The Journal of Neuroscience, October 15, 2014 • 34(42):14115–14127 • 14115

Behavioral/Cognitive

Manipulating a “Cocaine Engram” in Mice
Hwa-Lin (Liz) Hsiang,1,2,3 Jonathan R. Epp,1,2,3 Michel C. van den Oever,1,2,3 Chen Yan,1,2,3 Asim J. Rashid,1,2,3
Nathan Insel,1,2,3 Li Ye,4 Yosuke Niibori,1,2,3 Karl Deisseroth,4 Paul W. Frankland,1,2,3 and Sheena A. Josselyn1,2,3
1

Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada, 2Institute of Medical Sciences, and 3Departmentof Psychology, Physiology, University of Toronto, Toronto, ON, M5G 1X8, Canada, and 4Department of Bioengineering and Psychiatry, Stanford University,
Stanford, California 94305

Experience with drugs of abuse (such as cocaine) produces powerful, long-lasting memories that may be important in the development
and persistence of drug addiction. The neural mechanisms that mediate how and where these cocaine memories are encoded, consolidated and stored are unknown. Here we used conditioned place preference in mice to examine the precise neural circuits that support the
memory of a cocaine-cue association (the “cocaine memory trace” or “cocaine engram”). We found that a small population of neurons(⬃10%) in the lateral nucleus of amygdala (LA) were recruited at the time of cocaine-conditioning to become part of this cocaine engram.
Neurons with increased levels of the transcription factor CREB were preferentially recruited or allocated to the cocaine engram. Ablatingor silencing neurons overexpressing CREB (but not a similar number of random LA neurons) before testing disrupted the expression ofa previously acquired cocaine memory, suggesting that neurons overexpressing CREB become a critical hub in what is likely a larger
cocaine memory engram. Consistent with theories that coordinated postencoding reactivation of neurons within an engram or cell
assembly is crucial for memory consolidation (Marr, 1971; Buzsáki, 1989; Wilson and McNaughton, 1994; McClelland et al., 1995;
Girardeau et al., 2009; Dupret et al., 2010; Carr et al., 2011), we also found that post-training suppression, or nondiscriminate activation,of CREB overexpressing neurons impaired consolidation of the cocaine memory. These findings reveal mechanisms underlying how andwhere drug memories are encoded and stored in the brain and may also inform the development of treatments for drug addiction.
Key words: amygdala; cocaine; conditioning; memory; place preference

Introduction
Drug addiction is a chronically relapsing brain disease. One obstacle to the treatment of drug abuse (including cocaine abuse) isthe high incidence of relapse to drug-taking following months, oreven years, of abstinence (Dackis and O’Brien, 2001; Wagner andAnthony, 2002). In currently abstinent, former-cocaine users,
mere exposure to environmental cues present at the time of previous cocaine use may evoke powerful memories of the rewarding
properties of cocaine, induce drug craving, and precipitate relapse to cocaine-seeking and consumption (Childress et al.,Received Aug. 11, 2014; revised Sept. 9, 2014; accepted Sept. 10, 2014.
Author contributions: P.W.F. and S.A.J. designed research; H.-L.H., J.R.E., M.C.v.d.O., C.Y., A.J.R., and Y.N. performed research; L.Y. and K.D. contributed unpublished reagents/analytic tools; H.-L.H., J.R.E., M.C.v.d.O., C.Y.,
A.J.R., N.I., P.W.F., and S.A.J. analyzed data; H.-L.H., J.R.E., M.C.v.d.O., N.I., L.Y., Y.N., K.D., P.W.F., and S.A.J. wrote
the paper.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP-74650 to S.A.J.,
MOP-86762 to P.W.F.), Natural Science and Engineering Research Council (NSERC), Brain and Behavior Foundation(formerly NARSAD), EJLB Foundation, Human Frontiers Program Research Grant and Ontario Mental Health Foundation (OMHF) (S.A.J.); H.-L.H. was supported by a Vanier Canada Scholarship from CIHR, M.C.v.d.O. was supported
by an EMBO Long-Term Fellowship, C.Y. was supported by a CIHR training fellowship and a Restracomp Fellowships(Hospital for Sick Children), and A.J.R. was supported by a Restracomp Fellowship. We thank A. De Cristofaro, R.
Braybon-Smith, and M. Yamamoto for excellent technical assistance.
The authors declare no completion financial interests.
Correspondence should be addressed to Dr Sheena Josselyn, Hospital for Sick Children Research, 555 University
Avenue, Toronto, M5G 1X8 ON, Canada. E-mail: sheena.josselyn@sickkids.ca.
M.C. van den Oever’s present address: Department of Molecular and Cellular Neurobiology, VU University, Amsterdam, 1081 HV, The Netherlands.
DOI:10.1523/JNEUROSCI.3327-14.2014
Copyright © 2014 the authors 0270-6474/14/3414115-13$15.00/0

1999). Similarly, recall of a cocaine-related memory may besufficient to induce relapse to cocaine-seeking and/or cocainetaking in drug-free rodents with a history of cocaine administration (Shaham et al., 2003; Stefanik et al., 2013). These findings
indicate that conditioned cues are important factors in the ongoing cycle of relapse in addiction. Therefore, understanding how
the brain associates initially motivationally neutral environmental cues with the rewarding properties of cocaine may inform thedevelopment of novel strategies aimed at treating or preventing
relapse in humans by inhibiting drug-seeking urges evoked bydrug-associated environmental cues.
Despite considerable progress in identifying the neural mechanisms mediating the acute unconditioned rewarding effects ofcocaine and other drugs of abuse (Nestler, 2005; Koob andVolkow, 2010; Mameli and Lüscher, 2011), relatively little is
known about the molecular, cellular, and circuit mechanisms
mediating the persistent conditioned rewarding effects of cocaine. The amygdala, including the lateral amygdala (LA), hasbeen implicated in the process by which an initially neutral cue
acquires conditioned rewarding properties by virtue of being
paired with a rewarding stimulus, such as food or cocaine (Hiroi
and White, 1991; Everitt et al., 2000; Baxter and Murray, 2002;
Tye et al., 2008; Morrison and Salzman, 2010). For instance, lesioning (Fuchs et al., 2002) or disrupting synaptic plasticity(Heldt et al., 2014) in the lateral/basolateral amygdala region inrodents, impairs the acquisition of cocaine-conditioned place
preference (CPP), in which an initially neutral environmental cue

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is paired with cocaine administration. Moreover, functional
magnetic resonance imaging (fMRI) data from humans with ahistory of cocaine use shows that presentation of cues previously
associated with cocaine not only induce craving but increase activation in a number of brain regions, including the amygdala(Chase et al., 2011; Prisciandaro et al., 2014). Together, thesefindings indicate that the amygdala may play an important role in
the formation and expression of cocaine-cue memories that are
relevant for addiction. It is of interest to note, however, thatelectrophysiological recording studies found that only a portion(but not all) neurons in the lateral/basolateral amygdala in either
monkeys or rats respond to cues associated with food or cocaine
reward (Muramoto et al., 1993; Carelli et al., 2003; SugaseMiyamoto and Richmond, 2005; Bermudez et al., 2012). Here we
investigated which particular neurons in the LA are critical for
encoding and storing a memory of the cocaine-cue association in
mice. That is, in this series of studies, we searched for the “cocaine
memory engram.”

