The role of ventral and dorsal striatum mGluR5 in relapse to cocaine-seeking and extinction learning

The role of ventral and dorsal striatum mGluR5 inrelapse to cocaine-seeking and extinction learning

Lori A. Knackstedt, Heather L. Trantham-Davidson & Marek Schwendt*Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA

ABSTRACT

Cocaine addiction is a chronic, relapsing disease characterized by an inability to regulate drug-seeking behavior. Herewe investigated the role of mGluR5 in the ventral and dorsal striatum in regulating cocaine-seeking following bothabstinence and extinction. Animals underwent 2 weeks of cocaine self-administration followed by 3 weeks of home-cage abstinence. Animals were then reintroduced to the operant chamber for a context-induced relapse test, followedby 7–10 days of extinction training. Once responding was extinguished, cue-primed reinstatement test was conducted.Both drug-seeking tests were conducted in the presence of either mGluR5 negative allosteric modulator, MTEP orvehicle infused into either the nucleus accumbens (NA) core or dorsolateral striatum (dSTR). We found that MTEPinfused in the NA core attenuated both context-induced relapse following abstinence and cue-primed reinstatementfollowing extinction training. Blocking dSTR mGluR5 had no effect on context- or cue-induced cocaine-seeking.However, the intra-dSTR MTEP infusion on the context-induced relapse test day attenuated extinction learning for 4days after the infusion. Furthermore, mGluR5 surface expression was reduced and LTD was absent in dSTR slices ofanimals undergoing 3 weeks of abstinence from cocaine but not sucrose self-administration. LTD was restored by bathapplication of VU-29, a positive allosteric modulator of mGluR5. Bath application of MTEP prevented the induction ofLTD in dSTR slices from sucrose animals. Taken together, this data indicates that dSTR mGluR5 plays an essential rolein extinction learning but not cocaine relapse, while NA core mGluR5 modulates drug-seeking following both extinc-tion and abstinence from cocaine self-administration.

Keywords Abstinence, cocaine, dorsal striatum, extinction, long-term depression, mGluR5, nucleus accumbens,surface expression.

Correspondence to: Marek Schwendt, Psychology Department, College of Liberal Arts & Sciences, University of Florida, 114 Psychology Building, 945Center Drive, Gainesville, FL 32611-2250, USA. E-mail: [email protected]

INTRODUCTION

Cocaine addiction is a compulsive and chronically relaps-ing disorder (McLellan et al. 2000; O’Brien 2001). Therisk of relapse remains high even after months or years ofabstinence and represents a major challenge in the suc-cessful treatment of drug addiction. Animal models ofrelapse have been developed to study the neural circuitryand molecular substrates underlying persistent drug-seeking and ultimately to screen targeted pharmacologi-cal treatments to prevent relapse. In these models,animals do not relapse to drug-taking (e.g. intravenousdrug delivery) but instead relapse is considered to be aresumption of the drug-seeking response (e.g. lever press-ing). One such model is the extinction-reinstatementparadigm, in which animals are trained to self-administer

drug in an operant chamber and then undergo extinctiontraining during which previously reinforced behaviors nolonger result in drug infusion and said behavior decreases(de Wit & Stewart 1981). Once behavioral responding islow, the drug-seeking response is reinstated with stimuliknown to cause relapse in humans, including stress, dis-crete and contextual cues previously associated withdrug delivery, and/or the drug itself (for review seeEpstein et al. 2006). A second animal model is theabstinent-relapse model in which animals do not undergoextinction training following self-administration butinstead experience abstinence in the home cage withdaily handling. Animals are then re-exposed to the drug-taking environment (operant chamber) for a context-induced relapse test, which is also Day 1 of extinctiontraining (for review see Reichel & Bevins 2009). Both

PRECLINICAL STUDY

bs_bs_bannerAddiction Biologydoi:10.1111/adb.12061

© 2013 Society for the Study of Addiction Addiction Biology, 19, 87–101

models have been judged to possess face validity for dif-ferent facets of addiction and are valuable tools forscreening potential pharmacotherapies for their ability toattenuate drug-craving and relapse (Epstein et al. 2006;Reichel & Bevins 2009).

The extinction-reinstatement model has been exten-sively used to identify the neural circuitry involved inrelapse, with the ventral striatum (in particular nucleusaccumbens core) being identified as a key structurein mediating stress- and drug-primed reinstatement(McFarland & Kalivas 2001; McFarland, Lapish & Kalivas2003; McFarland et al. 2004). Reversible inactivation ofboth the nucleus accumbens (NA) core and the dorsalmedial prefrontal cortex (dmPFC) projection to the NAcore attenuate drug-primed reinstatement followingextinction training (McFarland & Kalivas 2001). Further-more, stress and cocaine-primed reinstatement are drivenby a release of glutamate along this pathway (McFarlandet al. 2003, 2004).

Using the abstinent-relapse model, it has been foundthat inactivation of the lateral subregion of dorsal stria-tum (or dorsolateral striatum—dSTR) attenuatescontext-induced drug-seeking following 2–3 weeks ofabstinence (Fuchs, Branham & See 2006). Interestingly,neither the dmPFC nor the NA are necessary for context-induced relapse following abstinence (Fuchs et al. 2006;See, Elliott & Feltenstein 2007), although both have pre-viously been shown to be necessary for explicit cue-induced reinstatement of extinguished cocaine-seeking(McLaughlin & See 2003; Fuchs et al. 2004). It has beensuggested that both the dmPFC and NA are incorporatedinto the reinstatement neurocircuitry through theprocess of extinction learning (Peters, LaLumiere &Kalivas 2008). Furthermore, See et al. (2007) deter-mined that while reversible inactivation of the NA coredid not affect abstinent-relapse, extinction learning wasattenuated on subsequent days following the inactiva-tion. Conversely, reversible inactivation of the dSTR sig-nificantly attenuated abstinent-relapse but did not affectsubsequent extinction learning.

