance the amplitude of evoked responses in the tetanized
pathway for hours or days thereafter.
 The fact that it occurs in the hippocampus has done much to
stimulate interest in LTP as a synaptic model of memory

PROCEDURE
•Transverse slices, are cut from the hippocampus of guinea pigs which are
incubated for 90–120 min in the recording chamber to allow equilibration
with artificial cerebrospinal fluid.
• They are submerged, placed on a nylon mesh and perfused at a flow rate of
2–2.5 ml/min with oxygenated cerebrospinal fluid having the following
composition (in mM): NaCl 124, KCl 3.3, CaCl2 2.5, KH2PO4 1.25, MgSO4 2,
NaHCO3 25,7,glucose 10.
• The electrodes are placed into the stratum pyramidale of CA1 or CA3.
• The signal is amplified and stored on magnetic discs for later analysis.
•After the baseline is recorded for 10–20 min, LTP is induced by repetitive
stimulation in CA1 and in CA3 at the same rate and are recorded 0, 10, 20 and
30 min after repetitive stimulation.
EVALUATION
The time course of LTP is registered for CA1 and CA3.The mean percent
increase in the amplitude of the population spike from baseline responses
after drug application is compared with controls.

•Fujii studied the effects of an adenosine a1 receptor antagonist, 8
cyclopentyltheophylline, on the reduction of LTP in ca1 neurons of
hippocampal slices.
•Behnisch and reymann employed slices of hippocampal area ca1 in the rat to
test the hypothesis that the activation of metabotropic glutamate receptors
during tetanization is necessary for the maintenance of long-term
potentiation.
LIMITATION
•They lack normal synaptic connections with other neurons, may be
functioning under conditions greatly different than normally seen in the
living organism
• It is not certain that the "extracellular" and "intracellular" media used do not
constitute a totally artificial environment that would not be relevant to a
living neuron in vivo.
• The lack of normally circulating agents such as steroids, hormones, plasma
proteins, and other colloidal substances could lead to drastic changes in the
function of the molecules and channels under study

ANIMALS WITH MEMORY DEFICIT
MEMORY DEFICIT AFTER CEREBRAL LESIONS
 Cerebral lesions have been used as a method of determining the
involvement of particular brain area in performing a particular
function
 By training an animal for a certain task, then lesioning a specific
area(inj of ibotenic acid in basal forebrain),one can determine whether
that area of brain is necessary to perform that function
 Mostly neurotoxins are tested esp excitatory amino acid
neurotransmitters such as glutamate.

OTHER FORMS OF THE SAME TEST-
•Cognitive deficit after cerebral ishemia
•Strains with hereditary memory deficit
•Genetically modified animals
LIMITATION
•It requires a lot of skill and expertise.
•Takes a lot of time
•Specificity of lesion and co relating with effects is difficult

In vitro methods
In vitro inhibition of acetylcholine-esterase
activity in rat striatum
 The purpose of this assay is to screen drugs for inhibition of
acetylcholine-esterase activity. Inhibitors of this enzyme may be
useful for the treatment of Alzheimer’s disease.
 Acetyl cholinesterase (Ache) which is sometimes called true or
specific cholinesterase, is found in nerve cells, skeletal muscle etc
 Its distribution in brain roughly correlates with cholinergic
innervations and sub fractionation shows the highest level in nerve
terminals.
 Recent studies have suggested that Ache inhibitors may also be
beneficial in the treatment of Alzheimer’s dementia

PROCEDURE
•A 2 mM stock solution of the test drug is made up in a suitable solvent
and q.s. to volume with 0.5 mM DTNB (di thio nitro benzoic acid)
•Drugs are serially diluted (1 : 10) such that the final concentration (in
cuvette) is 10–4 M and screened for activity.
•If active, IC50(half maximal inhibitory conc) values are determined
from the inhibitory activity of subsequent concentrations
TISSUE PREPARATION
•Male Wistar rats are decapitated, brains rapidly removed, corpora
striata dissected free, weighed and homogenized in 19 volumes.
•A 25 μl aliquot of this suspension is added to 1 ml of the vehicle or
various concentrations of the test drug and reincubate for 10 min

ASSAY
•Enzyme activity is measured with the Beckman DU-50
spectrophotometer.
•This method can be used for IC50 determinations and for measuring
kinetic constants
EVALUATION
For IC50 determinations: Substrate concentration is 10 mM diluted 1 : 2
in an assay yielding a final concentration of 5 mM.DTNB concentration
is 0.5 Mm yielding 0.25 mM final concentration
%inhibition=slope control-slope drug x 100
slope control
LIMITATION
•Good lab set up is required which is costly
•It requires lot of skill and precision