Materials and Methods
Mice. Adult (at least 10 weeks of age) wild-type (WT) male and female F1
hybrid mice (C57BL/6NTac ⫻ 129S6/SvEvTac) were used for all experiments, except where noted (see Fig. 2C–E). For the targeted neuronalablation experiments (see Fig. 2C–E), transgenic mice which express asimian diphtheria toxin receptor (DTR) in a cre-recombinase-inducible
fashion were used (iDTR transgenic mice, The Jackson Laboratory, stock
no. 007900; Buch et al., 2005; Han et al., 2009). In these mice, the geneencoding a DTR (simian Hbegf, heparin-binding epidermal growth
factor-like growth factor) is under the control of the ubiquitous ROSA26
locus promoter, but expression of the DTR transgene is dependent on thecre recombinase-mediated removal of a transcriptional STOP cassette.
Mice heterozygous for this mutation were maintained on a C57BL/
6NTac genetic background. WT littermates were used as controls. Genotypes were determined by PCR analysis of tail DNA samples as previously
described (Buch et al., 2005; Han et al., 2009).
Mice were bred at the Hospital for Sick Children and group housed(3–5 mice per cage) on a 12 h light/dark cycle with food and water
available ad libitum. Behavioral experiments were conducted during thelight-phase of the cycle. All procedures were conducted in accordancewith the policies of the Hospital for Sick Children Animal Care and Use
Committee and conformed to both the Canadian Council on Animal
Care and National Institutes of Health Guide for the Care and Use ofLaboratory Animals.
HSV vectors. cDNAs for wild-type full-length CREB fused to
N-terminal GFP (kindly provided by Dr Satoshi Kida, Tokyo Universityof Agriculture, Tokyo, Japan), GFP (subcloned from pEGFP-N1, Clontech Laboratories), cre recombinase (cre, kindly provided by Dr Andras
Nagy, Samuel Lunenfeld Research Institute, Toronto, Canada), hM3Dq
or hM4Di (kindly provided by Dr Bryan Roth, University of North Carolina) were subcloned into bicistronic HSV vectors containing the constitutive HSV promoter IE4/5 and a CMV promoter. The first namedtransgene (GFP-CREB or GFP) was driven by the IE 4/5 promoter, while
the second (cre, hM3Dq, or hM4Di) was driven by the CMV promoter.
For vectors containing CREB alone, GFP-CREB fusion expression was
driven by the IE4/5 promoter.
HSV containing these vector amplicons was packaged using a
replication-defective helper virus, purified on a sucrose gradient, pelleted, and resuspended in 10% sucrose, as previously described (Carlezon and Neve, 2003; Han et al., 2008). The average titer of the virus stocks
was 4.0 ⫻ 10 7 infectious U/ml. Transgene expression using this viralsystem typically peaks 3 d, and dissipates within 10 –14 d, following microinjection (Josselyn et al., 2001; Barrot et al., 2002; Vetere et al., 2011;
Cole et al., 2012).
Surgery. Mice were pretreated with atropine sulfate (0.1 mg/kg, i.p.),anesthetized with chloral hydrate (400 mg/kg, i.p.), and placed in a stereotaxic frame. Skin was retracted and holes drilled in the skull bilaterally
above the LA (AP ⫽ ⫺1.3, ML ⫽ ⫾3.4, V ⫽ ⫺5.0 mm from bregma)

Hsiang et al. • Amygdala and Cocaine Reward

according to (Paxinos and Franklin, 2001). Viral vector (1.5 ␮l/side) was
microinjected through glass micropipettes connected via polyethelene
tubing to a microsyringe (Hamilton) at a rate of 0.1 ␮l/min. Micropipettes were left in place an additional 10 min following microinjection toensure diffusion. Micropipettes were slowly retracted, the incision closed
and mice treated with analgesic (ketoprophen, 5 mg/kg, s.c.).
Verifying location of vector microinjection and extent of viral infection.
Following the final CPP test, mice were transcardially perfused with 0.1 M
PBS followed by 4% PFA. Brains were postfixed for 2 h (4°C) and transferred to a 30% sucrose solution. Coronal brain slices (50 ␮m) across theanterior–posterior extent of LA were collected using a cryostat (Leica
CM1850). Every second section was mounted on a gel-coated glass slideand coverslipped with Vectashield fluorescence mounting medium containing DAPI (Vector Laboratories). Consistent with previous reports
from several laboratories (Carlezon et al., 1998; Wallace et al., 2004;
Brightwell et al., 2005; Airan et al., 2007; Han et al., 2009; Vetere et al.,2011; Cole et al., 2012) microinjection of HSV vectors produced robust
localized transgene expression with minimal tissue damage around thesite of microinjection. Native GFP-immunofluorescence (which did not
differ across vectors) was used to determine placement and extent of theviral infection for each mouse. Based on this, each mouse was classified asa “hit” or “miss” by an examiner unaware of the treatment condition andbehavioral results. Mice were defined as hits if robust bilateral GFP expression was observed in LA in at least five consecutive brain sections(across the anterior–posterior plane). All other mice were classified as
miss (including those with unilateral, weak or no transgene expression in
the LA). Only mice determined to be a bilateral hit were included insubsequent data analysis.
CLARITY. To visualize the placement and extent of viral infection inthree dimensions, a set of viral infused brains were processed using the
CLARITY technique as described previously (Chung and Deisseroth,2013; Chung et al., 2013). Briefly, mice were perfused with ice-cold saline
followed by a hydrogel solution (consisting of 4% formaldehyde, 4%acrylamide, 0.025% bis-acrylamide, and 0.25% VA044 thermal initiator)and brains were then postfixed in the same solution for 48 h at 4°C. The
brains and hydrogel solution were polymerized by increasing thetemperature to 37°C. Tissue was cleared for several days using SDS electrophoresis. Once clear, brains were cut on a vibratome (Leica) into
1-mm-thick coronal sections and mounted on glass slides in FocusClear(CelExplorer Labs). Finally, the 1-mm-thick sections were imaged usinga Zeiss LSM 710 laser scanning confocal microscope with a 10⫻ 0.3 NA
air-objective. Image stacks (150 images with a 6.0 ␮m step size) werestitched together using Zen imaging software (Zeiss) and 3D reconstructions were assembled using ImageJ and VAA3D.
Stereological counting. To estimate the percentage of total LA neurons
infected by the viral vectors, we used unbiased stereology and systematic
sampling techniques. Stereological counting was performed using Stereology Investigator software (MBF) on every third section (50 ␮m thickness, at least 8 sections per mouse) from a subset of mice microinjectedwith CREB vector (n ⫽ 6). The entire LA (from ⫺0.70 to ⫺2.5 mm frombregma in the anterior–posterior direction) was traced and the optical
fractionator probe was used to randomly place sampling boxes (250 ⫻250 ␮m) throughout the LA (West et al., 1991) and the number of
DAPI ⫹ and GFP ⫹ cells per section assessed. Small, bright uniformly
DAPI stained nuclei (⬃5 ␮m diameter) from putative glial cells were notincluded in DAPI counts. A counting frame of 5 ⫻ 50 ␮m was used forassessing DAPI and a counting frame of 120 ⫻ 120 ␮m was used forassessing GFP. The total number of DAPI ⫹ cells (putative pyramidal
neurons) in the LA was estimated to be 200,000 (sum of left ⫹ right LA),
consistent with previous reports (Tuunanen and Pitkänen, 2000; vonBohlen und Halbach and Unsicker, 2002). Using this stereologicalmethod, we estimate that between 9.84% (⫾0.89%) of all principal neurons in LA were infected by our viral manipulations.
Drugs. Cocaine HCl (Health Canada) was dissolved in sterile PBS anddelivered intraperitoneally at the appropriate dose. Because the behavioral response to cocaine may differ depending on background genetics
of mice (Eisener-Dorman et al., 2011), we varied the dose of cocaine insome experiments (see below for precise dosing information). DT was
dissolved in sterile PBS and systemically administered (16 ␮g/kg, i.p.). As