As evidenced by a number of studies using theextinction-reinstatement model, dysregulation of gluta-mate homeostasis in the NA is the primary driver ofdrug-seeking behavior during reinstatement (seeKnackstedt & Kalivas 2009 for review). Metabotropicglutamate receptors of subtype 5 (mGluR5) are highlyenriched in the striatum and mediate long-term synapticplasticity, such as long-term depression (LTD; Sung, Choi& Lovinger 2001; Fourgeaud et al. 2004; Moussawi et al.2009). Systemic pharmacological or genetic disruptionof mGluR5 function attenuates the reinstatement ofextinguished cocaine-seeking (Chiamulera et al. 2001;Bäckström & Hyytiä 2006; Kumaresan et al. 2009;Martin-Fardon et al. 2009). Specific blockade of NA core

(Wang et al. 2013) and NA shell (Kumaresan et al. 2009)mGluR5 receptors also attenuates cocaine reinstatement.Moreover, in rats with a history of cocaine self-administration followed by 2–3 weeks of extinction train-ing, the ability to induce mGluR5-dependent LTD in theNA core is lost (Knackstedt et al. 2010). The loss of LTD isaccompanied by a decrease in mGluR5 surface expressionand changes in the mGluR5 signaling complex in thisbrain region (Knackstedt et al. 2010). Interestingly,mGluR5 surface levels as well as mGluR5-dependentLTD in the NA core are not significantly altered inanimals which underwent cocaine self-administrationand extended abstinence but not extinction training(Knackstedt et al. 2010; McCutcheon et al. 2011a). Ingeneral, there is a distinctive pattern of glutamatergicneuroadaptations found in the striatum, depending onpost-cocaine experience (abstinence versus extinction)and the length of drug-free interval (days versus weeks;Sutton et al. 2003; Ghasemzadeh et al. 2009; Knackstedtet al. 2010; Edwards et al. 2011; McCutcheon et al.2011b). While there is strong support for the theory thatglutamate (including mGluR5-dependent) signaling inthe NA core is essential for reinstatement followingextinction training (Cornish & Kalivas 2000; McFarlandet al. 2003; Wang et al. 2013), the identity of the neuro-transmitters and receptors in the dorsal and ventral stria-tum involved in context-induced relapse followingabstinence remain poorly understood. Our initial studieshave indicated that in the dSTR, several proteins withinthe mGluR5 receptor signaling complex (such as RGS4,Gaq subunit and PLCb1) are dysregulated followingcocaine self-administration and prolonged (3 week) absti-nence, (Schwendt et al. 2007a; Schwendt, Knackstedt &McGinty 2007b). However, it remains unclear whethermGluR5 function in the dSTR is altered following absti-nence from cocaine self-administration, and whethermanipulation of mGluR5 receptor in this brain regionwould interfere with relapse and or extinction learning.

Here, we investigated the role of mGluR5 in relapsefollowing both abstinence (without extinction) andextinction training. We utilized an animal model whichplaces animals into forced abstinence followed by reintro-duction to the self-administration environment for acontext-induced relapse test. This test day is also Day 1 ofextinction training as presses on the previously activelever do not yield drug or presentation of drug-pairedcues. Extinction training continues until responding isreduced and animals are then tested for cue-primed rein-statement. Using this model, we tested the ability of3-[(2-methyl-1, 3-thiazol-4-yl) ethynyl] pyridine (MTEP),a highly specific negative allosteric modulator (NAM) ofmGluR5 (Anderson et al. 2002, 2003; Cosford et al.2003), to attenuate both context- and cue-inducedrelapse when infused into either the dSTR or NA core. We

88 Lori A. Knackstedt et al.


also assessed potential changes in mGluR5 function asmeasured by slice electrophysiology and surface bioti-nylation in dSTR slices.

MATERIALS AND METHODS

Drugs

Cocaine hydrochloride was acquired from the NIDAControlled Substances Program (Research TriangleInstitute, NC, USA). The selective mGluR5 NAM, MTEPhydrochloride, was purchased from Abcam Biochemicals(Cambridge, MA, USA). The selective mGluR5 posi-tive allosteric modulator 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (VU-29) was obtained from Dr.Foster Olive (Arizona State University, Tempe, AZ, USA).The cocaine used for intravenous self-administrationwas dissolved in 0.9% physiological saline. MTEP andVU-29 were dissolved in 1% Tween-80 in artificial cer-ebrospinal fluid (aCSF; in mM: 125 NaCl, 1 CaCl2, 2.5KCl, 4 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, 0.4 ascorbicacid and 10 d-glucose). Tween-80 (1%) in combinationwith aCSF was used as vehicle control for these agents.Solutions of all drugs were neutralized to pH 7.2–7.4with 1N NaOH, if necessary. The concentrations of thedrugs used were determined based on the results of pre-viously published studies (Ayala et al. 2009; Gass & Olive2009b; Schwendt & Olive 2012; Sinclair et al. 2012;Wang et al. 2013), as well as on our preliminary behav-ioral experiments (Hearing, Knackstedt, Schwendt,unpublished data).

Animals

Adult male Sprague Dawley rats (Charles River Labora-tories, Wilmington, MA, USA), weighing 275–300 gwere single-housed in a temperature- and humidity-controlled vivarium on a reversed 12-hour light/darkcycle with water available ad libitum. Animals were food-restricted to 85% of free-feeding weight. All animal pro-cedures were approved by the Institutional Animal Careand Use Committee of the Medical University of SouthCarolina and were performed in accordance with theGuide for the Care and Use of Laboratory Animals (Insti-tute of Laboratory Animal Resources, National AcademyPress 1996).

Catheter and stereotaxic surgery

Rats were anesthetized with ketamine HCl (87.5 mg/kg,IM) and xylazine (5 mg/kg, IM). Ketorolac (3 mg/kg, IP)was administered pre-operatively to provide analgesia.SILASTIC tubing (0.51 mm inner diameter, 0.94 mmouter diameter; Dow Corning, Midland, MI, USA) wasimplanted into the jugular vein. The tubing exited the

vein and traveled subcutaneously between the shoulderblades to exit from the skin on the back. The tubing wasattached to a stainless-steel cannulae which was heldwithin a harness (Instech, Plymouth Meeting, PA, USA).Animals were then placed into a small animal stereotaxicinstrument (Kopf Instruments, Tujunga, CA, USA) andguide cannulas were implanted and secured with dentalcement. Cannulas (20 gauge; Plastics One, Roanoke, VA,USA) were implanted 2 mm above the infusion targetaccording to following coordinates: dSTR (+1.2 mmAP, � 3.4 mm ML and -3.4 mm DV), NA core (+1.8 mmAP, �1.6 mm ML and -5.5 mm DV) relative to bregmaaccording to (Paxinos & Watson 2007). Animals wereallowed to recover for 5 days prior to the initiation ofself-administration. Catheters were flushed with100 U/ml heparinized saline (Elkins-Sinn, Cherry Hill,NJ, USA) daily throughout self-administration.