In vitro inhibition of butyrylcholine-esterase
activity in human serum
 This assay can be used in conjunction with the acetylcholine-esterase assay
to determine the enzyme selectivity of various cholinesterase inhibitors.
 Butyrylcholine-esterase (BChE), which is called pseudocholinesterase,
preferentially hydrolyzes butyrylcholine. It is found in the highest
amounts in serum, but its physiological role is not known
PROCEDURE
Enzyme Preparation
 A vial of lyophilized human serum is reconstituted in 3 ml of distilled
water. A 25 ml aliquot of this suspension is added to 1 ml of the vehicle or
various concentrations of test drug and pre-incubated for 10 min at 37 °C.
EVALUATION AND LIMITATION
 Same as previous experiment

Ex vivo cholinesterase inhibition
 This assay is used to determine the dose-response relationship and duration
of action of cholinesterase inhibitors in vivo. Cholinesterase inhibitors,
including physostigmine and tacrine have been shown to improve cognitive
functions in Alzheimer’s disease.
 Physostigmine is a potent, but nonselective inhibitor of cholinesterase and
has a short duration of action. Tacrine also inhibits both acetylcholine-
esterase (true) and butyrylcholine-esterase, but is more potent as an
inhibitor of the pseudo-enzyme .
 Ideally, a dose-response for cholinesterase inhibition is determined first.
 Then a dose which gives a reasonable effect (>50% inhibition if possible) is
chosen to do the time-course experiment.
 The effects on brain acetyl cholinesterase activity are examined in striatal
tissue, using 5 mM acetylthiocholine as a substrate

PROCEDURE
Substrate in buffer
a) 198 mg acetylthiocholine chloride (10 mM)
b) q.s. to 100 ml with 0.05 M phosphate buffer, pH 7.2 (reagent 1)
DRUG TREATMENT
•Groups of four male Wistar rats are dosed i.p. with vehicle or the test
drug.
•For the initial dose response study, the rats are given varying doses of drug
based on toxicity reported in primary overt effects testing and sacrificed at
1 h after dosing.
•The animals are observed and the occurrence of cholinergic signs is noted
( salivation, diarrhea and chromodacryorrhea).
•For the time-course study, a dose of the test drug is given which gave
significant inhibition of cholinesterase activity
TISSUE PREPARATION
Same as previous experiment

ASSAY
•Enzyme activity is measured with the Beckman DU-50
spectrophotometer.
•Substrate concentration is 10 mM diluted 1 : 2 in the assay yielding final
concentration of 5 mM.
EVALUATION
The percent inhibition at each dose or time is calculated by comparison
with the enzyme activity of the vehicle control group.
%inhibition=slope control-slope drug x 100
slope control
•Antagonism of physostigmine-induced lethality in mice has been used by
Gouret as a general indicator of central or peripheral anticholinergic
activity.
•A low dose of physostigmine can be used for detecting procholinergic
activity

EXPERIMENTS AT MOLECULAR LEVEL
Molecular forms of acetyl cholinesterase from rat
frontal cortex and striatum
 Different molecular forms of acetyl cholinesterase can be
isolated from animal tissues.
 The number of forms isolated, their relative amounts and
molecular characteristics depend on the tissue source and the
conditions used for solubilization of the membrane bound
enzyme
[3H]Oxotremorine-M binding to muscarinic
cholinergic receptors in rat forebrain
 The purpose of this assay is to determine the binding affinity
of potential cholinomimetic drugs for muscarinic receptors in
brain, using an agonist ligand

[3H]-N-Methylscopolamine binding in the presence and
absence of Gpp(NH)p
The assay differentiates the interaction of muscarinic agonists and
muscarinic antagonists with 3H-N-methylscopolamine (3H-NMS)-labeled
receptors in cerebellar tissue based on the selective effect of guanine
nucleotides on the affinity of muscarinic agonists for the receptor
STIMULATION OF PHOSPHATIDYLINOSITOL TURNOVER IN RAT
BRAIN SLICES
•The purpose of this assay is to determine the ability of test compounds to
stimulate the turnover of phosphatidylinositol (PI) in brain tissue.
• This assay can be used to determine agonist activity at a number of CNS
receptors known to be linked to the PI response. A major interest is the
evaluation of muscarinic cholinergic receptors

RELEASE OF [3H]ACH AND OTHER TRANSMITTERS FROM RAT
BRAIN SLICES
•Electrically stimulated release of [3H]ACh is used as a biochemical screen for
agents which may possibly enhance or inhibit release of [3H]ACh through a
direct muscarinic interaction or other indirect interactions.
•Muscarinic autoreceptors have been shown to have a role in the regulation of
ACh release in several areas of the CNS
• Direct stimulation of muscarinic receptors with muscarinic agonists or
indirect stimulation with acetyl cholinesterase inhibitors decreases ACh
release evoked by either increased potassium concentration or electrical
stimulation.