Hsiang et al. • Amygdala and Cocaine Reward

a control, we systemically administered vehicle (PBS). Clozapine-Noxide (CNO, Toronto Research Chemicals) was made in a stock solutionof 10 mg/ml in DMSO and then diluted in saline to desired concentration. CNO was injected at a dose of 1 mg/kg, intraperitoneally, 1 h before
testing, immediately after training, or 18 – 48 h after training (depending
on experiment).
CPP. CPP was performed using an unbiased, counterbalanced protocol (Josselyn and Beninger, 1993; Prus et al., 2009). The CPP apparatus
consisted of two 15 ⫻ 20 cm Plexiglas chambers connected by a guillotine
door. Each chamber had a unique combination of visual, tactile, andolfactory properties (one side had white walls and a transparent roughfloor, whereas the other side had black and white striped walls, a smoothwhite floor that was wiped with 0.2 ml of 3% acetic acid before eachconditioning and test trial). We balanced the two chambers in terms ofinitial, baseline preference such that during the pretraining (habituation)phase, all groups of mice spent similar time in each chamber. In all
experiments conducted herein, we failed to observe a group-wide bias for
one chamber before training. The two chambers were separated by aguillotine door (which was open during the pretest habituation and test
sessions, but closed during the conditioning sessions). The activity andlocation of mice was monitored by an overhead CCD camera, connected
to a computer running Limelight software (Colbourn Instruments).
The CPP procedure consists of three sequential phases: habituation(pretest), conditioning, and postconditioning test (test). In the habituation phase, drug-free mice were allowed access to the two chambers of the
CPP apparatus for 10 min. Time spent in each chamber (defined as thetime when mice fully entered the chamber) was calculated for eachmouse. To measure this, we used Limelight software to visually divide the
CPP apparatus into three zones; chamber1 zone, middle zone, and chamber2 zone (15 ⫻ 18 cm; 15 ⫻ 4 cm; 15 ⫻ 18 cm), thus eliminating thetime mice spent in the center of the apparatus from our calculations.
Conditioning sessions took place twice daily for 1, 2, or 3 d, with aminimum of 5 h between each session. We chose this method of inducing
CPP because of the short time course of transgene expression using theseviral vectors. On each conditioning day, mice were confined to one
chamber for 15 min immediately following saline (morning) or cocaine(afternoon) administration. The location of the cocaine-paired chamber
was randomized and counterbalanced across groups. Eighteen hours following the final training session, CPP memory was assessed in a test
session, in which drug-free mice were given free access to the two chambers (as in the pretest). Only the first 5 min of data were used for analysis
to exclude possible effects of within-session extinction. We calculated a
CPP score for each mouse (time, in seconds, spent in cocaine-paired zone
minus time spent in saline-paired zone during the test).
CPP scores were analyzed using one- (vector) or two-way (vector ⫻drug) ANOVAs.
catFISH. We identified neurons specifically activated by exposure tocues previously paired with cocaine using arc (activity-regulated
cytoskeleton-associated protein) mRNA, as previously described (Han etal., 2007, 2009). Following the completion of training, mice were reexposed to the chamber previously paired with cocaine for 7 min, and 5
min later, brains were removed. Under basal conditions, neurons express
very low levels of arc mRNA. Approximately 2 min after the type ofstimulation that is associated with synaptic plasticity, arc RNA is observed in the nuclei of activated neurons (Guzowski et al., 2001, 2005).
However, this robust burst of nuclear arc is transient and arc RNA is
rapidly transported to the cytoplasm, where it can be detected 20 – 45 minafter induction (Guzowski et al., 2001, 2005). Because the time course of
the nuclear signal is distinct from the cytoplasmic signal, the subcellular
distribution of arc mRNA provides a time-stamp of neural activity for aparticular neuron, with arc mRNA expression in the nucleus being amolecular signature of a neuron that was active ⬃5 min before. In nonseizure conditions, arc mRNA is expressed exclusively in neurons, primarily in principal neurons (Vazdarjanova et al., 2006).
Coronal sections (20 ␮m) were cut and thaw-mounted on slides, suchthat each slide contained sections from different treatment conditions(to maximize relevant between-group comparisons within each slide).
Digoxigenin (DIG)-labeled antisense and sense riboprobes for arc
mRNA were generated using a commercial transcription kit (MaxiScript,

J. Neurosci., October 15, 2014 • 34(42):14115–14127 • 14117

Ambion) and RNA labeling mix (Roche Diagnostics) from a plasmid
encoding arc ORF. arc antisense riboprobe was detected with an anti-DIG
antibody conjugated with horseradish peroxidase (HRP) FAB fragments(1/300; JacksonImmunoResearch) and visualized with an AlexaFluor 488conjugated tyramide amplification system (PerkinElmer).
Briefly, sections were fixed with 4% PFA, washed in 2⫻ saline–sodium
citrate (SSC) buffer, treated with 0.5% acetic anhydrate/1.5% triethanolamine, and 50% methanol/50% acetone before being incubated in hybridization buffer (with 50% formamide, 5⫻ SSC, 0.1 mg/ml yeasttransfer RNA, 0.1% Tween 20, and 50 mg/ml heparin) for 30 min at room
temperature. Sections were incubated with arc riboprobe (1 ng/␮l in
hybridization buffer) at 56°C overnight. Twenty-four hours later, sections were treated sequentially with RNaseA (1 ␮g/ml in 2⫻ SSC at 37°C
for 30 min), hydrogen peroxide (1% for 30 min), blocking reagent(Roche Diagnostics) with goat serum (4%, 30 min) and incubated with ananti-DIG HRP-conjugated antibody (described above) in blocking solution
for 2 h at room temperature. Tyramide amplification was performed for 30
min at room temperature. Nuclei were visualized with Hoechst 33258(Sigma-Aldrich) counterstain.
Images were captured using a confocal microscope (Zeiss LSM 710
equipped krypton-argon laser) and acquired using a 40⫻ oil-immersion
lens and z-sectioned in 1-␮m-thick optical sections. Manual cell counts
were performed by two observers unaware of the treatment condition.
Small, bright uniformly DAPI-stained nuclei (⬃5 ␮m diameter) fromputative glial cells were not counted. All other whole nuclei cells (putative
neurons) in the field-of-view within the LA were counted. Neurons wereassigned to one of the following four categories: (1) GFP ⫹ (infected byvector)/arc ⫺ (not active), (2) arc ⫹ in the nucleus (recently activated,memory trace/GFP ⫺ (not infected), (3) both GFP ⫹ and arc ⫹ in the
nucleus, or (4) both GFP ⫺ and arc ⫺.
Five to 10 slices were analyzed for each mouse and each treatmentgroup contained three to six mice. The portion of neurons positive for arc
in the nucleus in the infected (GFP ⫹) or noninfected (GFP ⫺) population was calculated by slice. The data were analyzed with an ANOVA with
between-factor, vector, and a within-factor, neuronal infection status,(infected, noninfected neuron).

Specific methods
Figure 1A–C. One day after habituation, WT hybrid mice were trained forcocaine-cue CPP over 1, 2 or 3 consecutive days. In the morning of eachtraining day (between 9:00 and 11:00 A.M.), saline (SAL) was administered directly before mice were placed in one chamber (counterbalanced)
and in the afternoon (between 3:00 and 5:00 P.M.), mice either received
SAL or COC (15 mg/kg, i.p.) before being placed in the other chamber.
Figure 1E,F. One day after habituation, WT hybrid mice were microinjected with CREB or GFP vector. Mice recovered for 1 d before cocainecue CPP training (for 2 or 3 d). Saline was administered before chamber
placement in morning and COC (15 mg/kg, i.p.) was administered before chamber placement in afternoon.
Figure 1G. One day after habituation, WT hybrid mice were trained in
cocaine-cue CPP for 3 consecutive days (as above) and the following day,microinjected with CREB or GFP vector. Mice were tested 3 d later (tomimic microinjection-test time course in Fig. 1F ).
Figure 1H. One day after habituation, WT hybrid mice were trained in
cocaine-cue CPP over 3 consecutive days. As before, mice were administered
SAL in morning and either SAL or COC (1.25, 3, or 15 mg/kg) in afternoon.
Figure 1I. One day after habituation, WT hybrid mice were microinjected with CREB or GFP vector. Mice recovered for 1 d before training as
above [for 3 d, saline in morning, COC (1.25 mg/kg, i.p.) in afternoon].
Figure 2A. One day after habituation, WT hybrid were microinjectedwith CREB or GFP vector and allowed to recover for 1 d before being
trained for 1 d. Mice were administered SAL in morning and COC (30mg/kg) in afternoon. One day later, drug-free mice were confined in the
cocaine-paired chamber for 7 min and brains removed 5 min later.
Figure 2C. One day after habituation, iDTR transgenic or WT littermate control mice were microinjected with CREB-cre vector. Two days
later, mice were trained for 1 d [SAL in morning and COC (15 mg/kg) inafternoon]. One day after training, DT or PBS was administered once per
day for 2 d.

