Pain-Induced Negative Affect Is Mediated via Recruitment

of The Nucleus Accumbens Kappa Opioid System Pdf

Download

https://ebookmass.com/product/pain-induced-negative-affect-is-
mediated-via-recruitment-of-the-nucleus-accumbens-kappa-opioid-
system/

★★★★★
4.6 out of 5.0 (74 reviews )

Instant PDF Download

ebookmass.com
 Pain-Induced Negative Affect Is Mediated via Recruitment of
The Nucleus Accumbens Kappa Opioid System Pdf Download

EBOOK

Available Formats

■ PDF eBook Study Guide Ebook

EXCLUSIVE 2025 EDUCATIONAL COLLECTION - LIMITED TIME

INSTANT DOWNLOAD VIEW LIBRARY

Collection Highlights

How We Hurt: The Politics of Pain in the Opioid Epidemic

Melina Sherman

The Right to Pain Relief and Other Deep Roots of the

Opioid Epidemic Mark D. Sullivan

Fighting the Opioid Epidemic: The Role of Providers and

the Clinical Laboratory in Understanding Who is Vulnerable
1st Edition Amitava Dasgupta

Mediated Shame of Class and Poverty Across Europe Irena

Reifová
Office-Based Buprenorphine Treatment of Opioid Use
Disorder Second Edition

The Rhetorical Power of Popular Culture: Considering

Mediated Texts 3rd Edition, (Ebook PDF)

Laser-Induced Breakdown Spectroscopy 2nd Edition Jagdish

P. Singh (Editor)

Emotional Choices: How the Logic of Affect Shapes Coercive

Diplomacy 1st Edition Robin Markwica

Circuit directionality for motivation: lateral accumbens-

pallidum, but not pallidum-accumbens, connections regulate
motivational attraction to reward cues Elizabeth B.
Smedley & Alyssa Dileo & Kyle S. Smith
 Report

Pain-Induced Negative Affect Is Mediated via

Recruitment of The Nucleus Accumbens Kappa
Opioid System
Graphical Abstract Authors
Nicolas Massaly, Bryan A. Copits,
Adrianne R. Wilson-Poe, ...,
Ream Al-Hasani, Michael R. Bruchas,
Jose A. Morón

Correspondence
[email protected] (R.A.-H.),
[email protected] (M.R.B.),
[email protected] (J.A.M.)

In Brief
Massaly et al. identify a pain-induced
enhancement in the kappa opioid system
within nucleus accumbens, which drives
pain-associated negative emotional
states. These results provide a functional
substrate for therapies that would
circumvent pain-induced affective
disorders.

Highlights
d Pain recruits the dynorphin-kappa opioid receptor system in
the nucleus accumbens

d Inhibitory inputs onto dynorphin cells are reduced during

inflammatory pain

d Increase in dynorphin tone mediates inflammatory pain-

induced negative affect

Massaly et al., 2019, Neuron 102, 1–10

May 8, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.neuron.2019.02.029
 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Neuron

Report

Pain-Induced Negative Affect Is Mediated

via Recruitment of The Nucleus Accumbens
Kappa Opioid System
Nicolas Massaly,1 Bryan A. Copits,1 Adrianne R. Wilson-Poe,1 Lucia Hipólito,2 Tamara Markovic,1 Hye Jean Yoon,1
Shiwei Liu,3 Marie C. Walicki,1,4,5 Dionnet L. Bhatti,1 Sunil Sirohi,6 Amanda Klaas,7 Brendan M. Walker,6 Rachael Neve,8
Catherine M. Cahill,3 Kooresh I. Shoghi,7,9 Robert W. Gereau IV,1,9 Jordan G. McCall,1,4,5 Ream Al-Hasani,1,4,5,*
Michael R. Bruchas,1,9,* and Jose A. Morón1,9,10,*
1Department of Anesthesiology, Washington University Pain Center, Washington University in St. Louis, School of Medicine, St. Louis, MO

63110, USA
2Department of Pharmacy and Pharmaceutical Technology and Parasitology, University of Valencia, Valencia 46100, Spain
3Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, Los Angeles, CA 90095, USA
4Department of Pharmaceutical and Administrative Sciences, St. Louis College of Pharmacy, St. Louis, MO 63110, USA
5Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University in St. Louis School of Medicine, St. Louis, MO

63110, USA
6Department of Psychology, Washington State University, Pullman, WA 99164-4820, USA
7Department of Radiology, Washington University in St. Louis, School of Medicine, St. Louis, MO 63110, USA
8Department of Brain and Cognitive Science, Viral Gene Transfer Core, MIT, Cambridge, MA 02139-4307, USA
9Department of Neuroscience, Washington University in St. Louis, St. Louis, MO 63110, USA
10Lead Contact

*Correspondence: [email protected] (R.A.-H.), [email protected] (M.R.B.), [email protected] (J.A.M.)

https://doi.org/10.1016/j.neuron.2019.02.029

SUMMARY states largely drives anxiety- and stress-induced disorders and

involuntary opioid overdoses (Elman and Borsook, 2016; Volkow
Negative affective states affect quality of life for and McLellan, 2016). Despite the drive to improve pain treatment
patients suffering from pain. These maladaptive and prevent tragic outcomes, the mechanisms underlying these
emotional states can lead to involuntary opioid over- negative affective states are not yet clear. Recent human studies
dose and many neuropsychiatric comorbidities. show reduced nucleus accumbens (NAc) activity, alterations
Uncovering the mechanisms responsible for pain- in reward evaluation, decision making, and motivation in pain
patients (Apkarian et al., 2004; Verdejo-Garcı́a et al., 2009). In
induced negative affect is critical in addressing these
rodent models, transient activation of kappa opioid receptors
comorbid outcomes. The nucleus accumbens (NAc)
(KORs) in a discrete subregion of the NAc abates the reinforcing
shell, which integrates the aversive and rewarding and motivational values of rewards through presynaptic inhibi-
valence of stimuli, exhibits plastic adaptations in tion of dopamine, glutamate, and serotonin release (Crowley
the presence of pain. In discrete regions of the and Kash, 2015; Narita et al., 2005; Tejeda et al., 2017). This
NAc, activation of the kappa opioid receptor (KOR) behavioral adaptation is also observed during inflammatory
decreases the reinforcing properties of rewards and pain, which represents a characteristic feature of negative affec-
induces aversive behaviors. Using complementary tive states (Al-Hasani et al., 2015; Castro and Berridge, 2014;
techniques, we report that in vivo recruitment of Narita et al., 2005; Shippenberg et al., 1988; Verdejo-Garcı́a
NAc shell dynorphin neurons, acting through KOR, et al., 2009). We hypothesized that KORs in a subregion of the
is necessary and sufficient to drive pain-induced NAc (NAc shell cold spot [NAcShCS] for rats and ventromedial
NAc shell [vNAcSh] for mice) represent a key mechanism for
negative affect. Taken together, our results provide
generating pain-induced negative affective states.
evidence that pain-induced adaptations in the kappa
opioid system within the NAc shell represent a func- RESULTS
tional target for therapeutic intervention that could
circumvent pain-induced affective disorders. Kappa Opioid Receptors Are Both Necessary and
Sufficient to Drive Pain-Induced Negative Affect
Recent evidence from pain and depression studies report that
INTRODUCTION decreased motivation in goal-directed behavior represents a
characteristic feature of pain-induced negative affect (Hipólito
Pain is a major, growing epidemic in the U.S., afflicting more than et al., 2015; Schwartz et al., 2014). Therefore, to determine the
30% of the population (Elman et al., 2013). In this maladaptive role of the KORs in these maladaptive states, we combined local
condition, the presence of under- or untreated negative affective microinjection of the long-acting selective KOR antagonist

Neuron 102, 1–10, May 8, 2019 ª 2019 Elsevier Inc. 1

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

(legend on next page)