Experiments on nootropics on
humans
 The use of a scopolamine model to study the potential nootropic
effects of aniracetam and piracetam in healthy volunteers
ABSTRACT-
 In this study 26 healthy volunteers received scopolamine 0.7 mg
subcutaneously on seven occasions at least a week apart.
 Following this, over the seven occasions, a range of oral and intravenous
dose regimens were administered including aniracetam 2 mg
intravenously, 100 mg intravenously, 200 mg intravenously, 1500 mg per
os and piracetam 2400 mg per os
 At 60 min, scopolamine produced marked and significant decrements in
all of the measures of memory and information processing.
 Aniracetam 1500 mg was able to significantly antagonize decrements on
both memory and information processing tasks.
 Overall, the findings demonstrate that aniracetam 1500 mg can
antagonize cognitive decrements produced by cholinergic blockade in
healthy volunteers, and suggest that the drug possesses nootropic
properties.

Giaguinto and colleagues gave 12 healthy humans 5mg Valium orally at
10PM the night before their experiment.
• The next morning they were given either I.V. Oxiracetam or saline in a
double blind crossover experiment.
•Oxiracetam strongly decreased the excessive delta activity while
simultaneously strongly increasing alpha activity, and also induced a modest
increase in beta activity.
 Itil and co-workers (1986) treated four groups of 15 patients suffering mild
to moderate dementia with either Oxiracetam or placebo for three months.
•The double blind study used Oxiracetam in doses of 800, 1600 and 2400mg
daily. Quantitative EEG data indicated that in patients with dementia,
Oxiracetam had a mode of action similar to other vigilance enhancing
compounds. The majority of patients who had abnormal slow EEG patterns
before treatment showed a "normalization" of their brain waves- i.e. a
decrease in slow (delta and theta) and an increase in alpha waves.

Mindus and colleagues reported the results of a double blind crossover trial
with 18 healthy middle aged people based on medical records and
administration of several intelligence tests
•Most of the subjects were in intellectually demanding jobs, but had reported
a slight reduction for some years in their capacity to retain or recall
information.
•After four weeks of 4.8 grams per day Piracetam, subjects were switched to
placebo for four weeks, while the original placebo group then received
Piracetam for four weeks.
•Results of a series of paper and pencil tests, as well as computerized tests to
measure perceptual motor reactions, showed a clear benefit of Piracetam over
placebo.
LIMITATION
•A major complication that is raised in memory research is its fallible nature
in humans.
• Our ability to store and process what is going on around us relies on memory
being a constructive process.
• The technologies explained above may show areas of activation associated
with certain behaviors, but without any idea of lesion location, it is difficult to
pinpoint exactly what part of the brain relates to which behavioral deficits
observed

Indications of cognitive enhancers
 Degenerative changes with ageing
 For treating diseases-alzheimer disease,huntington
disease,adhd,parkinson disorder
 Memory loss due to brain injury
 As a study aid
 For work and business
 For social acuity
 For health
 Eugeroics (armodafinil)– wakefulness promoting agents they are
clinically prescribed for narcolepsy,shift work disorder
 It can even reduce the risk of stroke and repair brain damage
from physical or chemical trauma like alcohol consumption and
concussion
 Myoclonus-piracetam is approved in UK

BIBLIOGRAPHY
 HANS GERHARD VOGEL 3RD EDITION;DRUG DISCOVERY AND EVALUATION:DRUG EFFECTS ON PHARMACOLOGICAL
ASSAYS; LEARNING AND MEMORY ;PAGE 878-942
 SK GUPTA 1ST EDITION;DRUG SCREENING METHODS;DRUGS FOR LEARNING AND MEMORY;PAGE 117-130
 KD TRIPATHI 7TH EDITION;ESSENTIALS OF MEDICAL PHARMACOLOGY;CNS STIMULANTS AND COGNITION
ENHANCERS;PAGE488-491
 HL SHARMA 2ND EDITION;PRINCIPLES OF PHARMACOLOGY;DRUG THERAPY FOR NEURODEGENERATIVE
DISEASES;PAGE 532-543
 SK SRIVASTAVA 1ST EDITION VOL1;MEDICAL PHARMACOLOGY;CNS STIMULANTS,ANALEPTICS AND COGNITIVE
ENHANCERS;PAGE 763-767
 BRANDEN MAHER ET AL;POLL RESULTS –LOOK WHO’S DOPING;NATURE INTERNATIONAL JOURNAL OF SCIENCE

Presentation on experimental evaluation of memory enhancers

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Presentation on experimental evaluation of memory enhancers