Self-administration and abstinence

Rats were trained to press a lever to receive an intra-venous infusion of cocaine in operant chambers(30 ¥ 24 ¥ 30 cm; Med Associates, St. Albans, VT, USA)under an FR1 schedule of reinforcement for 2 hours dailyduring the dark cycle for a minimum of 12 days.Responses on the active lever resulted in the delivery ofcocaine (0.2 mg in 50 ml saline) accompanied by a5-second presentation of a light + tone conditioned cue.Each cocaine infusion was followed by a 20-second ‘time-out’ period during which presses on the drug-paired leverdid not yield drug or cue presentation. Responses on theinactive lever had no programmed consequences butwere recorded. Catheter patency was periodically verifiedwith methohexital sodium (10 mg/ml; Eli Lilly, Indiana-polis, IN, USA), a short-acting barbiturate that produces arapid loss of muscle tone when administered intrave-nously. Control animals received sucrose pellets and cuepresentations on an FR-1 schedule of reinforcement.Data collection and reinforcer delivery were controlledusing Med PC software version IV (Med Associates). Fol-lowing the completion of 12-day self-administrationregimen (minimum of 12 infusions or 50 sucrose pellets/day) all animals underwent 20 days of home cage absti-nence. During the last 10 days of abstinence, rats werehandled and habituated for 2 hour/day to another proce-dure room. At the end of abstinence, animals wereassigned to one of three cohorts receiving the followingprocedures: (1) animals were euthanized and the dSTRslices were collected for surface biotinylation and Westernblot analysis; (2) animals were euthanized and slices ofthe dSTR were made for electrophysiology, or (3) animalswere tested to evaluate the effects of intra-NA core orintra-dSTR vehicle versus MTEP microinjections on drug-seeking and extinction.

mGluR5 and cocaine-seeking 89


Intracranial microinjections

Two 33-gauge microinjectors that extended 2 mmbeyond the tip of the guide cannula were bilaterallyinserted into the NA core or dSTR. Vehicle (VEH) or MTEPwas infused at a rate of 0.5 ml/minute in a total volume of0.5 ml/side (NA core) or 1 ml/side (dSTR) using an infu-sion pump (Harvard Apparatus, Holliston, MA, USA).After the infusion was completed, the microinjectors wereleft in place for an additional 1 minute to allow for diffu-sion of the drugs. Only rats with correct cannula place-ments were included in the data analysis.

Relapse, extinction and reinstatement procedures

On day 21 of abstinence, animals received a single bilat-eral microinjection of VEH or MTEP into the dSTR (5 mg/side) or NA core (1 mg/side) using the proceduredescribed above and were placed into the operant cham-bers for a 2-hour context-induced relapse test duringwhich lever presses were recorded but had no pro-grammed consequences. After the test, rats underwentdaily 2-hour extinction sessions until extinction criterion(a minimum of seven extinction sessions and �25 activelever responses per session for the last 2 consecutive days)was reached. Upon completion of extinction training,animals underwent a cue-induced relapse test in thesame manner as described above. A cross-over design wasused so that animals received infusion of one substance(MTEP or VEH) prior to the context-induced relapse testand the other substance prior to the cue-primed rein-statement test. During the cue-primed reinstatement test,active lever presses resulted in discrete cue presentation(light + tone) in the absence of cocaine delivery. At theend of this final test, animals were euthanized and thebrains harvested for histological verification of cannulaesites. Animals received vehicle and MTEP infusions in acounterbalanced manner and at least 1 week separatedtwo consecutive tests.

Locomotor testing

We quantified locomotor behavior (total distancetraveled) after infusion of MTEP and VEH into the dSTRand NA core. To do so, we used a subset of the animalswhich underwent cocaine and sucrose relapse testing;locomotor testing was completed 5–7 days following thelast reinstatement test. Animals were placed into thelocomotor apparatus (Accuscan Instruments, Columbus,OH, USA) and baseline locomotion was recorded for 1hour, at which point animals received microinjection ofeither VEH or MTEP and behavior was recorded for 2hours. Animals receiving intra-dSTR infusions were alsotested in the same manner but without intra-dSTR infu-sions on the 2 days following the first test.

Histology

In some experiments, following the last reinstatement orlocomotor testing, animals were rapidly euthanized andbrains collected, frozen and stored until analysis ofmicroinjection placements. Brains were blocked andsliced in coronal sections (50 mm) through the striatalsubregions analyzed. Sections were mounted on posi-tively charged Superfrost slides (Fisher Scientific Co.,Houston, TX, USA), stained with cresyl violet, and exam-ined for cannula placement by an individual unawareof each subject’s behavior. All placements were deter-mined using the rat brain atlas by Paxinos & Watson(2007).

Slice electrophysiology

Brain slices were prepared from cocaine and sucrose self-administering animals. Coronal slices (350 mm) were cutin ice-cold modified artificial cerebrospinal fluid (aCSF)containing the following (in mM): 200 sucrose, 1.9 KCl,6 MgCl2, 33 NaHCO3, 1.2 Na2HPO4, 0.5 CaCl2, 0.4 ascor-bic acid, 10 d-glucose and adjusted to pH 7.4 by bubblingwith 95% O2/5% CO2. Slices containing the cortex andthe anterior striatum were completely submerged andcontinuously bathed in incubation aCSF (in mM: 125NaCl, 1 CaCl2, 2.5 KCl, 4 MgCl2, 25 NaHCO3, 1.25NaH2PO4, 0.4 ascorbic acid and 10 d-glucose). RecordingaCSF contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1.2MgCl2, 25 NaHCO3, 0.4 ascorbic acid, 10 d-glucose.Recording aCSF and drugs (MTEP 1 mM, VU-29 1 mM)were bath applied to slices by gravity flow. Superfusatewas passed through an in-line heater at a flow rate of 1.5-2.5 ml/minute and temperature was held constantat 34°C.