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Hsiang et al. • Amygdala and Cocaine Reward

Figure 1. Increasing CREB function in a small population of LA neurons during training enhances cocaine-cue memory formation. A–C, Repeated pairing of cocaine (COC), but not saline (SAL),
with initially motivationally neutral cues (chamber) induces a CPP [CPP score ⫽ Time (seconds) in COC-paired chamber ⫺ time in SAL-paired chamber during the drug-free test]. One pairing:
SAL-SAL (n ⫽ 8), SAL-COC (n ⫽ 8); two pairings: SAL-SAL (n ⫽ 6), SAL-COC (n ⫽ 7); three pairings: SAL-SAL (n ⫽ 8), SAL-COC (n ⫽ 9). D, Using CLARITY to clear brain chunk and examine the patternand extent of viral infection in three dimensions. Microinjecting CREB vector infects a small, random population of neurons that are largely restricted to the LA. 3D rendering of 900-␮m-thick coronalbrain section containing the LA 5 d postmicroinjection of CREB vector showing distribution of native GFP expression. Scale bar, 50 ␮m. E, F, Increasing CREB in ⬃10% of LA principal neurons before
training enhances CPP memory formation. Two pairings: CREB vector (n ⫽ 8), GFP (n ⫽ 8); three pairings: CREB (n ⫽ 8), GFP (n ⫽ 12). G, Microinjecting CREB vector after CPP training has no effecton CPP memory expression. CREB vector (n ⫽ 7), GFP (n ⫽ 7). H, Dose–response curve for COC-induced CPP. SAL (n ⫽ 8), COC 1.25 mg/kg (n ⫽ 8), 3 mg/kg (n ⫽ 11), 15 mg/kg (n ⫽ 9). I, Increasing
CREB in LA before training does not induce CPP in mice trained with a subthreshold dose of COC (1.25 mg/kg), suggesting that increasing CREB in LA does not enhance CPP by increasing theunconditioned rewarding properties of COC. CREB vector (n ⫽ 8), GFP (n ⫽ 8). In this and all other figures, data presented are mean ⫾ SEM. n.s. denotes not significantly different, * denotes p ⬍0.05. Data presented are mean ⫾ SEM.

Hsiang et al. • Amygdala and Cocaine Reward

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Figure 2. LA neurons with increased CREB function during CPP training become a critical component of a cocaine-cue memory engram. A, B, Neurons overexpressing CREB (but not thoseexpressing GFP only) during training are more likely to be allocated to the cocaine-cue memory trace (arc⫹) than their noninfected neighbors. Mice were microinjected with CREB or GFP vector 2 d
before training and catFISH for nuclear localized arc conducted 5 min after drug-free re-exposure to the chamber previously paired with cocaine. Active neurons (part of memory trace) ⫽ neurons
with nuclear localized arc RNA. B, Example images from results quantified in A. In mice microinjected with CREB vector, arc RNA is preferentially localized in GFP ⫹ (infected) neurons (rather thanneighboring uninfected GFP ⫺ neurons), whereas in mice microinjected with GFP vector, arc is observed equally in infected and uninfected neurons. CREB vector n ⫽ 31 slices (5 mice), GFP n ⫽ 21
slices (3 mice). C, D, Post-training ablation of neurons overexpressing CREB (but not a similar number of random neurons expressing GFP alone) disrupts subsequent expression of cocaine-cue
memory. C, iDTR transgenic or WT littermate mice microinjected with CREB-cre vector before training and administered DT (or PBS) after training to ablate CREB-overexpressing neurons. WTmice-PBS (n ⫽ 12), WT mice-DT (n ⫽ 13), iDTR mice-PBS (n ⫽ 11), and iDTR mice-DT (n ⫽ 12). D, iDTR mice microinjected with CREB-cre or GFP-cre vector before training and administered DT(or PBS) after training. No effect of ablating neurons expressing GFP alone, whereas ablating neurons overexpressing CREB disrupts cocaine-cue memory expression. GFP vector-PBS (n ⫽ 9), GFP
vector-DT (n ⫽ 10), CREB vector-PBS (n ⫽ 12), and CREB vector-DT (n ⫽ 11). E, In contrast to behavioral extinction, COC administration before a memory test fails to reinstate a cocaine-cue memory
after ablation of neurons overexpressing CREB. iDTR mice were microinjected with CREB-cre or GFP-cre vector before training and administered DT after training (as above). Ablation of CREBoverexpressing (but not GFP-expressing) neurons disrupted expression of cocaine-cue memory (Test 1, as above). Mice with GFP-cre vector were given extinction training (both chambers paired withSAL) until these mice no longer preferred the chamber originally paired with COC (extinction group). During this time, CREB-cre mice (neuronal ablation group) remained drug-free in homecage.
Immediately before a final memory test (Test 4), all mice were administered COC. Cocaine-cue memory was reinstated in the extinction group only (not in the neuron ablation group), suggesting that
ablating CREB-overexpressing neurons effectively degrades the cocaine-cue memory trace. GFP-cre⫹DT (n ⫽ 13), CREB-cre⫹DT (n ⫽ 11). F, Similar to permanent ablation, temporarily silencing
neurons overexpressing CREB (using hM4Di DREADD⫹CNO) before a memory test disrupted cocaine-cue memory expression. GFP vector: CNO (n ⫽ 9), VEH (n ⫽ 7); CREB vector: CNO (n ⫽ 8), VEH(n ⫽ 9); GFP-hM4Di vector: CNO (n ⫽ 9), VEH (n ⫽ 8); CREB-hM4Di vector: CNO (n ⫽ 8), VEH (n ⫽ 7). Data presented are mean ⫾ SEM. n.s. denotes note significantly different, * denotes p ⬍ 0.05,*** denotes p ⬍ 0.001.
Figure 2D. One day after habituation, iDTR transgenic mice weremicroinjected with CREB-cre or GFP-cre vector and allowed to recover for 2 d. Mice were trained for 1 d [SAL in morning, COC (30mg/kg) in the afternoon, such that mice with GFP-cre vector would
show robust CPP]. One day after training, DT or PBS was administered once per day for 2 d.
Figure 2E. One day after habituation, iDTR transgenic mice were microinjected with CREB-cre or GFP-cre vector and allowed to recover for
2 d. Mice were trained for 1 d [SAL in morning and COC (30 mg/kg) inafternoon, as above]. One day after conditioning, DT or PBS was administered (once per day for 2 d). A drug-free test (Test 1) was conducted
24 h later. For the extinction group (iDTR mice with GFP-cre vector),mice received extinction training of SAL-SAL training sessions for 2 d (inwhich each chamber was paired with SAL administration). Extinction
tests (without prior drug injection, Test 2, Test 3) took place at the end ofeach extinction-training day. During this time, iDTR mice with CREB-

cre vector (neuron ablation group) were maintained drug-free in their
homecage. Before the final (reinstatement) test (Test 4) all mice received
COC (15 mg/kg) immediately before being tested.
Figure 2F. One day after habituation, WT hybrid mice were microinjected with CREB, GFP, CREB-hM4Di, or GFP-hM4Di vector. Mice
were allowed to recover for 2 d. On day 3, mice were given a single
training day [SAL in morning, COC (30 mg/kg) in afternoon]. One day
later, mice were tested 1 h following CNO or VEH administration.
Figure 3A. One day after habituation, WT hybrid mice were microinjected with CREB-hM4Di vector and allowed to recover for 2 d. One day
later, mice were trained [for 1 d, SAL in morning, COC (10 mg/kg) inafternoon]. Immediately after this, CNO or VEH was administered. A
drug-free test (no COC or CNO, Test) was conducted 24 h later.
Figure 3B. One day after habituation, WT hybrid mice were microinjected with CREB, GFP, CREB-hM4Di or GFP-hM4Di vector. Mice recovered for 2 d. On day 3, mice were given a single training day [SAL inHsiang et al. • Amygdala and Cocaine Reward

14120 • J. Neurosci., October 15, 2014 • 34(42):14115–14127

Figure 3. Consolidation of cocaine-cue memory is disrupted by interfering with normal coordinated postencoding activity of LA neurons overexpressing CREB. A–C, Post-training silencing of
neurons overexpressing CREB disrupts subsequent cocaine-cue memory expression in a time-dependent manner. A, In mice microinjected with CREB-hM4Di vector, CNO administered immediately
after training (to silence CREB-overexpressing neurons) produced a small disruption in subsequent cocaine-cue memory expression (CNO, n ⫽ 7; VEH, n ⫽ 7). B, Administering CNO both
immediately and 18 h after training in mice with CREB-hM4Di vector robustly impaired subsequent cocaine-cue memory expression. Silencing a similar number of random neurons (GFP-hM4Di
vector) had no effect on memory consolidation. CREB-hM4Di vector: CNO (n ⫽ 11), VEH (n ⫽ 11); GFP-hM4Di vector: CNO (n ⫽ 8), VEH (n ⫽ 8). C, In contrast, memory was intact in mice
microinjected with CREB-hM4Di vector if CNO treatment began 18 h after training. CNO (n ⫽ 6), VEH (n ⫽ 7). D, E, Nondiscriminately artificially enhancing activity of neurons (using hM3Dq
DREADD⫹CNO) overexpressing CREB (but not a similar number of random neurons expressing GFP) after training disrupts subsequent cocaine-cue memory expression. E, Mice microinjected
CREB-hM3Dq vector (GFP-hM3dq used as a control vector). CNO administered 24 h after training disrupts cocaine-cue memory in mice microinjected with CREB-hM3Dq vector only. CREB-hM3Dq
vector: CNO (n ⫽ 7), VEH (n ⫽ 9); GFP-hM3Dq vector: CNO (n ⫽ 9), VEH (n ⫽ 9). F, Memory disruption is pronounced in mice microinjected with CREB-hM3Dq vector administered additional CNO
injections (2 injections/d/2 d beginning 18 h after training). Cocaine-cue memory is not reinstated by subsequent administration of COC before a final memory test, suggesting memory trace isdegraded. CNO (n ⫽ 6), VEH (n ⫽ 6). In this and all other figures, data presented are mean ⫾ SEM. n.s. denotes not significantly different, * denotes p ⬍ 0.05.