2 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

norbinaltorphimine (norBNI) with a progressive ratio (PR) tory pain without directly impacting the nociceptive component
schedule of reinforcement for sucrose rewards (Figure 1A). In of pain (Figures 1B–1H). In order to examine whether activation
agreement with previous literature (Leitl et al., 2014; Narita of KOR within the NAc would be sufficient to drive a decrease
et al., 2005; Schwartz et al., 2014), inflammatory pain decreased in motivation, rats received a local bilateral micro-injection of
the motivational state of rats as measured by a lower number of the short-acting KOR agonist U50,488 (1 mg/side) (Figure 1I).
rewards earned (Figures 1B and 1C). Local pharmacological Selective engagement of KOR signaling within the NAcShCS
blockade of KORs, using bilateral micro-injections of norBNI induced a significant decrease in motivation for sucrose self-
(2 mg/side) into the NAcShCS, was sufficient to prevent this administration in naive rats (Figures 1J and 1K). Moreover, resto-
pain-induced decrease in motivation (Figures 1B and 1C). This ration in sucrose seeking was observed during a third PR test run
result cannot be attributed to an intrinsic effect of norBNI as 24 h after pharmacological treatment, a time point at which
blockade of KORs alone, in sham pain conditions, did not alter U50,488 is washed off (Figures 1J and 1K). Interestingly, local
the rat’s motivational state (norBNI-Saline, Figures 1B and 1C). KOR stimulation did not impact sucrose self-administration on
Furthermore, inflamed rats did not exhibit any difference in a fixed ratio schedule of reinforcement or impact the initial shape
reward acquisition when lower efforts were required (i.e., the of the PR test (Figures S1A–S1C and Figure 1K, respectively).
initial slope of reward earning during PR test [Figure 1C] or in a These data further support the specific involvement of KORs in
fixed ratio schedule of reinforcement [Hipólito et al., 2015]). the NAcShCS in decreasing motivated behavior rather than
This demonstrates that inflammatory pain did not affect the abil- causing a generalizable behavioral impairment.
ity for rats to interact with the reward-paired lever but rather
impaired their motivation when high efforts were required to Activation of Dynorphin-Containing Neurons in the
obtain the reward. Additionally, while injection of norBNI in the vNAcSh Is Sufficient to Drive Negative Affective States
NAcShCS reversed pain-induced negative affective state, com- and Aversive Behavior
plete Freund’s adjuvant (CFA)-induced hyperalgesia, measured In the NAcSh, a large population of MSNs contain dynorphin and
using Hargreaves thermal test, was not affected (Figure 1D). locally control presynaptic neurotransmitter release (Al-Hasani
These results demonstrate that KOR blockade in the NAcShCS et al., 2015; Nestler and Carlezon, 2006). To investigate
reverses pain-induced negative affective states without affecting the role of these dynorphin-containing neurons on negative
thermal hyperalgesia. It has been widely shown that pain- affective states, we selectively expressed channelrhodopsin-2
induced decrease in motivated behavior occurs across species (ChR2) in the vNAcSh of dynorphin-cre+ mice (Figure 2A). Here
(Okun et al., 2016; Schwartz et al., 2014). Therefore, to determine we observe that stimulation of dynorphin-containing neurons in
the extent to which these findings apply to other species, we the vNAcSh is sufficient to decrease motivation to self-admin-
conducted additional studies to further evaluate a potential ister sucrose (Figures 2B–2D). In addition, we previously re-
role for KOR in pain-induced negative affect in both male and ported that stimulation of dynorphin-containing neurons in the
female mice (Figures 1E–1H). Local micro-injection of norBNI vNAcSh was sufficient to drive a KOR-dependent real-time place
(2 mg/side) in the vNAcSh reversed the pain-induced decrease aversion (RTPA) (Al-Hasani et al., 2015). We combined RTPA-
in motivation observed 2 days after CFA treatment (Figures induced photo-stimulation of dynorphin-containing neurons in
1E–1H) in both sexes. Importantly, this result emphasizes a the vNacSh with local KOR blockade using norBNI (2 mg and
KOR-dependent mechanism in the vNAcSh to drive pain- 4 mg in 0.5 mL) to test their involvement in mediating negative
induced negative affect across species and sexes. Overall, our affective states and aversive behavior during inflammatory
data suggest that KORs are necessary within the NAcShCS/ pain. We found that local NAc norBNI pretreatment (2 mg
vNAcSh for reducing motivated behavior induced by inflamma- and 4 mg per side) blocks dynorphin-neuron-stimulated RTPA

Figure 1. Kappa Opioid Receptors Are Both Necessary and Sufficient to Drive Pain-Induced Negative Affect
(A) Schematic representation of the behavioral methodology.
(B) Blockade of KORs in the NAcShCS prevented pain-induced decrease in motivation (two-way ANOVA for repeated measures: time: F2,116 = 69.51, p < 0.0001;
interaction: F6,116 = 7.349, p < 0.0001. Post hoc during PR3: aCSF-Saline [n = 16] versus aCSF-CFA [n = 19], p < 0.0001, norBNI-Saline [n = 9] versus aCSF-CFA
[n = 19], p < 0.0001 and aCSF-CFA [n = 19] versus norBNI-CFA [n = 18], p = 0.0225).
(C) Representation of the average number of rewards obtained every minute across the 2-h PR3 test session.
(D) Paw withdrawal latency was unchanged in inflamed animals treated with norBNI (two-way ANOVA for repeated measures: time: F2,116 = 35.95, p < 0.0001;
treatment: F3,58 = 25.08, p < 0.0001; interaction: F6,116 = 74.93, p < 0.0001. Post hoc during PR3: aCSF-CFA [n = 19] versus norBNI-CFA [n = 18], p = 0.9998).
(E) Schematic representation of the behavioral methodology.
(F) Representation of training process across experimental days.
(G) Blockade of KORs in the vNAcSh prevented the pain-induced decrease in motivation (two-way ANOVA for repeated measures: time: F2,76 = 0.5201,
p < 0.0001; treatment: F2,38 = 3.432, p = 0.0426. Post hoc: norBNI-Saline [n = 14] versus aCSF-CFA [n = 13], p = 0.0023, and aCSF-CFA [n = 13] versus
norBNI-CFA [n = 14], p = 0.0408).
(H) Representation of the average number of rewards obtained every minute across the PR3 session.
(I) Schematic representation of the behavioral methodology.
(J) Stimulation of KORs in the NAcShCS by U50,488 injection is sufficient to decrease motivation (two-way ANOVA for repeated measures: time: F2,24 = 4.767, p =
0.0181; treatment: F1,12 = 6.005, p = 0.0306. Post hoc after treatment [PR2]: aCSF [n = 7] versus U50,488 [n = 7], p = 0.0064, and 24 h after injection: p > 0.9999).
(K) Representation of the average number of rewards obtained every minute across the 2-h test PR2 session.
(L) During Hargreaves test, the paw withdrawal latency is slightly increased after U50,488 injection in the NAcShCS (two-way ANOVA for repeated measures: time
F2,28 = 9,246, p = 0.0008; interaction F2,28 = 4.060, p = 0.0283; Post hoc test: aCSF [n = 7] versus U50,488 [n = 7] on PR2: p = 0.0326).

Neuron 102, 1–10, May 8, 2019 3

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Figure 2. Activation of Dynorphin-Containing Neurons in the vNACSh Is Sufficient to Drive Negative Affective States and Aversive Behavior
(A) Schematic representation of the behavioral paradigm.
(B) Representation of the training process across experimental days for mice.
(C) Photo-stimulation of dynorphin-containing neurons in the vNAcSh is sufficient to decrease motivation for sucrose self-administration. Mann-Whitney: Ctrl
(n = 7) versus Chr2 (n = 6), p = 0.0385.
(D) Representation of the average number of rewards obtained every minute across the 1-h test session (Kolmogorov-Smirnov: p < 0.0001).
(E) Schematic representation of the behavioral paradigm for real-time place preference testing (RTPT).
(F) Representative heatmaps of the animal activity during the 20 min RTPT.
(G) During baseline test (no pain), aCSF-dyn-cre+ mice demonstrated an aversion for the photo-stimulated compartment (one-sample t test compared to 50%:
n = 13, p = 0.0352). This effect was fully prevented by norBNI pretreatment (one-sample t test compared to 50%: norBNI-dyn-cre+ 2 mg: n = 9, p = 0.6833, norBNI-
dyn-cre+ 4 mg: n = 7, p = 0.3014). Two days after pain induction, 2 mg of norBNI did not reverse the aversive properties of dynorphin-containing neurons
stimulation (one-sample t test compared to 50%: n = 9, p = 0.0122). In contrast, 4 mg of norBNI successfully prevented photo-stimulation-induced aversion
(one-sample t test compared to 50%: n = 7, p = 0.0807).
(H) Locomotor activity was similar throughout all groups of mice during RTPT (two-way ANOVA for repeated measures: time: F1,33 = 30.16, p < 0.0001; treatment:
F3,33 = 0.7857, p = 0.5105; interaction: F3,33 = 1.342, p = 0.5722).

(Figures 2E–2G). Interestingly, 48 h after CFA injection, pretreat- ditions. Importantly, KOR blockade did not impact locomotor
ment with 2 mg of norBNI no longer prevented KOR-mediated activity (Figure 2H) or have an effect on RTPT behavior in
aversion (Figures 2F and 2G). However, local vNAcSh infusion eYFP-expressing controls (Figures S1D–S1F). Furthermore, we
of 4 mg of norBNI blocked the aversion in inflammatory pain con- demonstrated that norBNI micro-injection in the vNAcSh did

4 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Figure 3. Inflammatory Pain Increases KOR Functional Activity and Recruits Dynorphin-Containing Neurons in the NAcSh through a
Disinhibition Mechanism
(A) Dynorphin A stimulation dose-dependently increases GTPgS incorporation in NAc tissue from CFA-injected (n = 6) and saline-injected (n = 6) rats (two-way
mixed-model ANOVA: dose: F1,30 = 8.717, p = 0.0066). GTPgS incorporation is significantly higher in CFA-treated animals, suggesting an increase in KOR
functional activity (two-way mixed-model ANOVA: pain effect: F2,30 = 29.98, p < 0.0001).
(B) Representative GTPgS autoradiography of slices incubated with U69,593 in saline (top) and CFA ± JdTic (bottom) injected animals.
(C and D) In conditions of pain, KOR functional activity was increased in the NAc shell (C; unpaired two-way t test: p = 0.0027, n = 8) but not in the NAc core
(D; unpaired two-way t test: p = 0.1450, n = 8).
(E) Representative pictures of dynorphin A expression in the NAcShCS in either saline-injected (top) and CFA-injected (bottom) animals. ACA, anterior
commissure; NAcSh, nucleus accumbens shell; NAcCr, nucleus accumbens core.
(F) Dynorphin A content in the NAcShCS is increased after CFA-induced inflammation (Mann-Whitney test for unpaired values; p = 0.0022, n = 6).
(G) Schematic representation of electrophysiology methodology. Lower panel: representative picture of a patch pipette onto the somatic area of an Ai14+
dynorphin neuron in the vNACsh.
(H) Representative traces of current response from vNacSh dynorphin neurons obtained from either saline or CFA mice.
(legend continued on next page)