Extracellular recordings were obtained using pipettesmade from borosilicate glass. Pipette resistance rangedfrom 0.9 to 1.1 MW when filled with recording aCSF solu-tion. Signals were amplified using an Axon 700B(Molecular Devices, Sunnyvale, CA, USA) recorded usinga Windows PC running Axograph X and MulticlampCommander. The stimulus intensity was set to the level atwhich an evoked population spike (PS) was approxi-mately one-half of the amplitude of the maximal obtain-able response prior to high-frequency stimulation (HFS).Stimulus intensity was adjusted to a level evoking amaximal response during HFS. Stimulus intensity rangedfrom 15 to 120 V while the stimulus duration was set to0.1 and delivered using a Grass S-88 Stimulator. Themajority of the slices were also subjected to maximalstimulation at the end of an experiment to ensure that nosignificant deterioration of the slices occurred during theexperiment, as determined by the presence of a maximalPS in response to such stimulation. LTD was induced by



the following HFS protocol: four 1-second duration,100-Hz trains, delivered at a frequency of one train every10 seconds (Partridge, Tang & Lovinger 2000).

Slice biotinylation

Slice preparation and surface biotinylation was con-ducted as described in our previous studies (Knackstedtet al. 2010; Hearing, Schwendt & McGinty 2011) withfew modifications. The dSTR was punched from a 2-mmcoronal section (Fig. 6) and cut into 250-mm slices usinga McIlwain Tissue Chopper (Stoelting, Wood Dale, IL,USA). Slices were immediately biotinylated with EZlinkNHS-SS-Biotin (1 mg/ml in aCSF; Thermo Fisher Scien-tific, Rockford, IL, USA) for 1 hour at 4°C. Next, biotinyla-tion reaction was quenched with 100 mM glycine/aCSFbuffer followed by two washes with aCSF. Biotinylatedslices were homogenized by a brief sonication in a HEPES-Triton-sodium dodecyl sulfate (SDS) lysis buffer (25 mMHEPES, 150 mM NaCl, 1% Triton X-100, 0.1% SDS) sup-plemented with protease inhibitors and phosphataseinhibitors. Following incubation for 1 hour at 4°C,insoluble debris was removed by centrifugation. An equalaliquot of pre-cleared solubilizate (total protein fraction,‘T’) was saved and stored at -80°C. Biotinylated proteinswere captured by incubating with streptavidin agarosebeads (Thermo Fisher Scientific) overnight at 4°C. Aftera brief centrifugation, supernatants (non-biotinylated,intracellular proteins, ‘I’) were saved and beads werewashed three times with lysis buffer. Biotinylated(surface, ‘S’) proteins were eluted off the beads withLaemmli sample buffer [62.5 mM Tris-HCl, pH 6.8, 20%glycerol, 2% SDS, and 50 mM dithiothreitol (DTT)] andincubation at 95°C for 10 min.

Immunoblotting

Proteins of interest in total (T), intracellular (I) andsurface (S) fractions were analyzed by immunoblotting.Equal aliquots from each fraction separated by SDS-PAGE(4–15%) and transferred to polyvinylidene fluoride mem-brane. Membranes were blocked for 1 hour in 5% milk/Tris-buffered saline and probed overnight at 4°C withprimary antibodies diluted in 5% milk/Tris-bufferedsaline with 0.1% Tween 20. The following primary antis-era were used: anti-calnexin (Enzo Life Sciences,Farmingdale, NY, USA), anti-mGluR1 and anti-mGluR5(Millipore, Billerica, MA, USA), anti-cannabinoid CB1receptor, anti-b-tubulin and anti-SNAP25 (all Abcam).Anti-DexRas1 antibody, a gift from Dr. Stephen Lanier(MUSC, Charleston, SC, USA), was characterized previ-ously (Vaidyanathan et al. 2004). After incubation withan appropriate horseradish peroxidase-conjugated sec-ondary antiserum (all from Jackson Immuno Research,West Grove, PA, USA), immunoreactive bands on the

membranes were detected by ECL+ chemiluminescencereagents on an X-ray film (GE Healthcare, Piscataway,NJ, USA). Subsequently, the blots were stripped andre-probed with anti-DexRas1 antibody to monitor bioti-nylation of intracellular proteins and with anti-calnexinantibody to normalize for unequal loading and/or trans-fer of proteins. The integrated band density of eachprotein sample was measured using NIH Image J softwareversion 1.32j (http://rsb.info.nih.gov/ij/).

Statistical analysis

For all statistical analyses used, the alpha level was set atP < 0.05. Relapse tests were analyzed with independentsample t-tests. Both extinction training and locomotordata were analyzed with mixed-factorial two-way analy-ses of variance (ANOVAs), with time as the repeatedmeasure. Independent sample t-tests were used toexamine group differences only if a significant Group ¥Time interaction was obtained. Immunoblotting data,represented by integrated density of individual proteinbands, were normalized for the density of calnexin immu-noreactivity within the same sample and the two groups(sucrose and cocaine) were analyzed by t-test.

Electrophysiology data were analyzed as follows: PSamplitude was measured using Axograph X usingmethods of Partridge et al. (2000). The magnitude of LTDwas normalized to baseline on a single-recording basis.The data were analyzed with independent sample t-testsand the time-course data were analyzed with a mixed-factorial two-way ANOVA with repeated measures ontime. LSD post hoc tests were used when appropriate.