morning, COC (10 mg/kg) in afternoon for mice that were microinjectedwith CREB or CREB-hM4Di vector and COC (30 mg/kg) for mice with
GFP or GFP-hM4Di vector. Different doses of COC were used to equalize the resulting CPPs in VEH-treated mice]. Immediately and 18 h after
the last training session, CNO or VEH was administered. A drug-free test
(Test) was conducted 24 h later.
Figure 3C. One day after habituation, WT hybrid mice were microinjected with CREB-hM4Di vector. Mice recovered for 2 d. On day 3, mice
were given a single training day [SAL in morning, COC (10 mg/kg) inafternoon]. Eighteen hours after the last training session, CNO or VEH
was administered twice daily for 2 d, with a minimal of 8 h between
injections. Drug-free test (Test) was conducted 24 h later.
Figure 3D. One day after habituation, WT hybrid mice were microinjected with CREB, GFP, CREB-hM3Dq, or GFP-hM3Dq vector. Micerecovered for 2 d. On day 3, mice were given a single training day [SAL inmorning, COC (10 mg/kg) in afternoon for mice microinjected with
CREB or CREB-hM4Di vector, whereas a higher dose of COC (30 mg/kg)
was administered to mice microinjected with GFP or GFP-hM4Di vector]. Different doses of COC were used to equate the resulting CPPs inmice microinjected with GFP or CREB vector. Eighteen hours after thelast training session, CNO or VEH was administered. A drug-free test was
conducted 24 h later.

Figure 3E,F. One day after habituation, WT hybrid mice were microinjected with CREB-hM3Dq vector. Mice recovered for 2 d. On day 3,
mice were given a single training day [SAL in morning, COC (10 mg/kg)
in afternoon]. Eighteen hours after last training session, CNO or VEH
was administered (2 times per day for 2 d). A drug-free test was conducted 24 h after last injection of CNO or VEH. Figure 3F, 3 h after thetest session, a reinstatement test was conducted immediately following
administration of COC (5 mg/kg).

Results
Assessing cocaine-cue memory in mice using CPP
To examine the neural mechanisms mediating the formation of a
cocaine-cue memory in mice, we used CPP. Mice were initially
exposed to two distinct, motivationally neutral experimental
chambers separated by a removable guillotine door. One day after
this habituation session, the guillotine door was closed and one
chamber was paired with cocaine administration and the second
chamber was paired with saline administration (using an unbiased, counterbalanced design; Josselyn and Beninger, 1993; Prus
et al., 2009). Cocaine-cue memory was assessed during a test inwhich drug-free mice were allowed access to both chambers (withHsiang et al. • Amygdala and Cocaine Reward

guillotine door removed) and the amount of time mice spent inboth chambers compared. Mice that learned and remembered
the cocaine-cue association subsequently spent more time in thechamber previously paired with cocaine administration, thereby
exhibiting a CPP (Carlezon et al., 1998; Miller and Marshall,
2005; Airan et al., 2009; Heldt et al., 2014).
We first verified that cocaine reliably induced CPP in adult
WT mice in a hybrid genetic background (see Materials andMethods). Mice were trained for 1, 2, or 3 d. On each training day,saline was paired with one chamber (A.M.) and cocaine waspaired with the second chamber (P.M.; Fig. 1A–C). Control
groups received saline administration before placement in bothchambers (A.M. and P.M.). To assess cocaine-cue memory, we
calculated a CPP score in which we subtracted the amount of time(in seconds) mice spent in the chamber previously paired withsaline from time spent in the chamber previously paired withcocaine. Control groups, that received 1, 2, or 3 training days
during which saline was paired with both chambers, spent equal
time in each chamber during the test (showing no CPP), indicating that mice had no innate preference for either chamber. We
observed that two cocaine-cue pairings induced statistically reliable CPP and three cocaine-cue pairings induced robust CPP[Figure 1A, 1 pairing; one-way ANOVA (saline, cocaine), F(1,14) ⫽0.43, p ⬎ 0.05; Figure 1B, 2 pairings; F(1,11) ⫽ 7.93, p ⬍ 0.05;
Figure 1C, 3 pairings; F(1,15) ⫽ 13.93, p ⬍ 0.05]. The current
protocol, therefore, induced a graded CPP, the magnitude ofwhich was related to training intensity.
Increasing CREB function in a random, small population of LA
pyramidal/principal neurons enhanced the formation of a cocainecue memory
In addition to being implicated in reward-related learning [inwhich an initially neutral cue is paired with an appetitive unconditioned stimulus, such as food or drugs of abuse; (Hiroi andWhite, 1991; Everitt et al., 2000; Baxter and Murray, 2002; Tye etal., 2008; Morrison and Salzman, 2010; Heldt et al., 2014), the LA
has also been shown to be critical for fear/threat-related learning(LeDoux, 2014), in which an initially neutral cue (typically atone) is paired with an aversive unconditioned stimulus (typically a shock); Davis, 1992; Fanselow and Gale, 2003; Maren,
2003; Duvarci and Pare, 2014]. Previous results show that thefear/threat memory is sparsely encoded within a small number ofneurons in the LA. Specifically, electrophysiological data indicatethat although ⬎70% of pyramidal/principal neurons in the rodent LA respond to both tone and shock presentation (suggestingthese neurons are “correctly wired” and presumably eligible tobecome part of the fear/threat memory engram), only a small
portion (⬃10 –30%) of these eligible neurons seem to become
part of any one fear/threat memory engram (Repa et al., 2001;
Rumpel et al., 2005; Reijmers et al., 2007). Similarly, fMRI datasuggest that in the human amygdala, fear/threat memories are
similarly encoded in a sparse trace (Bach et al., 2011). We (Han etal., 2007, 2009) and others (Zhou et al., 2009; Kim et al., 2014)
found that LA neurons compete against one another for recruitment (or allocation) to a fear/threat engram. Furthermore, we
found it was possible to bias the outcome of this competition by
manipulating CREB function in individual LA neurons. Neurons
with relatively higher CREB function were more likely to be included, whereas neurons with relatively lower CREB function
were more likely to be excluded, from the fear/threat memory
engram. Moreover, increasing CREB in a small population of LA
neurons before training was sufficient to enhance fear/threat
memory and these neurons were critical for subsequent expres-J. Neurosci., October 15, 2014 • 34(42):14115–14127 • 14121