Neuron 102, 1–10, May 8, 2019 5

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

not impact the inflammatory pain-induced anxiety as measured this increase in dynorphin A expression was correlated with an
in an open field chamber (Figures S1G–S1I), consistent with a increase in dynorphin neuron excitability, we conducted whole-
role of KORs in anxiety in the amygdala (Al-Hasani et al., 2015; cell patch-clamp electrophysiological recordings on dynorphin-
Bruchas et al., 2009; Crowley et al., 2016; Knoll and Carlezon, containing neurons in the vNAcSh in dynorphin-cre+ reporter
2010). Our results indicate that pain does not potentiate the aver- mice (Figure 3G) (Al-Hasani et al., 2015; Krashes et al., 2014).
sive behavior induced by dynorphin-containing neuron photo- Dynorphin-containing neurons from CFA-treated reporter mice
stimulation but that KORs are required for the modulation of exhibited enhanced excitability, as measured by a depolarized
pain-induced negative affect. These findings identify a key role resting membrane potential and a lower rheobase (Figures 3H–
for the KOR-dynorphin system in pain-induced negative affect. 3J). These results demonstrate that inflammatory pain signifi-
cantly increases the excitability of dynorphin-containing vNAcSh
Inflammatory Pain Increases KOR Functional Activity neurons (Figures 3H–3J). To determine the potential cellular
and Recruits Dynorphin-Containing Neurons in the mechanisms underlying this increase in dynorphinergic tone
NAcSh through a Disinhibition Mechanism within the NAcSh, we examined spontaneous synaptic input to
In order to determine how inflammatory pain engages the KOR the vNAcSh using dynorphin-cre reporter mice. In inflammatory
system in the NAcShCS, we used in situ hybridization in rat tissue pain, as compared to sham pain controls, a decrease in the fre-
to measure the expression of KOR mRNA (Oprk1) (Tejeda et al., quency and amplitude of spontaneous inhibitory postsynaptic
2017). No significant differences in KOR mRNA expression were currents was specifically observed in dynorphin-containing
observed between saline- and CFA-injected animals 48 h after neurons (Figures 3K–3P). Interestingly, a shift in the distribution
pain induction (Figures S2A and S2B). In addition, we assessed toward increased inhibitory tone onto non-dynorphin neurons
KOR functional activity using a radiolabeled non-hydrolysable in vNAcSh was also observed (Figures 3N–3P). Together, these
[35S] GTPgS to measure KOR-dependent G-protein coupling. results support the conclusion that a pain-induced selective
Dynorphin-A-stimulated [35S] GTPgS binding was significantly disinhibition of dynorphin-containing neurons leads to an
elevated in the NAc of CFA-injected animals compared to con- enhancement of their excitability in the vNAcSh.
trols (Figure 3A), indicating enhanced KOR G-protein coupling
in inflammatory pain. For additional anatomical resolution, we Inflammatory Pain Mediates Negative Affective States
utilized [35S] GTPgS autoradiography in brain slices (Liu et al., through Recruitment of Dynorphin-Containing Neurons
2016). In CFA-injected rats, KOR-induced G-protein activation in the NAcShCS
was enhanced in the NAc shell (Figures 3B–3D). This increase To further determine whether pain-induced increase in dynor-
was dependent on KOR activation, as no radioactive incorpora- phin cell excitability results in an enhancement in dynorphin
tion was visualized when slices were with both KOR agonist tone, we used in vivo positron emission tomography (PET) imag-
and a selective KOR antagonist. Interestingly, only a slight ing (Figure 4A). In anesthetized rats, the binding efficacy of a
non-significant increase in [35S] GTPgS radiolabeling was radioactive competitive antagonist for KORs, 11C-LY2795050
observed in the NAc core and in the band of Broca, suggesting (Zheng et al., 2013), was measured before (baseline) or 48 h
that enhanced KOR-mediated activation of Ga signaling in pain after a saline or a CFA injection as an indirect measure of recep-
conditions is tightly localized to the NAc shell (Figure 3D). tor occupancy. Compared to baseline, the distribution volume of
11
Compensatory changes in the KOR system may also be medi- C-LY2795050 was significantly decreased when rats were
ated via recruitment of dynorphin-containing medium spiny neu- in pain while it remained unchanged in sham pain control animals
rons (MSNs). We hypothesized that changes in NAc dynorphin (Figures 4B and 4C). This selective reduction in 11C-LY2795050
tone may underlie the KOR-dependent decrease in motivation binding suggests an elevation in endogenous dynorphin onto
and adaptive changes in KOR activity observed during pain KORs during inflammatory pain.
(Liu et al., 2016; Muschamp and Carlezon, 2013; Narita et al., We then examined whether dynorphin-containing neuronal
2005). To test this, we examined the expression of the dynorphin activation is necessary for the observed effects of inflammatory
A peptide in the NAc shell using immunohistochemistry. Rats pain on motivated behavior, using a chemogenetic approach
experiencing inflammatory pain exhibited a robust increase in (Roth, 2016) to selectively silence the activity of NAc dynor-
dynorphin A expression within the NAcShCS (Figures 3E and phin-containing neurons. Following a baseline PR test, wild-
3F) compared to sham pain controls. To investigate whether type rats were bilaterally micro-injected in the NAcShCS with a

(I and J) CFA-injected animals display a higher resting membrane potential (I; unpaired two-way t test: p = 0.032, ncells/animals = 7–9/4) and (J) a lower rheobase
(J; unpaired two-way t test: p = 0.019, ncells/animals = 7–9/4).
(K) Representative traces of sIPSCs from dyn+ neurons.
(L and M) Amplitude (L) and frequency (M) of sIPSC onto dyn+ neurons are decreased in conditions of inflammatory pain as compared to saline control (frequency:
two-tailed t test for unpaired values p = 0.0406; amplitude: two-tailed Mann-Whitney for unpaired values, p = 0.0140, ncells/animals = 7–8/4). The cumulative
probability plots demonstrate a significant shift toward smaller and less frequent events in dynorphin+ neurons after CFA (Kolmogorov-Smirnov test, p < 0.0001,
ncells/animals = 7–8/4).
(N) Representative traces of sIPSCs from Dyn– neurons.
(O and P) Neither the mean amplitude (O) nor frequency (P) of sIPSCs onto dyn– neurons are affected by inflammatory pain (frequency: two-tailed Mann-
Whitney for unpaired values, p = 0.6277; amplitude: two-tailed Mann-Whitney for unpaired values, p = 0.4242, ncells/animals = 5–6/4). A plot of the cumulative
probability revealed a significant shift toward larger and more frequent events in dynorphin-negative neurons after CFA (Kolmogorov-Smirnov test,
p < 0.0001, ncells/animals = 5–6/4).

6 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Figure 4. Inflammatory Pain Mediates Negative Affective States through Recruitment of Dynorphin-Containing Neurons in the NAcShCS
(A) Schematic representation of the PET imaging methodology in rats.
(B) Inflammatory pain, but not saline control, decreases the distribution volume of 11C-LY2795050 observed in rat’s brain 2 days after CFA injection (two-way
ANOVA for repeated measures: time: F1,12 = 26.88, p = 0.0002; interaction: F1,12 = 10.02, p = 0.0081; post hoc tests: baseline CFA versus test CFA: p < 0.0001;
baseline saline versus test saline: p = 0.3260; test CFA versus test saline: p = 0.0282, n = 7).
(C) Representative images of PET imaging before (left) and after (right) inflammation.
(D) Schematic representation of the behavioral methodology.
(E) Silencing dynorphin neurons in the NAcShCS prevented the inflammatory pain-induced decrease in motivation for sucrose self-administration (two-way
ANOVA for repeated measures: time: F1, 38 = 75.81, p < 0.0001; interaction: F4,38 = 15.84, p < 0.0001. Post hoc test: aCSF-Veh.-CNO versus hM4Di-CFA-Sal.:
p = 0.0006; aCSF-Veh.-CNO versus Ctrl-CFA-CNO: p = 0.0003; Ctrl-CFA-CNO versus hM4Di-CFA-CNO: p = 0.0255; hM4Di-CFA-Sal. versus hM4Di-CFA-CNO:
p = 0.0417; hM4Di-CFA-Sal. versus hM4Di-Veh.-CNO: p = 0.0046; Ctrl-CFA-CNO versus hM4Di-Veh.-CNO: p = 0.0027; hM4Di-CFA-CNO versus
hM4Di-Veh.-CNO: p = 0.9658; hM4Di-CFA-Sal. versus Ctrl-CFA-CNO: p = 0.9988; aCSF-Veh.-CNO versus hM4Di-Veh.-CNO: p = 0.9706; and aCSF-Veh.-CNO
versus hM4Di-CFA-CNO: p = 0.7173, n = 8–9).