RESULTS

Animals self-administered cocaine (Figs 1a & 2a) orsucrose (Fig. 3a) for 12 days on an FR-1 schedule of rein-forcement. Following 3 weeks of abstinence in the homecage (without extinction training), animals were placedinto the operant boxes for a 2-hour context-inducedrelapse test following infusion of MTEP or vehicle (VEH)into either the NA core or dSTR. Animals which receivedintra-accumbens MTEP (1 mg/side) displayed signifi-cantly less presses on the previously active lever(t(1,10) = 5.597, P < 0.05; Fig. 1b). There was no effectof intra-accumbens MTEP during the abstinent relapsetest on subsequent extinction learning (Fig. 1c). A two-way ANOVA conducted on these data revealed no effect ofGroup (F(1,10) = 1.121, n.s.) or Group ¥ Day interaction(F(1,10) = 1.959, n.s.); however, there was a significanteffect of Day (F(3,30) = 7.360, P < 0.05), as both groupsdecreased lever presses over days. Intra-accumbensMTEP significantly attenuated cue-primed reinstatementfollowing extinction training (t(1,13) = 7.040, P < 0.05;



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Figure 1 MTEP infused into the NA core attenuates relapse after both abstinence and extinction. (a) Mean number infusions attained duringcocaine self-administration. (b) Intra-NA core MTEP attenuates context-induced relapse following abstinence. (c) Intra-NA core MTEP on testday has no lasting effects on extinction learning. (d) Intra-NA core MTEP significantly attenuates cue-primed reinstatement following extinction.n = 4–8/group. (e) Diagrams of the rat brain coronal sections, adapted from Paxinos & Watson (2007), show the distribution microinjectionsites in the NA core. Numbers indicate distance from bregma in the anteroposterior plane. * = P < 0.05 in comparison to vehicle

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Figure 2 MTEP infused into the dSTR does not affect relapse but has lasting effects on extinction learning. (a) Mean number infusionsattained during cocaine self-administration. (b) Intra-dSTR MTEP does not affect context-induced relapse following abstinence. (c) Intra-dSTRMTEP prior to the context-induced relapse test (extinction Day 1) has lasting effects on extinction learning in subsequent days. (d) Intra-dSTRMTEP does not affect cue-primed reinstatement following extinction. (e) Diagrams of the rat brain coronal sections, adapted from Paxinos& Watson (2007), show the distribution microinjection sites in the dSTR. Numbers indicate distance from bregma in the anteroposteriorplane. * = P < 0.05 in comparison to vehicle. n = 10–12/group



Fig. 1d). Histological analysis of microinjection place-ments confirmed that microinjection sites clusteredwithin the core subregion or on the border with NAshell (Fig. 1e). We found no effect of MTEP on inactivelever pressing during either the abstinent-relapse test(t(1,10) = 0.218, n.s.) or cue-primed reinstatement(t(1,13) = 0.87, n.s.). Mean inactive lever presses were asfollows, abstinent-relapse test: MTEP: 24.8 � 7.6, VEH:31.1 � 9.3; cue-primed reinstatement: MTEP: 1.6 �

0.9, VEH: 3.43 � 1.0.Intra-dSTR MTEP (5 mg/side) had no effect on

context-induced relapse following 3 weeks of abstinence(t(1,21) = 0.834, n.s.; Fig. 2b). However, animals receiv-ing MTEP infusions only on the day of the context-induced relapse test subsequently displayed attenuatedextinction learning (Fig. 2c). A two-way ANOVA revealedsignificant effects of Day (F(3,60) = 37.632, P < 0.001)and Group (F(1,20) = 5.569, P < 0.05) and a significantGroup x Day interaction (F(1,20) = 2.948, P < 0.05).Intra-dSTR MTEP had no effect on cue-induced reinstate-ment following extinction training (t(1,21) = 0.254, n.s;Fig. 2d). Histological analysis of microinjection place-ments confirmed, that microinjection sites were locatedwithin the dSTR subregion (Fig. 2e). We found no effect ofintra-dSTR MTEP on inactive lever pressing during either

the abstinent-relapse test (t(1,21) = 1.643, n.s.) or cue-primed reinstatement (t(1,21) = 1.794, n.s.). Mean inac-tive lever presses were as follows, abstinent-relapse test:MTEP: 14.7 � 3.4, VEH: 22.9 � 8.1; cue-primed rein-statement: MTEP: 5.33 � 2.1, VEH: 3.08 � 0.7.

An alternate explanation for the elevation in activelever pressing observed following MTEP infusions in thedSTR (relative to VEH-infused animals) is that extinctionlearning occurred at an enhanced rate in the VEH-infused animals. Comparing active lever pressingbetween animals infused with VEH in NA core (Fig. 1c)versus the dSTR (Fig. 2c), it is evident that dSTR-infusedanimals show less absolute active lever pressing on Day 2.However, it is important to consider Day 2 behavior inrelation to Day 1 behavior for each cohort. In order toinvestigate this further, we calculated the ratio of Day 2lever pressing to Day 1 lever pressing and found no sig-nificant difference in extinction learning between VEH-infused dSTR and NA core animals (t(1, 14) ) = 0.05,n.s.; Supporting Information Fig. S1). In agreement withour conclusion that MTEP infusion in the dSTR depressesextinction learning, we found a significant difference inthe rate of extinction when comparing animals infusedin the dSTR with VEH versus MTEP (t(1,18) = 5.718,P < 0.05; Supporting Information Fig. S1).

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Intra-dSTR MTEP had no effect on context-inducedrelapse of sucrose-seeking (t(1,22) = 2.037; Fig. 3b) andno effect on subsequent extinction learning (Fig. 3c). Atwo-way ANOVA revealed a significant effect of Day(F(3,63) = 69.830, P < 0.001), but no effect of Group(F(1,21) = 2.133, n.s.) and no Group ¥ Day interaction(F(1,21) = 0.159, n.s.). Histological analysis of microin-jection placements confirmed that microinjection siteswere located within the dSTR subregion (Fig. 3d). Therewas no effect of MTEP on inactive lever presses (t(1,22) =2.463). Mean inactive lever presses for the MTEP-treatedanimals was 20.3 � 6.7 while the mean for the VEH-treated animals was 9.1 � 1.7.