sion of that memory, suggesting that they became necessary
members of the engram (Josselyn et al., 2001; Han et al., 2007,2009; Zhou et al., 2009; Rexach et al., 2012; Cowansage et al.,2013). Therefore, the formation of a fear/threat memory involves
neuronal competition in the LA, the outcome of which is sensitive to relative neuronal CREB function.
We investigated whether similar neural mechanisms underlie
the formation of a cocaine-cue memory engram in the LA. To thisend, we used replication-defective HSV vectors to increase CREB
levels in a small but random population of LA neurons. To subsequently identify infected neurons, we used a virus that also
expressed GFP. A virus encoding GFP alone served as a control.
Microinjection of HSV overwhelmingly (⬃99%) infects excitatory, pyramidal/principal neurons in the LA (Cole et al., 2012;
Yiu et al., 2014). By titrating the concentration and volume of
HSV microinjected into the LA, we infected a small number ofseemingly random LA neurons [⬃10% of LA pyramidal/principal neurons (9.84 ⫾ 0.89%, n ⫽ 6), as determined by stereological counting].
To better visualize the pattern and extent of neurons infectedby intra-LA microinjection of HSV, we took advantage of therecently developed CLARITY (lipid-exchanged, anatomicallyrigid, imaging/immunostaining compatible, tissue hydrogel) tissue processing technique which permits examination of thickvolumes of fixed tissue in three dimensions without the need for
sectioning or reconstruction (Chung and Deisseroth, 2013;
Chung et al., 2013). Microscopic imaging of brain tissue is normally severely constrained by light scattering caused, in largepart, by the fatty lipids that make up the cellular membranes. This
results in reduced resolution of images collected from thick brainslices. By replacing the lipids in brain tissue with a clear hydrogel,
CLARITY converts an optically opaque brain into a transparent
structure. This allows for high cellular resolution images to becollected from relatively thick sections without the need of sectioning. The hydrogel preserves the fine structure of the brain
including the localization of proteins and does not reduce thestability of fluorescent signals, such as GFP. We used CLARITY toclear the brains of mice microinjected with our viral vectors andimaged endogenous HSV-GFP signal in 1-mm-thick coronal brain
sections. Using this technique, we verified that our vectors infected asmall, seemingly random population of neurons in the LA and thatthe majority of infection was limited to the LA (Fig. 1D).
Transgene expression using this type of HSV peaks 2–3 d after
microinjection (Josselyn et al., 2001; Barrot et al., 2002; Vetere etal., 2011; Cole et al., 2012). To examine the effects of increasing
CREB in a small population of LA neurons during training on
formation of a cocaine memory, we microinjected CREB or control vector 2 d before training mice with 2 or 3 cocaine-cue pairings. One day after the final training session, drug-free mice were
tested. Increasing CREB in a small, random subpopulation of LA
neurons enhanced the CPP produced by either two or threecocaine-cue pairings (Figure 1E; F(1,14) ⫽ 7.42, p ⬍ 0.05; Figure
1F; F(1,18) ⫽ 5.71, p ⬍ 0.05). Therefore, similar to a fear/threat
memory, enhancing CREB function increased memory for a
cocaine-cue association.
To more thoroughly investigate this effect, we conducted aseries of control experiments. First, we examined the effects ofincreasing CREB on the expression of a previously formed cocaine memory, by microinjecting CREB or control vector into the
LA 1 d after training. Expression of a previously acquired
cocaine-cue memory was not affected by increasing CREB (Figure 1G; F(1,12) ⫽ 0.002, p ⬎ 0.05), indicating that CREB enhances
encoding (rather than expression) of a cocaine-cue memory. Pre-14122 • J. Neurosci., October 15, 2014 • 34(42):14115–14127

vious results indicate that similar manipulations of CREB function in the nucleus accumbens alter the (unconditioned)
motivational/rewarding properties of cocaine itself (Carlezon etal., 1998). We investigated whether the enhancement of CPP weobserved in the present experiments were due to our LA manipulation similarly increasing the unconditioned rewarding properties of cocaine. To test this, we microinjected CREB vector into
the LA 2 d before training mice with a subthreshold dose of
cocaine (1.25 mg/kg; Carlezon et al., 1998), that is normally not
sufficient to support CPP (Figure 1H; F(3,32) ⫽ 3.63, p ⬍ 0.05;
Newman–Keuls post hoc tests showed CPP was produced by 3
mg/kg or 15 mg/kg, but not 1.25 mg/kg, of cocaine). If increasing
CREB in the LA increased the unconditioned rewarding properties of cocaine, we would predict that this low, subthreshold, doseof cocaine might become sufficient to support CPP. However, weobserved no evidence of CPP in mice microinjected with CREB
vector in the LA before training with this low dose of cocaine(Figure 1I; F(1,14) ⫽ 0.61, p ⬎ 0.05). This finding is consistent with
the interpretation that increasing CREB in a small population of
LA principal neurons does not enhance the rewarding propertiesof cocaine, but instead enhances memory of the cocaine-cue
association.
Neurons overexpressing CREB during training are selectively
allocated to the cocaine-cue memory engram
LA neurons are thought to compete against one another for recruitment/allocation (Zhou et al., 2009) to a fear/threat memory
engram and neurons with relatively enhanced CREB function arecompetitively advantaged in this competition (Han et al., 2007,2009; Zhou et al., 2009). To examine whether CREB-mediatedneuronal competition underlies formation of a cocaine-cue
memory, we asked whether LA neurons with high levels of CREB
are over-represented in the cocaine-cue memory trace.
To visualize neurons that are part of the cocaine-cue memory
trace, we took advantage of the unique transcriptional timecourse of the activity-dependent gene arc (Guzowski et al., 1999,
2001). Under basal conditions, neurons typically have low levelsof arc RNA. Within 5 min of neuronal activity, though, there is abrief rapid burst of arc transcription. Within 40 min of this neuronal activity, arc RNA is transported from the nucleus to thedendrites. This stereotyped trafficking pattern allows the presence and localization of arc RNA to serve as an activity timestamp for individual neurons, with arc localized to the nucleus
being as a molecular marker of a recently active neuron. Therefore, to visualize neurons that may be part of the cocaine-cue
memory engram, we used nuclear arc to identify neurons specifically activated by cocaine-associated cues (Guzowski et al.,2001). Mice were microinjected with CREB or GFP vector andtrained as above. On the test day, drug-free mice were confined tothe chamber previously paired with cocaine. Five minutes later,
brains were removed and the overlap of active (arc localized to the
nucleus, arc ⫹) and infected (GFP ⫹) neurons assessed. The overall number of active (arc ⫹) LA neurons following the test was
similar in mice microinjected with CREB and GFP vector, indicating that increasing CREB function did not increase the overall
number of neurons expressing arc or size of the cocaine-cue
memory engram (CREB vector: 8.53 ⫾ 0.63% of LA principal
neurons; GFP vector: 8.76 ⫾ 0.52%; F(1,50) ⫽ 0.07, p ⬎ 0.05). In
contrast, the distribution of arc⫹ in infected versus noninfected
neurons differed markedly between vector groups. In mice microinjected with CREB vector, infected neurons with high levelsof CREB were ⬃3 times more likely to be arc ⫹ (part of the memory trace) than their noninfected neighbors. In mice microin-

Hsiang et al. • Amygdala and Cocaine Reward

jected with GFP vector, infected and noninfected neurons wereequally likely to be arc⫹ [Figure 2 A, B; significant vector (GFP,
CREB) ⫻ neuronal infection status (infected, noninfected) interaction; F(1,50) ⫽ 40.53, p ⬍ 0.001, post hoc Newman–Keuls tests].
Therefore, similar to a conditioned fear/threat memory (Han etal., 2007, 2009; Zhou et al., 2009), LA neurons with increased
CREB are preferentially allocated to what is likely a larger
cocaine-cue memory engram.
Neurons that overexpress CREB during training are essential for
subsequent expression of a cocaine-cue memory
To assess whether these neurons overexpressing CREB at the timeof training are necessary for subsequent expression of the resulting cocaine-cue memory, we selectively ablated just this population of neurons after training using a transgenic mouse whichtakes advantage of DT-induced ablation. We used iDTR mice, inwhich a functional DTR is expressed only in cells that have undergone Cre recombinase-induced recombination. Subsequentsystemic administration of DT induces apoptosis that is limited
to those cells which have undergone recombination and express
the functional DTR (Buch et al., 2005). To ablate neurons overexpressing CREB after training, we microinjected iDTR transgenic mice with a vector expressing both CREB and Cre
recombinase (CREB-cre vector). In these mice, neurons infectedby the CREB-cre vector have increased CREB levels and express
the DTR, thereby tagging these neurons for subsequent ablation
by systemic DT (Han et al., 2009). We microinjected iDTR micewith CREB-cre vector and, before a memory test, systemicallyadministered DT to delete only those neurons overexpressing
CREB. If these neurons with high levels of CREB during training
were necessary components of what is likely a larger engram of
the cocaine-cue association, we would expect that their ablation
after training would impair subsequent memory expression.
Three control groups were similarly treated, except that neurons
overexpressing CREB were not deleted: (1)WT littermate control
mice microinjected with CREB-cre vector and administered DT,or (2) PBS or (3) iDTR transgenic mice microinjected with
CREB-cre vector but systemically administered PBS rather thanDT. During the test, we observed robust CPP in these three control groups. In contrast, ablating neurons overexpressing CREB
impaired subsequent cocaine-cue memory expression (iDTR
mice microinjected with CREB-cre vector and administered DT
after training)[Figure 2C; significant iDTR Genotype (WT,
HET) ⫻ Drug (DT, PBS) interaction, F(1,44) ⫽ 5.15, p ⬍ 0.05;
Newman–Keuls post hoc test showed that iDTR transgenic
mice⫹CREB-cre vector ⫹ DT group had significantly lower CPP
than all control groups, which did not differ from each other].
The disruption of cocaine memory we observed in this experiment, though, could be due to a small lesion of the LA (in thatneurons with CREB-cre vector were ablated). To examine this,we conducted a similar experiment, but microinjected iDTR
mice with GFP-cre vector (rather than CREB-cre vector) to determine the effects of ablating a small, nonspecific population of
LA neurons. To equate the initial strength of the cocaine-cue
memory between CREB-cre and GFP-cre vector groups, wetrained mice with a higher dose of cocaine (30 mg/kg in single
conditioning trial). Indeed, we observed similar CPP scores in
iDTR mice microinjected with either CREB-cre or GFP-cre vector, which were administered PBS (rather than DT) before thetest. However, in contrast to the effects of ablating neurons overexpressing CREB, we found that ablating a similar number ofrandom neurons expressing GFP failed to impact subsequent
expression of the cocaine-cue memory [Figure 2D; significant