herpes simplex virus (HSV) containing inhibitory designer DISCUSSION

receptors exclusively activated by designer drugs (DREADDs)
(Dyn2.0-hM4Di-IRES-mCherry) or its control virus (Dyn2.0- In the present study, we identified a critical role for the kappa
IRES-mCherry) (Figure 4D). This approach allowed us to specif- opioid system in the modulation of negative affect associated
ically target NAcShCS dynorphin-containing neurons (Figures with inflammatory pain. We report that pain enhances dynorphin
S3A and S3B) and decrease their excitability upon CNO admin- expression and recruits dynorphin-containing neurons in a
istration (Figures S3C–S3F). Importantly, CNO alone did not discrete subregion of the NAc shell through a disinhibition mech-
impact neuronal excitability (Figures S3G–S3J). As previously anism. In addition, we further report that pain increases KOR
described, rats experiencing inflammatory pain display a signif- function and occupancy using both GTPgS binding and in vivo
icant decrease in motivation compared to sham pain animals. PET imaging. Finally, using a complementary series of pharma-
Chemogenetic silencing of NAcShCS dynorphin-containing cological, optogenetic, and chemogenetic approaches, we
neurons blocked the decrease in motivation observed in report that both dynorphin-containing neurons and KOR activity
CFA-treated animals (Figure 4E). Importantly, the sole expres- in the NAc shell are necessary and sufficient to drive pain-
sion of Gai-coupled DREADDs, not acute CNO administration induced negative affective states.
(1mg.kg 1), impacted pain-induced decrease in motivated Previous evidence has uncovered a role for the dynorphin
behavior (Figure 4E). Lastly, silencing dynorphin-containing neu- peptide in decreasing opioid-mediated goal-directed behaviors
rons did not increase motivated behavior in non-pain conditions and opioid-induced dopamine release in the NAc during pain
(Figure 4E). These results establish that neither DREADD states (Narita et al., 2005; Verdejo-Garcı́a et al., 2009). However,
expression nor CNO administration have non-specific actions the source of endogenous peptide and its consequent mecha-
on these behaviors (Figure 4E). Altogether, these results demon- nism of action have remained elusive. In the current work,
strate that dynorphin-containing neuronal activity is necessary we uncover that pain is sufficient to increase dynorphin expres-
for generating inflammatory pain-induced negative affect. sion in the NAcShCS (Figures 3E and 3F) and recruit local

Neuron 102, 1–10, May 8, 2019 7

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

dynorphin-containing neuron activity (Figures 3G–3I). We deter- sights into neurobiological targets for future pharmacother-
mine that this recruitment is due to a substantial decrease in apies that attenuate unwanted negative outcomes during
IPSCs onto these NAc dynorphin-containing neurons, leading pain. Emerging therapies such as focused ultrasound technol-
to a hyperexcitable state for those neurons (Figures 3G–3M). ogy (Elias et al., 2016), the development of photoactivable
We demonstrate for the first time using PET scan imaging in ro- compounds (Banghart and Sabatini, 2012), and drugs specif-
dents, that pain induces increases in dynorphin tone in vivo (Fig- ically targeting the activated-KOR structure (Che et al., 2018)
ures 4A–4C). In light of the overall increase in KOR occupancy could allow for selective and localized treatments. Together
observed in our study, it is important to consider the possibility with a better understanding of dynorphin-KOR system in
that several hubs of dynorphin-containing neurons, such as pain-induced negative affect, these studies open new avenues
ventral pallidum or lateral hypothalamus (Baldo et al., 2003; for targeting KOR as a site for treating pain-induced emotional
Peyron et al., 1998), are also recruited during pain conditions. states.
Through the development of a gen 2.0-specific and efficient
inhibitory DREADDs (Figure S3) in rats, we uncover the role STAR+METHODS
of the NAcShCS dynorphin-containing neuron recruitment in
pain-induced decreases in motivational states (Figures 4D and Detailed methods are provided in the online version of this paper
4E). Together, these results reveal a source of endogenous and include the following:
dynorphin in the vNAcSh/NAcShCS that drives pain-induced
negative affect, resolving an important gap in our understanding d KEY RESOURCES TABLE
of the impact of pain on the dynorphin-KOR system. d CONTACT FOR REAGENT AND RESOURCE SHARING
Recent studies have identified alterations in excitatory trans- d EXPERIMENTAL MODEL AND SUBJECT DETAILS
mission onto D2-expressing MSNs in the NAc during inflamma- d METHOD DETAILS
tory and neuropathic pain (Ren et al., 2016; Schwartz et al., B Surgeries
2014). While these studies describe increased excitatory inputs B Chemogenetics, Optogenetics and Behavioral Assays
(Ren et al., 2016) that can trigger long-term plasticity at synapses B Receptor Function Assessment
onto D2R-expressing neurons (Schwartz et al., 2014), we report B Electrophysiology
here that dynorphin-containing neurons are specifically disinhi- B Immunohistochemistry
bited in inflammatory pain (Figures 3K–3M). Furthermore, while B Positron Emission Tomography (Pet) Imaging
NAc receives KOR-enriched synaptic terminals from brain struc- B In Situ Hibridization
tures involved in affect and motivation (Bruchas et al., 2009; B Histology
Land et al., 2009; Margolis et al., 2003; Muschamp and Carlezon, d QUANTIFICATION AND STATISTICAL ANALYSIS
2013), we demonstrate here that local KOR blockade in the NAc d DATA AND SOFTWARE AVAILABILITY
reverses pain-induced negative affect in both mice and rats
(Figure 1). Local release of dynorphin, activating KORs in the SUPPLEMENTAL INFORMATION
NAc, may thus negatively regulate the release of serotonin,
dopamine, glutamate and/or GABA, leading to altered NAc func- Supplemental Information can be found with this article online at https://doi.
tion in pain-induced behaviors, including reward seeking. org/10.1016/j.neuron.2019.02.029.
Despite this evidence, the role of other peptides and neurotrans-
mitters in pain-induced negative affect cannot be ignored. ACKNOWLEDGMENTS
Similarly, activation of the KOR system located outside of the
NAc may be necessary to drive pain-induced adaptations in We would like to thank all members from the Moron-Concepcion, Bruchas and
Al-Hasani laboratories for their help throughout the completion of the current
negative affect. Nonetheless, in addition to previous reports
study and Lindsay Lueptow for the complementary analysis of GTPgammaS
(Ren et al., 2016; Schwartz et al., 2014), our work provides a slices. This work was supported by US National Institutes of Health (NIH) grant
novel allostatic mechanism through which pain impacts the DA041781 (J.A.M.), DA042581 (J.A.M.), DA042499 (J.A.M.), DA041883
nucleus accumbens microcircuitry to induce negative affective (J.A.M.), DA045463 (J.A.M.), NARSAD Independent Investigator Award from
states. the Brain and Behavior Research Foundation (J.A.M.), DA033396 (M.R.B.),
Using a combination of pharmacological studies in both rats DA037152 (M.R.B.), R01-NS106953 (R.W.G.), AA020394 (B.M.W.), K99/R00-
DA038725 (R.A.), K99-DA041467 (A.R.W.-P.), Philippe Foundation (N.M.),
and mice, we show that adaptations in the dynorphin-KOR sys-
the ‘‘Spanish Ministerio de Economia y Competitividad’’ MINECO PSI2016-
tem occur when aversive behaviors are measured in conditions
77895-R (L.H.), the Mallinckrodt Institute of Radiology at Washington Univer-
of pain (Figure 2). Lastly, a recent study from Yang and collabo- sity from pilot grant 16-014 to support the positron emission tomography
rators has uncovered two distinct accumbens shell subregions studies, the Hope Center Viral Vectors Core at Washington University School
projecting to the ventral tegmental area (VTA) to regulate motiva- of Medicine.
tional states (Yang et al., 2018). Future studies will further
examine how the dynorphin-containing neuronal population AUTHOR CONTRIBUTIONS
described in our findings integrates with those specific circuits
Conceptualization, N.M., L.H., R.W.G., K.I.S., R.A., M.R.B., and J.A.M.; Meth-
to drive motivational state adaptations in pain conditions.
odology, N.M., R.A., M.R.B., and J.A.M.; Formal Analysis, N.M., and J.A.M.;
While other studies describe a potential therapeutic role for Investigation, N.M., B.A.C., A.R.W.-P., M.C.W., J.G.M., T.M., L.H., H.J.Y.,
KOR antagonists (Al-Hasani et al., 2015; Castro and Berridge, B.M.W., S.S., S.L., C.M.C., D.L.B., R.A., and A.K.; Writing – Original Draft,
2014; Chavkin, 2011), our current findings provide further in- N.M., R.A., M.R.B., and J.A.M.; Writing – Review & Editing, N.M., R.A.,