To investigate whether the significant attenuation ofboth context- and cue-induced relapse occurred due tolocomotor-suppressive effects of intra-accumbens MTEP,animals were placed into a locomotor chamber for a60 min habituation period, followed by infusion of eitherMTEP or vehicle and a 2-hour test (Fig. 4a). A two-wayANOVA conducted on this data revealed a significanteffect of Time (F(17,170) = 12.545, P < 0.001) but noeffect of Group (F(1,10) = 0.244, n.s.) and no Group ¥Time interaction (F(1,10) = 0.881, n.s.). Because thecontext-induced relapse and cue-primed reinstatementtests were conducted using a counterbalanced, cross-overdesign all NA core animals had a prior history of bothMTEP and VEH infusions prior to locomotor testing. Toinvestigate whether attenuated extinction learning in thedays subsequent to MTEP infusion into the dSTR was anartifact of increased locomotion, animals were placedinto a locomotor chamber for a 60-minute habituationperiod, followed by intra-dSTR infusion of either MTEP orvehicle and a 2-hour test (Day 1; Fig. 4b). A two-wayANOVA conducted on this data revealed a significanteffect of Time (F(17,170) = 48.826, P < 0.001) but noeffect of Group (F(1,10) = 0.855, n.s.) and no Group ¥Time interaction (F(1,10) = 0.810, n.s.). Locomotoractivity was also recorded for 2 hours (without prior infu-sion) on the 2 days following Day 1 and there were nochanges in locomotion in animals which had receivedMTEP infusions on Day 1 (Fig. 4c,d). A two-way ANOVAconducted on Day 2 data revealed a significant effect ofTime (F(11,110) = 15.953, P < 0.001) but no effect ofGroup (F(1,11) = 0.611, n.s.) and no Group ¥ Timeinteraction (F(1,11) = 1.516, n.s.). A two-way ANOVAconducted on Day 3 data revealed a significant effect ofTime (F(11,110) = 22.125, P < 0.001) but no effect ofGroup (F(1,11) = 0.014, n.s.) and no Group ¥ Timeinteraction (F(1,11) = 0.788, n.s.). A subset of cocaine-and sucrose-self-administering animals with dSTR can-nulae were used to investigate the effects of MTEP onlocomotor activity: the cocaine animals all had a historyof both MTEP and VEH injections; however, the sucroseanimals were only tested once with either MTEP or VEH

prior to locomotor testing. We observed no effect of priortreatment on locomotor activity of the sucrose self-administering animals.

We have previously shown that LTD, a form ofmGluR5-dependent plasticity, cannot be induced inthe NA core of animals with a history of cocaineself-administration and extinction training (Knackstedtet al. 2010). Here, we wanted to investigate whether dis-rupted LTD in the dSTR could account for the effects ofMTEP on extinction learning. LTD was induced in slices

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Figure 4 Locomotion was not affected by MTEP infused into theNA core or the dSTR. Infusion occurred at Time 0 as indicated byarrows. (a) Intra-accumbens MTEP infusion did not affect locomotion(total distance traveled) immediately following infusion. n = 6/group.(b) Intra dSTR MTEP infusion did not affect locomotion immediatelyfollowing infusion or on the days following infusion (c,d).n = 5–7/group



taken from the dSTR following 3 weeks of abstinence fromeither sucrose or cocaine self-administration. Figure 5shows the field amplitudes (normalized to baseline).We found a loss of the ability to induce LTD in animalswith a history of cocaine self-administration (Fig. 5a).A two-way ANOVA revealed significant effects of Time

(F(59,826) = 10.04, P < 0.001), Group (F(1,826) =13.89, P < 0.01) and a significant Time ¥ Group interac-tion (F(59,826) = 4.37, P < 0.001). An independent-sample t-test conducted on the field amplitudes recorded30 to 45 minutes post-LTD induction (normalized tobaseline) revealed a significant difference between

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Figure 5 Cocaine self-administration followed by 3 weeks of abstinence impairs mGluR5-dependent LTD in the dSTR slices. Field amplitudesnormalized to baseline; LTD was induced atTime 0.The left panels show field amplitudes across time while the middle panels show the changein average amplitude during the 30–45 minutes after LTD induction.The right panels show representative traces both pre- (dashed line) andpost-LTD induction (solid line). (a) LTD could be induced only in slices taken from animals with a history of sucrose self-administration butnot in cocaine. (b) Applying MTEP (1 mM) to the recording bath abolished LTD in sucrose animals while having no additional effect in thecocaine group. (c) Treating slices with the mGluR5 PAM VU-29 (1 mM) restored LTD in slices taken from animals with a history of cocaine.n = 3–4/experiment. **=P < 0.01 in comparison to sucrose



sucrose and cocaine self-administering animals (t(1,14) =16.88, P < 0.001; Fig. 5a) indicating a loss of LTD in thecocaine group. Applying MTEP (1 mM) to the recordingbath abolished LTD in sucrose animals while having noadditional effect in the cocaine group (Fig. 5b; Group:F(1,826) = 0.29, n.s.; Time ¥ Group: F(59, 826) = 0.37,n.s.). Thus, after MTEP field amplitudes did not differbetween sucrose and cocaine groups (t(1,14) = 1.761).On the other hand, treating slices with the mGluR5 posi-tive allosteric modulator (PAM) VU-29 (1 mM) restoredLTD in slices taken from animals with a history of cocaineself-administration (Fig. 5c; t(1,12) = 0.1337). Compar-ing the field potentials over time following VU-29 applica-tion, a two-way ANOVA revealed no differences betweengroups (Fig. 5c left panel; Group: F(1,590) = 0.0, n.s.;Time ¥ Group interaction: F(59,590) = 0.4, n.s.).

In order to assess whether a loss of LTD in the dSTRobserved in animals with a cocaine history was due toa loss of mGluR5 availability, surface expression ofmGluR5 receptors was analyzed by biotinylation of slicesobtained from this brain region. Figure 6a (left panel)shows the rat brain atlas coordinates used as guidelinesfor preparation of dSTR slices. A representative immuno-blot analysis of biotinylated slices isolated from the dSTRis depicted in Fig. 6a (right panel). Signal correspondingto mGluR5 was detected in all three (total—T,intracellular—I and surface—S) fractions. Under the lowreducing conditions used in this study (5–10 mM DTT),mGluR5 were detected almost exclusively in a dimer form(~250 kDa, see also Romano, Yang & O’Malley 1996).Since dimerization/oligomerization of G-protein coupledreceptors is an essential step in the generation of ‘mature’functional complexes (Milligan et al. 2006) only dimericform of mGluR5 receptor was analyzed in the presentstudy. Two additional receptors participating in LTDinduction and maintenance, mGluR1 and cannabinoidreceptor 1 (CB1R; for review see Lovinger 2012), werealso detected in all three fractions. Control intracellularproteins b-tubulin and DexRas1 displayed only minimalor no presence in the surface fraction (Fang et al. 2000;Holman & Henley 2007), suggesting that non-specificbiotinylation of intracellular proteins was limited. On theother hand, loading control calnexin and synaptosome-associated protein SNAP-25 were present across all frac-tions in agreement with previous findings (Oyler et al.1989; Okazaki et al. 2000). It should be noted, that whilecalnexin is traditionally believed to be a resident endo-plasmic reticulum protein, it could be recruited to theplasma membrane of neuronal and non-neuronal cells(Chang et al. 1997; Tsujimura et al. 2002; Manganas andTrimmer 2004; Zhang et al. 2010).