Hsiang et al. • Amygdala and Cocaine Reward

vector (CREB-cre, GFP-cre) ⫻ drug (DT, PBS) interaction;
F(1,38) ⫽ 6.63, p ⬍ 0.05; Newman–Keuls post hoc test showed
iDTR mice microinjected with CREB-cre vector, but not GFP-cre
vector, administered DT after training had significantly lower
CPP than the other groups]. Therefore, ablating neurons overexpressing CREB after training impaired subsequent expression of
the cocaine-cue memory, consistent with the interpretation thatthese specific neurons are necessary for memory expression.
The expression of a cocaine-cue memory may also be decreased by extinction training, during which the conditioned cue
is repeatedly presented in the absence of cocaine (Mueller andStewart, 2000; Shaham et al., 2003; Ciccocioppo et al., 2004; Myers and Carlezon, 2010). Extinction training does not “erase” theoriginal cocaine memory as subsequent cocaine exposure is able
to reinstate the cocaine-cue memory (Mueller and Stewart, 2000;
Parker and Mcdonald, 2000; Shaham et al., 2003). Therefore, we
next asked whether the loss of the cocaine-cue memory we observed after deletion of neurons overexpressing CREB could be
similarly reinstated by cocaine exposure. We compared the effects of cocaine re-exposure before a memory test in two groupsof mice: (1) extinction group, iDTR mice microinjected with
GFP-cre vector and administered DT (“control mice”) that received extinction training; and (2) neuron-deletion group, iDTR
mice microinjected with CREB-cre vector and administered DTthat had neurons overexpressing CREB deleted after training but
did not receive extinction training. To extinguish the cocaine-cue
memory in the extinction group, we paired both experimental
chambers with saline administration (Mueller and Stewart, 2000;
Shaham et al., 2003) until mice no longer preferred the chamber
initially paired with cocaine [Figure 2E; repeated-measures
ANOVA for GFP-cre vector (extinction) group comparing Test 1
to Test 3, F(2,38) ⫽ 5.67, p ⬍ 0.05, Newman–Keuls post hoc test
showed mice had significant lower CPP scores during Test 3 thanduring Test 1]. During this time, mice in the neuron-deletiongroup remained drug-free in the homecage (and did not receive
extinction training). Importantly, both extinction and neurondeletion groups showed similarly low CPP scores during the last
test before cocaine re-exposure [CREB-cre vector (neurondeletion group) Test 1 versus GFP-cre vector (extinction group)
Test 3; F(1,22) ⫽ 0.08, p ⬎ 0.05]. Immediately before the final test
(Test 4) both the extinction and neuronal-deletion groups received systemic injection of cocaine. As expected based on previous findings, cocaine exposure was sufficient to reinstate thecocaine-cue memory in the extinction group. Strikingly, cocaine
exposure did not reinstate cocaine-cue memory in the neurondeletion group [Figure 2E; GFP-cre vector Test 3 vs Test 4:
F(1,24) ⫽ 5.00, p ⬍ 0.05; Test 4, CREB-cre vs GFP-cre vector: F(1,22) ⫽7.48, p ⬍ 0.05; CREB-cre vector, Test 1 vs Test 4: F(1,20) ⫽ 0.01,
p ⬎ 0.05]. Mice in the neuron-deletion group behaved as if they
had no prior experience with cocaine. Therefore, in contrast to
extinction training (which temporarily suppressed the cocainecue memory), post-training ablation of LA neurons overexpressing CREB may permanently degrade the cocaine-cue memory
engram.
Consolidation of a cocaine-cue memory is disrupted by posttraining silencing of neurons that overexpressed CREB during
training
Successful memory consolidation is thought to depend on postencoding reactivation of the activity patterns that were present
during the initial learning experience (Marr, 1971; Buzsáki, 1989;
Wilson and McNaughton, 1994; McClelland et al., 1995; Girardeau et al., 2009; Dupret et al., 2010; Ego-Stengel and Wilson,

J. Neurosci., October 15, 2014 • 34(42):14115–14127 • 14123

2010; Carr et al., 2011; Dudai, 2012). In vivo recording studies
show that the frequency of reactivation is highest in the minutes
following (Kudrimoti et al., 1999; Tatsuno et al., 2006) but may
persist for 18 –24 h (Kudrimoti et al., 1999; Karlsson and Frank,
2009) after an experience. Although largely studied in hippocampus and cortex, postencoding reactivation has also been described in other brain regions (Pennartz et al., 2004) and may
represent a ubiquitous phenomenon. For instance, Johnson et al.
(2009) observed that stimulation of the LA produced polysynaptic field potentials, consistent with the idea that reverberatory activity or reactivation may also occur in LA microcircuits.
We examined whether postencoding reactivation specificallyin neurons overexpressing CREB is important for the consolidation of a cocaine-cue memory. To disrupt activity specifically inthese neurons, we took advantage of genetically encoded mediators of neural excitability. Although optogenetic approaches are
remarkably valuable for controlling neural activity over shorttime periods (Airan et al., 2009; Gu et al., 2012; Tye and Deisseroth, 2012; Yiu et al., 2014), chemical genetic approaches using
DREADDs (designer receptors exclusively activated by designer
drug; Armbruster et al., 2007; Nichols and Roth, 2009), which areuseful for manipulating neuronal function for more prolonged
periods. DREADDS are modified G-protein-coupled receptors
which are insensitive to endogenous ligands but activated by asynthetic ligand (CNO, an otherwise pharmacologically inert
agent; Armbruster et al., 2007; Nichols and Roth, 2009). Following CNO binding, the Gi/o-coupled hM4Di DREADD activatesinwardly rectifying potassium 3 (Kir3) channels to hyperpolarize
and transiently silence neurons (Armbruster et al., 2007; Nichols
and Roth, 2009). Before we used this strategy to manipulate potential reverberatory activity, we first showed the effects of transiently silencing neurons overexpressing CREB immediately
before a memory test.
We microinjected WT hybrid mice with a vector expressing
GFP alone, CREB alone, GFP-hM4Di (to transiently silence a
random population of neurons) or CREB-hM4Di (to transiently
silence neurons overexpressing CREB). Mice were trained asabove and systemically administered CNO (1 mg/kg, i.p. or VEH)
1 h before the memory test. Although all three control groups
showed robust CPP, mice microinjected with CREB-hM4Di vector and administered CNO before the test (mice in which theneurons overexpressing CREB were silenced just before the test)
showed no evidence of CPP [Figure 2F; significant vector (GFP,
CREB, GFP-hM4Di, CREB-hM4Di) ⫻ drug (CNO, VEH) interaction: F(4,54) ⫽ 2.80, p ⬍ 0.05, post hoc Newman Keuls tests
showed only mice microinjected with CREB-hM4Di vector thatreceived CNO showed significantly lower CPP than othergroups]. Therefore, temporarily silencing neurons overexpressing CREB (but not a similar number of random LA neurons)
disrupted expression of the cocaine-cue memory. These results
are consistent with our above findings that post-training ablationof CREB-overexpressing neurons disrupted subsequent memory
expression and confirms the effectiveness of this DREADDmediated silencing approach (Richards et al., 2014).
Having established the utility of this DREADD approach, wenext asked whether silencing CREB-overexpressing neurons during the initial post-training period disrupted consolidation of thecocaine-cue memory. We microinjected WT mice with CREBhM4Di or GFP-hM4Di vector and administered a single injectionof CNO (or VEH) immediately after training. Silencing CREBoverexpressing neurons immediately after training (WT mice
microinjected with CREB-hM4Di vector systemically administered CNO) produced a mild deficit in subsequent cocaine-cue