8 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

M.R.B., and J.A.M.; Funding Acquisition, R.A., M.R.B., and J.A.M.; Resources, Knoll, A.T., and Carlezon, W.A., Jr. (2010). Dynorphin, stress, and depression.
R.N., R.A., M.R.B., and J.A.M.; Supervision, N.M., R.A., M.R.B., and J.A.M. Brain Res. 1314, 56–73.
Krashes, M.J., Shah, B.P., Madara, J.C., Olson, D.P., Strochlic, D.E., Garfield,
DECLARATION OF INTERESTS A.S., Vong, L., Pei, H., Watabe-Uchida, M., Uchida, N., et al. (2014). An excit-
atory paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature
The authors declare no competing financial interests. 507, 238–242.
Land, B.B., Bruchas, M.R., Schattauer, S., Giardino, W.J., Aita, M., Messinger,
Received: May 22, 2018 D., Hnasko, T.S., Palmiter, R.D., and Chavkin, C. (2009). Activation of the
Revised: September 20, 2018 kappa opioid receptor in the dorsal raphe nucleus mediates the aversive
Accepted: February 14, 2019 effects of stress and reinstates drug seeking. Proc. Natl. Acad. Sci. USA
Published: March 13, 2019 106, 19168–19173.
Leitl, M.D., Onvani, S., Bowers, M.S., Cheng, K., Rice, K.C., Carlezon,
REFERENCES W.A., Jr., Banks, M.L., and Negus, S.S. (2014). Pain-related depression
of the mesolimbic dopamine system in rats: expression, blockade by anal-
Al-Hasani, R., McCall, J.G., Shin, G., Gomez, A.M., Schmitz, G.P., Bernardi, gesics, and role of endogenous k-opioids. Neuropsychopharmacology 39,
J.M., Pyo, C.-O., Park, S.I., Marcinkiewcz, C.M., Crowley, N.A., et al. (2015). 614–624.
Distinct subpopulations of nucleus accumbens dynorphin neurons drive
Liu, S., Cook, C., Thai, E., Pickens, S., Taylor, A.M., Tea, V.D., Carroll, F.I.,
aversion and reward. Neuron 87, 1063–1077.
Leslie, F.M., Evans, C.J., and Cahill, C.M. (2016). Neuropathic pain alters
Apkarian, A.V., Sosa, Y., Krauss, B.R., Thomas, P.S., Fredrickson, B.E., Levy, reward and affect via kappa opioid receptor (KOR) upregulation. FASEB J.
R.E., Harden, R.N., and Chialvo, D.R. (2004). Chronic pain patients are 30, 928.5.
impaired on an emotional decision-making task. Pain 108, 129–136.
Logan, J. (2000). Graphical analysis of PET data applied to reversible and
Baldo, B.A., Daniel, R.A., Berridge, C.W., and Kelley, A.E. (2003). Overlapping irreversible tracers. Nucl. Med. Biol. 27, 661–670.
distributions of orexin/hypocretin- and dopamine-beta-hydroxylase immuno-
Margolis, E.B., Hjelmstad, G.O., Bonci, A., and Fields, H.L. (2003). Kappa-
reactive fibers in rat brain regions mediating arousal, motivation, and stress.
opioid agonists directly inhibit midbrain dopaminergic neurons. J. Neurosci.
J. Comp. Neurol. 464, 220–237.
23, 9981–9986.
Banghart, M.R., and Sabatini, B.L. (2012). Photoactivatable neuropeptides
McCall, J.G., Al-Hasani, R., Siuda, E.R., Hong, D.Y., Norris, A.J., Ford, C.P.,
for spatiotemporally precise delivery of opioids in neural tissue. Neuron 73,
and Bruchas, M.R. (2015). CRH engagement of the locus coeruleus noradren-
249–259.
ergic system mediates stress-induced anxiety. Neuron 87, 605–620.
Bruchas, M.R., Land, B.B., Lemos, J.C., and Chavkin, C. (2009). CRF1-R
Muschamp, J.W., and Carlezon, W.A., Jr. (2013). Roles of nucleus accumbens
activation of the dynorphin/kappa opioid system in the mouse basolateral
CREB and dynorphin in dysregulation of motivation. Cold Spring Harb.
amygdala mediates anxiety-like behavior. PLoS ONE 4, e8528.
Perspect. Med. 3, a012005.
Castro, D.C., and Berridge, K.C. (2014). Opioid hedonic hotspot in nucleus
Narita, M., Kishimoto, Y., Ise, Y., Yajima, Y., Misawa, K., and Suzuki, T. (2005).
accumbens shell: mu, delta, and kappa maps for enhancement of sweetness
Direct evidence for the involvement of the mesolimbic kappa-opioid system in
‘‘liking’’ and ‘‘wanting’’. J. Neurosci. 34, 4239–4250.
the morphine-induced rewarding effect under an inflammatory pain-like state.
Chavkin, C. (2011). The therapeutic potential of k-opioids for treatment of pain Neuropsychopharmacology 30, 111–118.
and addiction. Neuropsychopharmacology 36, 369–370. Nestler, E.J., and Carlezon, W.A., Jr. (2006). The mesolimbic dopamine reward
Che, T., Majumdar, S., Zaidi, S.A., Ondachi, P., McCorvy, J.D., Wang, S., circuit in depression. Biol. Psychiatry 59, 1151–1159.
Mosier, P.D., Uprety, R., Vardy, E., Krumm, B.E., et al. (2018). Structure of Okun, A., McKinzie, D.L., Witkin, J.M., Remeniuk, B., Husein, O., Gleason,
the nanobody-stabilized active state of the kappa opioid receptor. Cell 172, S.D., Oyarzo, J., Navratilova, E., McElroy, B., Cowen, S., et al. (2016).
55–67.e15. Hedonic and motivational responses to food reward are unchanged in rats
Crowley, N.A., and Kash, T.L. (2015). Kappa opioid receptor signaling in the with neuropathic pain. Pain 157, 2731–2738.
brain: circuitry and implications for treatment. Prog. Neuropsychopharmacol. Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcliffe,
Biol. Psychiatry 62, 51–60. J.G., and Kilduff, T.S. (1998). Neurons containing hypocretin (orexin) project to
Crowley, N.A., Bloodgood, D.W., Hardaway, J.A., Kendra, A.M., McCall, J.G., multiple neuronal systems. J. Neurosci. 18, 9996–10015.
Al-Hasani, R., McCall, N.M., Yu, W., Schools, Z.L., Krashes, M.J., et al. (2016). Ren, W., Centeno, M.V., Berger, S., Wu, Y., Na, X., Liu, X., Kondapalli, J.,
Dynorphin controls the gain of an amygdalar anxiety circuit. Cell Rep. 14, Apkarian, A.V., Martina, M., and Surmeier, D.J. (2016). The indirect pathway
2774–2783. of the nucleus accumbens shell amplifies neuropathic pain. Nat. Neurosci.
Elias, W.J., Lipsman, N., Ondo, W.G., Ghanouni, P., Kim, Y.G., Lee, W., 19, 220–222.
Schwartz, M., Hynynen, K., Lozano, A.M., Shah, B.B., et al. (2016). A random- Roberts, D.C., and Bennett, S.A. (1993). Heroin self-administration in rats un-
ized trial of focused ultrasound thalamotomy for essential tremor. N. Engl. J. der a progressive ratio schedule of reinforcement. Psychopharmacology
Med. 375, 730–739. (Berl.) 111, 215–218.
Elman, I., and Borsook, D. (2016). Common brain mechanisms of chronic pain Roth, B.L. (2016). DREADDs for neuroscientists. Neuron 89, 683–694.
and addiction. Neuron 89, 11–36. Schwartz, N., Temkin, P., Jurado, S., Lim, B.K., Heifets, B.D., Polepalli, J.S.,
Elman, I., Borsook, D., and Volkow, N.D. (2013). Pain and suicidality: insights and Malenka, R.C. (2014). Chronic pain. Decreased motivation during chronic
from reward and addiction neuroscience. Prog. Neurobiol. 109, 1–27. pain requires long-term depression in the nucleus accumbens. Science 345,
Hipólito, L., Wilson-Poe, A., Campos-Jurado, Y., Zhong, E., Gonzalez- 535–542.
Romero, J., Virag, L., Whittington, R., Comer, S.D., Carlton, S.M., Walker, Shippenberg, T.S., Stein, C., Huber, A., Millan, M.J., and Herz, A. (1988).
B.M., et al. (2015). Inflammatory pain promotes increased opioid self-adminis- Motivational effects of opioids in an animal model of prolonged inflammatory
tration: role of dysregulated ventral tegmental area m opioid receptors. pain: alteration in the effects of kappa- but not of mu-receptor agonists.
J. Neurosci. 35, 12217–12231. Pain 35, 179–186.
Kissler, J.L., Sirohi, S., Reis, D.J., Jansen, H.T., Quock, R.M., Smith, D.G., and Shoghi, K.I., and Welch, M.J. (2007). Hybrid image and blood sampling input
Walker, B.M. (2014). The one-two punch of alcoholism: role of central amyg- function for quantification of small animal dynamic PET data. Nucl. Med.
dala dynorphins/kappa-opioid receptors. Biol. Psychiatry 75, 774–782. Biol. 34, 989–994.

Neuron 102, 1–10, May 8, 2019 9

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Siuda, E.R., Copits, B.A., Schmidt, M.J., Baird, M.A., Al-Hasani, R., Planer, W.J., Wang, F., Flanagan, J., Su, N., Wang, L.-C., Bui, S., Nielson, A., Wu, X., Vo,
Funderburk, S.C., McCall, J.G., Gereau, R.W., 4th, and Bruchas, M.R. (2015). H.-T., Ma, X.-J., and Luo, Y. (2012). RNAscope: a novel in situ RNA analysis
Spatiotemporal control of opioid signaling and behavior. Neuron 86, 923–935. platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn.
Tejeda, H.A., Wu, J., Kornspun, A.R., Pignatelli, M., Kashtelyan, V., Krashes, 14, 22–29.
M.J., Lowell, B.B., Carlezon, W.A., Jr., and Bonci, A. (2017). Pathway- and
Yang, H., de Jong, J.W., Tak, Y., Peck, J., Bateup, H.S., and Lammel, S.
cell-specific kappa-opioid receptor modulation of excitation-inhibition
(2018). Nucleus accumbens subnuclei regulate motivated behavior via direct
balance differentially gates D1 and D2 accumbens neuron activity. Neuron
inhibition and disinhibition of VTA dopamine subpopulations. Neuron 97,
93, 147–163.
434–449.e4.
Verdejo-Garcı́a, A., López-Torrecillas, F., Calandre, E.P., Delgado-Rodrı́guez,
A., and Bechara, A. (2009). Executive function and decision-making in women Zheng, M.-Q., Nabulsi, N., Kim, S.J., Tomasi, G., Lin, S.F., Mitch, C., Quimby,
with fibromyalgia. Arch. Clin. Neuropsychol. 24, 113–122. S., Barth, V., Rash, K., Masters, J., et al. (2013). Synthesis and evaluation
Volkow, N.D., and McLellan, A.T. (2016). Opioid abuse in chronic pain–miscon- of 11C-LY2795050 as a k-opioid receptor antagonist radiotracer for PET
ceptions and mitigation strategies. N. Engl. J. Med. 374, 1253–1263. imaging. J. Nucl. Med. 54, 455–463.