Western blot analysis of total and biotinylated frac-tions was conducted on dSTR tissue of rats which hadself-administered cocaine or sucrose and underwent

Figure 6 Cocaine self-administration followed by 3 weeks of absti-nence reduces surface expression of mGluR5 receptors in the dSTR.(a) The rat brain atlas coordinates according to Paxinos & Watson(2007) used to conduct tissue dissections. Representative immuno-blot analysis of biotinylated slices isolated from the dSTR. (b) Theratio of protein density in the surface (biotinylated) fraction to thatin the total fraction was analyzed.We found a significant decrease insurface mGluR5s in cocaine relative to sucrose animals but nochange in mGluR1 or CB1 receptor surface expression. (c) In thetotal fraction, we found no differences in expression of mGluR5-associated proteins Homer 1b/c (H1b/c), calmodulin (CaM) orprotein kinase C isoform e (PKCe). * = P < 0.05 in comparison tosucrose. n = 6–8/group. T—total lysate, I—intracellular fraction,S—surface fraction



3 weeks of abstinence. The ratio of protein density in thesurface (biotinylated) fraction to that in the total fractionwas compared between cocaine and sucrose animals formGluR5, mGluR1 and CB1 receptors. There was a signifi-cant decrease in surface mGluR5 in following cocainerelative to sucrose (t(1,13) = 2.453, P < 0.05; Fig. 6b).Figure 6b also shows no significant differences in surface/total ratio for mGluR1 (t(1,13) = 1.366, n.s.) or CB1(t(1,13) = 0.688, n.s.). In addition, we examined totalfraction for levels of several intracellular proteins previ-ously shown to regulate mGluR5 surface expression(Ango et al. 2002; Lee et al. 2008; Fig. 6c). However,there were no differences in the expression of Homer1b/c (t(1,14) = 0.233, n.s.), calmodulin (CaM, t(1,14) =1.323, n.s.) and e subtype of the protein kinase C (PKCe,t(1,14) = 0.169).

DISCUSSION

Here, we sought to investigate the role of striatal mGluR5receptors in mediating context- and cue-induced relapseto cocaine-seeking after both abstinence and extinction.We used MTEP to target mGluR5 as it has been shown tobe highly selective for mGluR5 (versus mGluR1) and hasnegligible off target effects, as compared to older mGluR5blockers, such as MPEP (Anderson et al. 2002, 2003;Cosford et al. 2003). See et al. (2007) determined thattemporary inactivation of the NA core did not attenuatecontext-induced relapse after abstinence, but had lastingnegative effects on extinction learning. Conversely, theyfound that inactivation of the dSTR attenuated context-induced relapse but had no effect on subsequent extinc-tion learning. We hypothesized that intra-NA core anddSTR infusions of MTEP would provide similar effects onbehavior. Interestingly, we found the opposite. Blockade ofmGluR5 in the NA core significantly attenuated context-induced relapse after abstinence (Fig. 1b), with noobserved change in subsequent extinction learning(Fig. 1c). Intra-NA core MTEP also significantly attenu-ated cue-primed reinstatement after extinction training(Fig. 1d), in agreement with the results of Wang et al.(2013). Conversely, intra-dSTR MTEP had no effect oncontext-induced relapse but did significantly attenuateextinction learning on the days immediately followingMTEP infusion (Fig. 2b,c). There was also no observedeffect of intra-dSTR MTEP on cue-primed reinstatementfollowing extinction training (Fig. 2d). MTEP infused intothe dSTR of rats trained to self-administer sucrose pelletsdid not affect either context-induced relapse after absti-nence (Fig. 3a) or extinction learning following MTEPinfusion (Fig. 3b). These results imply that mGluR5blockade is not sufficient to mimic the effects of inacti-vation of the same brain regions and thus a lack of

signaling through these receptors may not mediate theeffects observed by See et al. (2007). An alternate expla-nation for the discrepancy in results is that See et al.(2007) trained animals to self-administer cocaine in theabsence of discrete cues while we paired both cocaine andsucrose delivery with cues so that we could later test forcue-primed reinstatement. Indeed, two studies havefound that training animals to self-administer in the pres-ence of discrete cues recruits dopamine and glutamatesignaling in the dSTR (Ito et al. 2002; Vanderschuren, DiCiano & Everitt 2005). This supports the idea that ourchoice to train animals to self-administer in the presenceof discrete cues altered the neurocircuitry involved incontext-induced relapse and extinction training. How-ever, further investigation is needed since (1) involvementof the dSTR in drug-cue associative processing clearlydepends on the extent of training (Gabriele & See 2010),and (2) only some (AMPA but not NMDA) glutamatereceptors in the dSTR seem to regulate drug-seeking con-trolled by discrete cues (Vanderschuren et al. 2005). Thepresent study is also limited in that only one dose of MTEPwas infused into both the NA core and dSTR.

MTEP infused into the NA core did not affect locomo-tor activity (Fig. 4a), indicating that the reduction inlever pressing during testing was not due to a generalsuppression of locomotion. Sinclair et al. (2012) showedthat a higher dose of MTEP (3 ug/side) infused in thisbrain region did not affect sucrose-seeking, providingfurther support for the specificity of the effect of mGluR5blockade on the attenuation of cocaine-seeking. It shouldbe noted that systemic MTEP has been found to attenuatethe cue-primed reinstatement of sweetened condensedmilk (Martin-Fardon et al. 2009) but not food pellets(Gass et al. 2009). We also determined that intra-dSTRMTEP infusion did not affect locomotor activity immedi-ately after infusion (Fig. 4b) or 24 and 48 hours post-infusion (Fig. 4c,d). Thus, the attenuation of extinctionlearning (higher levels of lever responding) observed inthe days subsequent to MTEP infusion in the dSTR wasnot a result of an increase in locomotor behavior.