Hsiang et al. • Amygdala and Cocaine Reward

14124 • J. Neurosci., October 15, 2014 • 34(42):14115–14127

memory expression (Figure 3A; F(1,12) ⫽ 5.69, p ⬍ 0.05). However, cocaine-cue memory expression was completely blocked if
we administered CNO both immediately after training and again
18 h later (at which time mice were in the homecage; Figure 3B;
significant Vector (GFP-hM4Di, CREB-hM4Di) ⫻ drug (CNO,
VEH) interaction: F(1,34) ⫽ 6.17, p ⬍ 0.05, post hoc Newman–
Keuls tests showed only mice microinjected with CREB-hM4Di
vector that received CNO showed significantly lower CPP thanother groups]. Interestingly, we observed no memory deficit if
CNO administration (2 injections/d for 2 d) began 18 h after
training was complete (Figure 3C; F(1,11) ⫽ 0.01, p ⬎ 0.05).
Therefore, disrupting activity in CREB-overexpressing neurons
starting immediately after training impaired memory consolidation, an effect that was amplified by repeating disruption 18 hlater. However, consolidation was not impaired if the silencing
started 18 h after training. These findings help identify the critical
temporal windows of consolidation during which ongoing neuronal reactivity may be important for memory consolidation andare consistent with previous estimates of this consolidation timewindow. For instance, Chawla et al. (2005) observed high Arc
protein in hippocampal CA1 and CA3 regions in the hours following behavioral experience (exploration), as well as the apparent rise of a second wave of activation 8 h later, but levels returned
to baseline 24 h later.
Consolidation of a cocaine-cue memory is disrupted by posttraining indiscriminate artificial activation of neurons
overexpressing CREB
In vivo electrophysiological data suggest that the postencoding
reactivation of activity patterns present during the initial learning
experience occurs with precise temporal fidelity and that the sequence of neuronal firing is important. Therefore, we next assessed the effects of disrupting the faithfulness of postencoding
neuronal reactivation by nondiscriminately enhancing activity in
CREB-overexpressing neurons after training. To nondiscriminately increase activity we used the DREADD hM3Dq, which is
coupled to Gq receptors. Activation of hM3Dq by CNO produces
Gq activation of phospholipase C and intracellular Ca 2⫹ release,which depolarizes neurons and increases action potential firing(Armbruster et al., 2007; Nichols and Roth, 2009). In this experiment, we microinjected WT mice with a vector expressing
CREB⫹hM3Dq or GFP⫹hM3Dq and trained as above. In thisway, systemic administration of CNO would increase activity in
CREB-overexpressing neurons (CREB-hM3Dq vector) or random neurons (GFP-hM3Dq vector). Interestingly, we observedthat a single systemic injection of CNO 18 h after training (while
mice were in the homecage) was sufficient to impair subsequent
expression of the cocaine-cue memory in mice with CREBhM3Dq vector. Memory was disrupted in mice microinjectedwith CREB-hM3Dq vector only; we observed normal CPP inmice microinjected with GFP-hM3Dq vector, indicating that artificially activating a random population of neurons does not
impair subsequent memory expression [Figure 3D; significant
vector (GFP-hM3Dq, CREB-hM3Dq) ⫻ drug (CNO, VEH) interaction: F(1,30) ⫽ 6.21, p ⬍ 0.05, post hoc Newman–Keuls tests
showed that mice microinjected with CREB-hM3Dq vector andadministered CNO had significantly lower CPP than the othergroups]. In contrast to the lack of effect produced by silencing
neurons with the hM4Di DREADD beginning 18 h after training(Fig. 3C), artificially activating neurons overexpressing CREB
and the hM3Dq DREADD with several injections of CNO beginning 18 h after training produced a pronounced memory deficit(Figure 3E; F(1,10) ⫽ 5.30, p ⬍ 0.05). This cocaine-cue memory

was not reinstated by subsequent cocaine administration (Figure
3F; F(1,10) ⫽ 6.71, p ⬍ 0.05), suggesting that post-training artificial activation of this key component of the memory trace degrades the integrity of the entire memory trace. Artificially
increasing firing in this population of LA neurons overexpressing
CREB only, independent from intra- or extra-LA components ofthe memory trace, which are likely at this time window to beinactive, may interfere with normal memory consolidation(“neurons that do not fire together may unwire together”).

Discussion
This series of experiments investigated the role of neurons in the
LA which become critical for encoding and storing cocaine-cue
memories in mice. Our findings indicate that LA neurons compete for allocation to a cocaine-cue memory trace and that neurons overexpressing CREB are competitively advantaged in thiscompetition. Specifically, we used viral vectors to increase CREB
levels in a random population of ⬃10% of LA principal neurons
and showed that this small group of neurons become a criticalhub of what is likely a larger neuronal cocaine-cue memory
network.
Several previous results show that artificially increasing theactivity of a similar small population of neurons thought to be ina memory trace impacts the formation and/or expression of thatmemory. For instance, Liu et al. (2012) reported that optogenetic
activation of a small number of dentate gyrus (DG) neurons thatwere active during context fear/threat training induces freezing(that is, artificially activating these cells served as a sufficient retrieval cue for expression of a conditioned fear/threat memory).
Using a similar activity tagging strategy, this group also showedthat artificial activation of a small number of DG neurons that
happened to be active during novel context exploration could
function as a conditioned stimulus in the formation of a new (butfalse) conditioned fear/threat memory (Ramirez et al., 2013). By
contrast, here we manipulated neurons we hypothesized are criticalcomponents of a larger memory trace by using CREB overexpression to allocate (or funnel) the memory to a small population of LA
neurons before training rather than “tagging” a population of neurons that happened to be active during training.
Using this technique to manipulate neurons which becomecomponents of an engram for a cocaine-cue association, we
found that post-training ablation or temporary silencing of thispopulation of CREB-overexpressing neurons before a memory
test disrupts subsequent expression of a cocaine-cue memory. In
contrast to extinction training (which temporarily suppresses
cocaine-conditioned memory), our findings suggest that posttraining ablation of this population of neurons may functionallyerase the cocaine-cue memory, as we observed no reinstatementof the cocaine-cue memory when mice were re-exposed to cocaine. Furthermore, we observed that interfering with the normal
post-encoding activity of these neurons (and not a similar number of random LA neurons) disrupts memory consolidation. Either silencing CREB-overexpressing neurons in the hours after
training or disrupting the ordered replay/reactivation of theseneurons by nondiscriminately activating these neurons impaired
cocaine-cue memory consolidation. Our results complement
previous findings showing that memory is disrupted by interfering with ordered postencoding reactivation. Although otherstudies have targeted larger networks during precise intervals ofreactivation (hippocampal sharp wave-ripple events; Girardeau
et al., 2009; Ego-Stengel and Wilson, 2010; Jadhav et al., 2012),
here we show that memory is disrupted by interfering with theactivity in a specific population of neurons during temporallyHsiang et al. • Amygdala and Cocaine Reward

extended consolidation windows. Our findings are consistent
with the hypothesis that long-term stabilization of the engram (orcell assembly) requires coordinated reactivation in the hours immediately after an experience.
A similar CREB-mediated neuronal selection process in the
LA also underlies the formation of a conditioned fear/threat
memory (Han et al., 2007, 2009; Zhou et al., 2009; Kim et al.,2014). Therefore, increasing CREB in a small seemingly random
population of LA pyramidal neurons enhanced memory for an
association between a cue and footshock (conditioned fear/threat
memory) as well for an association between a cue and cocaine(CPP). Together, these findings indicate that the LA plays a general role in assigning biological significance (be it appetitive or
aversive) to previously neutral cues. Although CREB is a ubiquitous transcription factor implicated in a range of diverse processes, we recently showed that the neuronal allocation effects ofincreasing CREB in a small portion of LA neurons were mimicked by increasing excitability and blocked by decreasing intrinsic excitability in CREB-overexpressing neurons (Yiu et al.,2014). These findings indicate that neuronal engram allocation isbased on relative neuronal excitability immediately before training. In this series of studies, we artificially manipulated intrinsic
excitability and memory allocation by overexpressing CREB.
However, endogenous changes in intrinsic excitability are linked
to learning in a variety of species [Hermissenda (Alkon, 1974) torodents (Thompson et al., 1996)], suggesting that the current
manipulations (CREB overexpression) tap into an underlying
fundamental memory process.

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