10 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit Anti-dynorphin A Peninsula Laboratories International Inc. Cat# T-4266.0500; RRID: AB_518292
Mouse Anti-mCherry DHBS Cat# DSHB-mCherry-3A11; RRID:
AB_2617430
Biotin conjugated anti-rabbit Bio-Rad Cat# 170-6515
Alexa 488 conjugated streptavidin Thermo Fisher Scientific Cat# S32354; RRID: AB_2336881
Goat anti-Mouse - Secondary Antibody, Thermo Fisher Scientific Cat# A-11019; RRID: AB_143162
Alexa Fluor 568
Endogenous Biotin Blocking Kit Thermo Fisher Scientific Cat# E21390
Goat anti-Rabbit, Biotin-XX Thermo Fisher Scientific Cat# B-2770; RRID: AB_2536431
Bacterial and Virus Strains
HSV-Dyn2.0-hM4Di-IRES-mCherry Obtained from Rachael Neve, M.I.T. N/A
HSV-Dyn2.0-IRES-mCherry Obtained from Rachael Neve, M.I.T. N/A
AAV-EF1a-DIO-hChR2(H134R)-EYFP UNC Vector Core N/A
AAV-EF1a-DIO-EYFP UNC Vector Core N/A
Chemicals, Peptides, and Recombinant Proteins
11
C-LY2795050 Washington University Cyclotron Facility N/A
Complete Freund’s adjuvant Thermo Fisher Scientific Cat# 77140
JDTic Obtained from Ivy Carroll, Reserach N/A
Triangle Institute
Nor-Binaltorphimine dihydrochloride Sigma-Aldrich CAS Number 105618-26-6; Cat# N1771
U-50488 Sigma-Aldrich Cat# D8040
U-69593 Sigma-Aldrich CAS Number 96744-75-1; Cat#U103
Clozapine-N-oxide Sigma-Aldrich CAS Number 34233-69-7; Cat# C0882
Experimental Models: Organisms/Strains
Rat: Sprague Dawley Envigo N/A
Mouse: C57BL/6J The Jackson Laboratory IMSR Cat# JAX:000664; RRID:
IMSR_JAX:000664
Mouse: C57BL/6J - preprodynorphin-IRES-cre Obtained from Bradford Lowell Laboratory N/A
(dyn-Cre)
Mouse: B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J - The Jackson Laboratory IMSR Cat# JAX:007909; RRID:
Ai9-reporter IMSR_JAX:007909
Mouse: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J - The Jackson Laboratory IMSR Cat# JAX:007914; RRID:
Ai14-reporter IMSR_JAX:007914
Software and Algorithms
GraphPad Prism 8 GraphPad RRID: SCR_002798
Illustrator Adobe RRID: SCR_010279
Ethovision XT 10.0 Noldus RRID: SCR_000441

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jose A.
Morón ([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

All procedures were approved by the Washington University Institutional Animal Care and Use Committee (IACUC) in accordance
with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Adult male Sprague Dawley rats

Neuron 102, 1–10.e1–e6, May 8, 2019 e1

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

(250-300 g), adult male and females dynorphin-cre (dyn-Cre) mice (25-30 g) (Al-Hasani et al., 2015), adult male and females Ai14 or
Ai9 preprodynorphin reporter mice (Al-Hasani et al., 2015; Krashes et al., 2014) (25-30 g) and adult C57BL/6J males and females mice
(25-30 g) were used for this study. All animals were 10 to 12 weeks at the beginning of the experiments. Two to three rats were housed
together with a 12/12 h dark/light cycle (lights on at 7:00 AM) and acclimated to the animal facility holdings for at least 7 days before
any manipulation. Rats received food ad libitum until 2 days before starting the behavioral studies, when food restriction (17 g of rat
chow per day) started and continued until the end of the experiment. Four to five mice were housed together, given access to food
pellets and water ad libitum, and maintained on a 12/12 h dark/light cycle (lights on at 7:00 AM). All animals were kept in a sound-
attenuated, isolated holding facility in the lab 1 week prior to surgery, post-surgery, and throughout the duration of the behavioral
assays to minimize stress.

METHOD DETAILS

Surgeries
All surgeries were performed under isoflurane (1.5/2 MAC) anesthesia under sterile conditions.
Intra cerebral injections
Rats were stereotaxically (World Precision Instruments, Sarasota, FL) injected with either norBNI (2 mg per side in 0.5 ml), HSV-
Dyn2.0-hM4di-IRES-mCherry (5x108 transducing units per ml - 0.5 mL per side), HSV-Dyn2.0-mCherry (5x108 transducing units
per ml - 0.5 mL per side) or implanted with guide cannula (Plastics One, Roanoke, VA) targeting the NAcShCS (stereotaxic coordinates
from Bregma: A/p = + 0.96mm, M/L = ± 0.8mm, D/V = – 6.5mm from the skull surface) (Castro and Berridge, 2014; Hipólito et al.,
2015). Animal’s skin was either sutured after bilateral injection using sterile nylon sutures or, when using guide cannula, the implants
were secured on the skull using two sterile bone screws and a dental cement head-cap (Lang Dental). Cannula were obstructed using
a dummy cannula and covered by a cannula cover until pharmacological injection.
Mice were anesthetized in an induction chamber (4 MAC Isoflurane) and placed into a stereotaxic frame (Kopf Instruments, Model
1900) where they were maintained at 1 MAC–2 MAC isoflurane. A craniotomy was performed and followed by a unilaterally
injection, using a blunt needle (86200, Hamilton Company), 300nl of AAV5-DIO-ChR2-eYFP or AAV5-DIO-eYFP controls (Hope
Center Viral Vector Core, viral titer 2 3 1013 vg/mL) into vNAcSh (stereotaxic coordinates from Bregma: A/P: + 1.30mm, M/L: ±
0.5mm, D/V: –4.75mm) (Al-Hasani et al., 2015). Two weeks after this injection mice underwent a second surgery where norBNI
(2 mg or 4 mg /0.5 ml) was locally injected and a fiber optic was placed in the vNAcSh (Al-Hasani et al., 2015; Siuda et al., 2015).
The implants were secured using two bone screws and a dental cement head-cap (Lang Dental). Mice were allowed to recover
one week before running any behavioral experiment, well within the limits of norBNI antagonism. Furthermore, the experiments
started 3 weeks after AAV5-DIO-ChR2-eYFP injection, permitting optimal expression of ChR2 in the dyn-Cre cell bodies.
A similar surgical procedure was used for pharmacological microinjection of norBNI (Figures 1E–1H). Briefly, following craniotomy
norBNI (2 mg or 4 mg /0.5 ml) was bilaterally injected into vNAcSh (stereotaxic coordinates from Bregma: A/P: + 1.30mm, M/L: ±
0.5mm, D/V: –4.75mm)5. Mice’s skin was sutured after bilateral injection using sterile nylon sutures and this animal was allowed
to recovery for a week before running any behavioral experiment.
CFA administration
After confirming sedation by the absence of reflex during a toe-pinch, rats and mice were injected with in the right hindpaw with
properly resuspended 150ul or 50ul of CFA solution (Thermo Fisher), respectively. Animal’ recovery and general behavior (feeding,
drinking, mobility) were monitored through the rest of the experiment.

Chemogenetics, Optogenetics and Behavioral Assays

Sucrose Self-administration
Rat operant-conditioning chambers (Med Associates, Fairfax, VT) were equipped with two retractable levers positioned on the right-
hand wall 12.5 cm apart and 5 cm above the floor, a food magazine connected to a food pellet dispenser, two cue lights positioned
2 cm above the levers, and one house light positioned on the top left-hand wall. At the beginning of the session, both levers (active
and inactive) were presented and a white light was on above the active lever. Pressing the active lever resulted in sucrose pellet
dispense and a 20 s’ retraction of both active and inactive levers, along with the turning off of the light cue above the active lever
after the retraction of the levers. Pressing the inactive lever had no effect. Animals were gently placed in the self-administration boxes
for 2 h sessions where a press on the active lever results in sucrose pellet dispense - fixed-ratio (FR) 1 schedule. Once rats acquired
the self-administration behavior (by obtaining 60 pellets/session on 5 consecutive sessions), the schedule of reinforcement was
changed to FR2 for the other 3 sessions, and then FR5 for another 3 sessions. Once they completed the training, the rat’s motivation
for sucrose self-administration was tested using a Progressive Ratio (PR) schedule of reinforcement (PR1). In the PR session, the
number of responses on the active lever to obtain the reward increased with the dose. The increase in the number of correct re-
sponses followed the equation response ratio = (5 3 e(0.2 3 infusion number) – 5 rounded to the nearest integer resulting in the following
PR steps: 1, 2, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95. (Hipólito et al., 2015; Roberts and Bennett, 1993; Schwartz et al., 2014).
For the studies on the necessity of KOR on pain effects on motivated behavior, rats were injected with either norBNI (2 mg per side)
or aCSF in the NAcShCS (A/P: + 0.96mm; M/L: +/– 0.8mm; D/V: - 6.5mm) (Castro and Berridge, 2014). A week after, to allow full
recovery, animals were tested on a second PR schedule of reinforcement (PR2) to assess any effect of KOR antagonism on

e2 Neuron 102, 1–10.e1–e6, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

motivation. Right after, rats were injected with 150 mL of saline or CFA solution in the hindpaw; and placed again in the self-
administration chambers 48 h after injection to undergo an additional PR session.
For the studies on the sufficiency of KOR on pain effects on motivated behavior, after training and a first PR ratio (PR1) as a baseline
measurement, rats were deeply anesthetized and guide cannula were placed bilaterally 1mm above the NAcShCS (A/P: - 0.96mm;
M/L: +/– 0.8mm; D/V: - 5.5mm) (Castro and Berridge, 2014). One week after surgery, to allow full recovery, animals were injected in
the NAcShCS with KOR agonist (U50,488 – 1 mg per side) or aCSF using a microinjection pump (rate 0.25 mg per minute) and the
injector, projecting 1mm below the cannula, was left in place for an additional 5 min to allow compound diffusion. 30 min after
U50,488 injection rats were tested for thermal hyperalgesia using Hargreaves test. 30 min later these animals were gently placed
in the self-administration boxes for a second PR test (PR2), thus assessing the role of KOR stimulation on sucrose self-administration.
Lastly, and because U50,488 is a reversible KOR agonist, a third PR test was performed 24 h later to assess if the effects of KOR
activation were transient or sustainable.
To determine the involvement of KOR stimulation during a FR schedule of reinforcement, animals were trained to self-administer
sucrose as pellets as mentioned above. After completion of three successive FR5 sessions, rats were deeply anesthetized and guide
cannula were placed bilaterally 1mm above the NAcShCS (A/P: - 0.96mm; M/L: +/– 0.8mm; D/V: - 5.5mm) (Castro and Berridge,
2014). A week after surgery, to allow full recovery, animals were injected in the NAcShCS with KOR agonist (U50,488 – 1 mg per
side) or aCSF using a microinjection pump (rate 0.25 mg per minute) and the injector was left in place for an additional 5 min to allow
compound diffusion. 1 h after U50,488 injection rats were gently placed in the self-administration boxes for new FR5 session, thus
assessing the role of KOR stimulation on a fixed ratio schedule of reinforcement for sucrose self-administration.
For the studies on the necessity of dynorphin containing neurons activity on pain effects on motivated behavior rats were injected
with either HSV-Dyn2.0-hM4Di-IRES-mCherry, HSV-Dyn2.0- mCherry (0.5 mL per side, viral titer: 5 3 108 transducing units per ml,
provided by Rachael Neve, MIT, Boston, Massachusetts) or aCSF in the NAcShCS (A/P: +0.96mm; M/L: +/–0.8mm; D/V: –6.5mm).
Three days after, animals were injected with 150 mL of saline or CFA solution in the hindpaw; and 48 h later, when inflammation was
stable, the rats were injected i.p with CNO (1 mg.kg-1) or saline as a control and placed again in the self-administration chambers to
undergo a second PR session 15 min after i.p. treatment.
For assessment of motivation in mice a PR schedule of reinforcement for sucrose pellet self-administration was also used and per-
formed as described above with slight modifications. Mice operant-conditioning chambers (Med Associates, Fairfax, VT) were equip-
ped with nose poke holes, both presenting a cue light, accessible to animals. An active nose poke resulted in a sucrose delivery in
the food magazine together with house light cue for 20 s (FR1). During this 20 s period, no further action had consequence (time out
period). Poking in the inactive hole had no consequence. Mice were trained to discriminate in between active and inactive lever
during 1 h long FR1 sessions. Once discrimination was acquired (less than 30% of total poking in the inactive hole), mice
underwent 3 consecutive sessions of FR2 and 3 consecutive sessions of FR5. Mice’s motivation was then assessed using a 1 h
session of progressive ratio schedule of reinforcement. To test the necessity of KOR to drive pain-induced negative affect, intra ac-
cumbal (AP: +1.3mm; L: +/– 0.5mm; DV: –4.75mm) injection of 0.5 mL of either aCSF or norBNI (2 mg per side) were then
performed. After surgery recovery, a second progressive ratio testing was performed to assess the consequences of kappa
opioid receptors antagonism on mice’s motivation (PR2). Following this, 50nl of either sterile saline or CFA solution was injected
the mice’ right hindpaw. 48 h later, when inflammation is stable, animals underwent a third progressive ratio session. To test
the sufficiency of dynorphin-containing neurons photostimulation to drive negative affective states, dynorphin-Cre mice received
an injection of AAV cre-dependent channelrhodopsin in the vNAcSh (AP: +1.3mm; L: +/– 0.5mm; DV: –4.75mm). After two weeks
recovery, using the same stereotaxic coordinates, the animals were implanted with fiber implant in the vNAcSh. Training period
was then performed as described above a week after surgery to allow full recovery. Following training the animals were exposed
to a PR session in which fiber implant was connected to a 473nm laser emitting constant photo-stimulation (20Hz, 10ms width) during
the test.
Plantar test for thermal sensitivity – Hargreaves test
The hyperalgesic effects induced by CFA injection in the rat’s hindpaw were examined using the thermal plantar test (Hargreaves
method, IITC Life Science). Animals were placed in Plexiglas boxes on top of a glass surface. After 30 min of habituation, a radiant
heat source was applied on the plantar surface of the right hindpaw, and the latency of paw withdrawal from the radiant heat stimulus
was recorded. Four measurements with at least a 5-min interval between trials were obtained for each session. The intensity of the
light beam was adjusted so that baseline latencies were 15 s in naive rats. A cutoff time of 30 s was imposed to prevent tissue
damage. Paw withdrawal thresholds (4 measurements with 5 min of resting period) were measured for 5 consecutive days during
the four last sucrose self-administration training days. These thermal sensitivity recordings were repeated 30 min before any progres-
sive ratio schedule of reinforcement test to confirm inflammation-induced hyperalgesia.
Real-Time Place Testing (RTPT)
All behaviors were performed within a sound-attenuated room maintained at 23 C at least 1 week after habituation to the holding
room and the final surgery. Lighting was stabilized at 1,500 lux for aversion behaviors, 250 lux for anxiety-like behaviors. Move-
ments were video recorded and analyzed using Ethovision XT 10.0 (Noldus Information Technologies).
For real-time place testing, after recovery from surgery (please refer to the surgeries section for further information on procedure)
mice were gently placed in a custom-made unbiased, balanced two-compartment conditioning apparatus (52.5 3 25.5 3 25.5 cm) as
described previously (Al-Hasani et al., 2015; McCall et al., 2015; Siuda et al., 2015). During a 20-min trial, entry into one compartment

Neuron 102, 1–10.e1–e6, May 8, 2019 e3

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

triggered photo-stimulation (20Hz, 10ms pulse width) while the animal remained in the light-paired chamber and entry into the other
chamber ended photo-stimulation.
Open Field Test
Open field testing was performed in a square enclosure (55 3 55 cm) within a sound attenuated room maintained at 23 C. Lighting
was measured and stabilized at 25 lux. Mice were placed in the center of the open field and allowed to roam freely for 21 min.
Throughout the 21 min mice received constant photo-stimulation (20Hz, 10ms pulse width). The open field was cleaned with 70%
ethanol between each trial. Movements were video recorded and analyzed using Ethovision XT 10.0 (Noldus Information Technol-
ogies, Leesburg, VA). The center was defined as a square comprised of 50% the total area of the open field chamber. Time in the
center was the primary measure of anxiety-like behaviors.

Receptor Function Assessment

Incorporation of GTPgS in membranes
NAc tissue was micro-dissected from saline and CFA-injected rat and tested in GTPyS coupling assay as described previously (Kiss-
ler et al., 2014), with slight modifications. Briefly, tissue was homogenized (40-45 strokes; glass homogenizer with Teflon plunger; on
ice) in 1.5 mL of membrane buffer (pH 7.4, 50.0 mM Tris–HCl, 3.0 mM MgCl2, and 1.0 mM EGTA). Homogenates were centrifuged
(15000rpm, 4 C for 30 min), re-suspended in 1.5 mL membrane buffer, homogenized (12-15 strokes; on ice) again and finally centri-
fuged. Pellet was homogenized (12-15 strokes; on ice) in 1.5 mL assay buffer (pH 7.4, 50.0 mM Tris–HCl, 3.0 mM MgCl2, 0.2 mM
EGTA, 100.0 mM NaCl). Protein estimation was conducted using BCA protein assay (Pierce). Protein (5.0 mg) was homogenized
(12-15 strokes; on ice) and incubated with Dynorphin A (0.1-1.0 uM), at least in duplicate, in assay buffer for 90 min at 25 C, with
10 mM GDP and 0.4 nM [35S] GTPgS in a total volume of 250.0 ml. Unlabeled GTPgS (10.0 mM) was used to assess nonspecific bind-
ing. Specific binding was obtained by subtracting nonspecific binding from total binding. The reaction was quickly terminated by
filtration through UniFilter-96 GF/B filter plates using a cell harvester (Brandel, Gaithersburg, MD), followed by 8-10 washes with
wash buffer (ice-cold phosphate buffer (pH 7.4), 5.0 mM MgCl2). Bound radioactivity on the filters was counted by Microbeta liquid
scintillation counter (PerkinElmer Life Sciences) on the following day. GTPyS coupling data were analyzed using a mixed-model
two-way ANOVA to compare changes in GTPgS signaling for the controls and CFA-treated animals. The within-subject variable
was DYN concentration and the between-groups variable was treatment condition.
Incorporation of GTPgS in slices (autoradiography)
Brains were collected from saline or CFA injected rats 48h post-injection and were snap-frozen with isopentane at –30 C, and stored
in –80 C until further processing. The brains were then coronal-sectioned via cryostat (20 mm thick) at –20 C, and thaw-mounted on
SuperFrost charged slides. Sections were pre-incubated in assay buffer (50mM Tris-HCl, 3mM MgCl2, 0.2mM EGTA, 100 mM NaCl,
2 mM GDP, 1 mM DPCPX, pH = 7.4) for 15 min. Agonist-stimulated KOR activity was determined by incubating brain sections in [35S]
GTPgS (40 pM) with U69,593 (10 mM) +/– JdTic (10 mM) for 1 h at RT. After incubation, slides were washed 2x in ice-cold wash buffer
(50 mm Tris-HCl, pH 7.4) followed by a brief wash in ice-cold deionized water (30 s). Slides were air-dried and exposed to Kodak
Biomax film together with [14C] standards for 2 days. Films were developed using Kodak GBX Developer and RapidFix solutions.
Films were digitally analyzed and quantified using MicroComputer Image Device (MCID) normalized to the [14C] standard curve,
measured in dpm/mg (MCID Imaging Research, St. Catherine, Ontario, Canada). The resulting agonist-stimulated samples were
compared to non-agonist-treated brain samples to determine the percent activation of KOR above basal.
Slices radiography for KOR occupancy
Brains were dissected and frozen for cryostat sectioning. Coronal, 50mm thick sections were thaw-mounted in pairs on poly-L-lysine
microscope slides and allowed to air-dry. The slides were then stored at –80 until used. Slides were removed from the –80 and
allowed to warm to room temperature. Paired sections were traced around using a hydrophobic pen. Each paired section was filled
with 1.0ml of either 50mci/mL of [11C]-LY2795050 and allowed to incubate for the allotted time (10-40 min). Once the incubation time is
complete, the radiopharmaceutical was removed and MilliQ water was replaced in the barrier area for approximately 10 s. The MilliQ
wash was repeated 3 times. Slides were allowed to air dry. The slides were then placed in a cassette and exposed to the phosphor
imaging screen overnight. The screen was then imaged using the typhoon imager.

Electrophysiology
In vitro electrophysiological experiments on brain slices were conducted 48 h after saline or CFA injection in the hindpaw.
To measure dynorphin neurons excitability, male and female Ai14-Dynorphin-Cre mice (5-10 weeks old) were deeply anesthetized
with isoflurane and perfused transcardially with 34 C aCSF (in mM as follows: 0.001 MK-801, 126 NaCl, 2.5 KCl, 1.4 NaH2PO4,
1.2 MgCl2, 2.4CaCl2, 11 glucose, and 25 NaHCO3) before being decapitated. Brains were quickly removed and submerged in
warm MK-801-buffered aCSF. Coronal forebrain sections (230–250 mm) that contained the NAc were cut using a vibratome
(VT1200S, Leica Microsystems) in warm aCSF containing 1 mM MK-801. NAc slices were submerged in aCSF (in mM as follows:
0.01 MK-801 126 NaCl, 2.5 KCl, 1.4 NaH2PO4, 1.2 MgCl2, 2.4CaCl2, 11 glucose, and 25 NaHCO3) at 34 C for 30 m, and equilibrated
with 95% O2 and 5% CO2 before maintenance at room temperature in aCSF without MK-801. Slices were then individually
transferred to the recording chamber (volume 0.8 ml) and superfused continuously (2.2 mL/min) with 34 C aCSF. NAc neurons
were visualized using differential interference contrast optics on an upright microscope (BX50WI, Olympus). Ai14 reporter
animals contain a cre-dependent tdTomato, which was visualized using a 530 nM light shone through the 40X objective.

e4 Neuron 102, 1–10.e1–e6, May 8, 2019

Random documents with unrelated
content Scribd suggests to you:
 Oceanography - Summary Sheet
Summer 2023 - Division

Prepared by: Lecturer Jones

Date: July 28, 2025

Results 1: Historical development and evolution

Learning Objective 1: Statistical analysis and interpretation
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Assessment criteria and rubrics
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Practical applications and examples
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 4: Practical applications and examples
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 5: Problem-solving strategies and techniques
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Comparative analysis and synthesis
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 6: Critical analysis and evaluation
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 7: Problem-solving strategies and techniques
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Problem-solving strategies and techniques
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Critical analysis and evaluation
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Part 2: Comparative analysis and synthesis
Example 10: Best practices and recommendations
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Experimental procedures and results
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Ethical considerations and implications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Study tips and learning strategies
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Historical development and evolution
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 15: Best practices and recommendations
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Historical development and evolution
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 17: Diagram/Chart/Graph]
Example 17: Research findings and conclusions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Important: Theoretical framework and methodology
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 19: Diagram/Chart/Graph]
Note: Statistical analysis and interpretation
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 20: Diagram/Chart/Graph]
Part 3: Current trends and future directions
Note: Comparative analysis and synthesis
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 21: Diagram/Chart/Graph]
Definition: Key terms and definitions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Assessment criteria and rubrics
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Case studies and real-world applications
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Study tips and learning strategies
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 25: Diagram/Chart/Graph]
Practice Problem 25: Experimental procedures and results
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Case studies and real-world applications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Assessment criteria and rubrics
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 28: Diagram/Chart/Graph]
Definition: Interdisciplinary approaches
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 29: Diagram/Chart/Graph]
Important: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Introduction 4: Ethical considerations and implications
Example 30: Case studies and real-world applications
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Current trends and future directions
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Case studies and real-world applications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Definition: Problem-solving strategies and techniques
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Definition: Historical development and evolution
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Theoretical framework and methodology
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 36: Case studies and real-world applications
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 37: Diagram/Chart/Graph]
Example 37: Case studies and real-world applications
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 38: Research findings and conclusions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Comparative analysis and synthesis
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Chapter 5: Comparative analysis and synthesis
Example 40: Fundamental concepts and principles
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 41: Diagram/Chart/Graph]
Example 41: Statistical analysis and interpretation
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 42: Literature review and discussion
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 43: Diagram/Chart/Graph]
Key Concept: Statistical analysis and interpretation
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Best practices and recommendations
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Interdisciplinary approaches
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Literature review and discussion
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 47: Diagram/Chart/Graph]
Note: Current trends and future directions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Key terms and definitions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 49: Diagram/Chart/Graph]
Key Concept: Historical development and evolution
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Appendix 6: Study tips and learning strategies
Practice Problem 50: Best practices and recommendations
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 51: Diagram/Chart/Graph]
Note: Ethical considerations and implications
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Definition: Assessment criteria and rubrics
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 53: Learning outcomes and objectives
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 54: Best practices and recommendations
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 55: Diagram/Chart/Graph]
Example 55: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 56: Assessment criteria and rubrics
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 57: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Problem-solving strategies and techniques
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Historical development and evolution
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Background 7: Ethical considerations and implications
Important: Theoretical framework and methodology
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Assessment criteria and rubrics
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Historical development and evolution
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Important: Comparative analysis and synthesis
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Research findings and conclusions
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Historical development and evolution
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Literature review and discussion
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Best practices and recommendations
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 68: Fundamental concepts and principles
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 69: Diagram/Chart/Graph]
Important: Ethical considerations and implications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Unit 8: Interdisciplinary approaches
Note: Case studies and real-world applications
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 71: Fundamental concepts and principles
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Problem-solving strategies and techniques
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Research findings and conclusions
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 74: Historical development and evolution
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Historical development and evolution
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 76: Diagram/Chart/Graph]
Practice Problem 76: Experimental procedures and results
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 77: Experimental procedures and results
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 78: Diagram/Chart/Graph]
Note: Study tips and learning strategies
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 79: Diagram/Chart/Graph]
Remember: Critical analysis and evaluation
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Test 9: Critical analysis and evaluation
Note: Statistical analysis and interpretation
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Key Concept: Case studies and real-world applications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Key terms and definitions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Critical analysis and evaluation
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 84: Diagram/Chart/Graph]
Note: Case studies and real-world applications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Problem-solving strategies and techniques
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 86: Diagram/Chart/Graph]
Remember: Research findings and conclusions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Experimental procedures and results
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Key terms and definitions
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 89: Practical applications and examples
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Lesson 10: Interdisciplinary approaches
Remember: Best practices and recommendations
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Statistical analysis and interpretation
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Statistical analysis and interpretation
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Historical development and evolution
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Fundamental concepts and principles
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 95: Fundamental concepts and principles
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Comparative analysis and synthesis
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Study tips and learning strategies
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 98: Research findings and conclusions
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Ethical considerations and implications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Summary 11: Practical applications and examples
Example 100: Critical analysis and evaluation
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 101: Diagram/Chart/Graph]
Definition: Practical applications and examples
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 102: Fundamental concepts and principles
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 103: Problem-solving strategies and techniques
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Critical analysis and evaluation
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Welcome to our website – the perfect destination for book lovers and
knowledge seekers. We believe that every book holds a new world,
offering opportunities for learning, discovery, and personal growth.
That’s why we are dedicated to bringing you a diverse collection of
books, ranging from classic literature and specialized publications to
self-development guides and children's books.

More than just a book-buying platform, we strive to be a bridge

connecting you with timeless cultural and intellectual values. With an
elegant, user-friendly interface and a smart search system, you can
quickly find the books that best suit your interests. Additionally,
our special promotions and home delivery services help you save time
and fully enjoy the joy of reading.

Join us on a journey of knowledge exploration, passion nurturing, and

personal growth every day!

ebookmasss.com