We hypothesized that blocking mGluR5 function withintra-dSTR MTEP impaired synaptic plasticity during thecontext-induced relapse test (Day 1 of extinction) whichprevented animals from consolidating the memory of thenon-reinforced session and thus attenuating extinctionlearning on subsequent days. The role of group I mGluRsin regulating striatal synaptic plasticity is well docu-mented. Either repeated paired-pulse stimulation orshort-term treatment with an mGluR1/5 agonist (such asDHPG) reliably induces cortico-striatal LTD (for reviewsee Lovinger 2012). A number of studies have shownthat chronic non-contingent or contingent cocaineexposure alters LTD in the NA (Thomas et al. 2001;Knackstedt et al. 2010; McCutcheon et al. 2011a; Huang



et al. 2011; Huang & Hsu 2012), but regulation of LTDby cocaine or other drugs of abuse in the dSTR has notbeen investigated. While Centonze et al. (2006) describeddepotentiation of cortico-striatal synapses in the dSTRfollowing non-contingent cocaine administration, thesefindings only suggest that cocaine can block the reversalof LTP rather induce LTD. Here, we show for the first timethat cocaine-self-administration impairs LTD in dSTR,demonstrated as a difference in baseline LTD betweencocaine and sucrose self-administering animals (Fig. 5a).We also found that MTEP only significantly impaired theability to induce LTD in slices from animals whichself-administered sucrose and not cocaine (Fig. 5b), likelybecause MTEP could not further impair LTD in slices fromcocaine self-administering animals. Bath application ofVU-29, a positive allosteric modulator of mGluR5,restored LTD in slices from cocaine self-administeringanimals (Fig. 5c), indicating that the loss of ability toinduce LTD stemmed from impairment of mGluR5 func-tion rather than from a general cocaine-induced disrup-tion of electrophysiological properties of neurons in thedSTR. In this paper, we attribute the impairment ofmGluR5-dependent LTD in the dSTR to a reduced cellsurface expression mGluR5 after cocaine (but no sucrose)self-administration. We have previously shown a similarreduction in surface mGluR5 in the NA core followingextinction from cocaine self-administration which wasaccompanied by an increase in Homer 1b/c expression.Based on work showing that Homer 1 b/c can internalizemGluR5 as well as serve as a component of the signalingscaffold (Ango et al. 2002), we hypothesized that theupregulation in Homer 1b/c levels caused a decrease insurface mGluR5. Here, we did not observe a similarincrease in Homer 1b/c (Fig. 6b), nor did we observe anychanges in several other intracellular proteins previouslydescribed to regulate mGluR5 trafficking such as PKCeand CaM (Fig. 6c, Roche et al. 1999; Lee et al. 2008).However, the mGluR5 intracellular tail interacts with ahost of scaffolding and signaling proteins (Mao et al.2008; Enz 2012), and some these proteins could enhanceor negate Homer-dependent regulation of mGluR5 traf-ficking (Hu et al. 2012). The cellular mechanism regulat-ing mGluR5 surface expression in animals with a cocainehistory will be evaluated in future studies.

mGluR1/5 work in concert with the endocanabinoidsystem to regulate synaptic depression that underliessome forms of learning and memory, including extinc-tion. Thus, systemic administration of a CB1 receptorantagonist or conditional deletion of mGluR5 attenuatesthe extinction of fear conditioning (Marsicano et al.2002; Xu et al. 2009). Blockade of CB1 receptors in thedSTR interferes with the extinction of a striatal-basedresponse-learning task (Rueda-Orozco et al. 2008).Because we found a decrease in surface mGluR5 (Fig. 6b)

accompanying a loss of the ability to induce LTD, wesuggest that the integrity of the mGluR5-dependent LTDin the dSTR is critical for post-cocaine extinction learn-ing. In support, we have previously shown that knock-down of the immediate early gene Arc in the dSTR doesnot alter context-induce relapse, but attenuates extinc-tion learning in a manner very similar to the presentstudy (Hearing et al. 2011). Arc protein is a regulator ofsynaptic and experience-dependent plasticity and is typi-cally activated during mGluR5-dependent LTD events(Waung et al. 2008). Interestingly, there seem to be acritical time period when inhibition of mGluR5 functionexerts lasting effects on extinction learning, as a singleinfusion of MTEP into dSTR attenuated extinction on thefollowing 4 days. As the identity of exact molecularmechanisms remains unknown, we can only speculate tothe causes of the duration of this effect. It has beenshown that in addition to LTD, stimulation of striatalmGluR1/5 receptors produces an insertion of GluR2-containing AMPA receptors into the membrane(McCutcheon et al. 2011a). Perhaps a long-lasting loss ofmGluR5-AMPA functional synergism contributes to apersistent behavioral impairment.

The electrophysiological data also indicate thatMTEP infusion into the dSTR likely impaired LTD insucrose animals during the context-induced relapse test,and yet this group exhibited normal extinction learningon subsequent days (Fig. 3b). Thus, a loss of LTD in thisregion is not the sole cause for the attenuation ofextinction learning observed in animals with a historyof cocaine self-administration. Instead, it is possible thatanimals with a history of cocaine self-administrationpossess deficits in additional brain regions involvedin extinction learning, while those regions remain‘healthy’ in sucrose animals. For example, the infram-limbic cortex, NA shell and basolateral amygdala areregions which regulate extinction learning followingboth drug self-administration and fear conditioning(Peters et al. 2008; Peters, Kalivas & Quirk 2009). It ispossible that cocaine induces pathologies in theseregions which then result in the dSTR exerting morecontrol over extinction learning than in sucrose animalswhich have intact extinction learning circuitry (forreview see Kalivas 2008). Importantly, the inability torecruit other circuits to compensate for striatal dysfunc-tion has been also observed in cocaine addicts (Hanlon,Wesley & Porrino 2009).

The context-primed relapse test likely engages extinc-tion learning as it is the first occasion on which animalsare placed into the self-administration environment andpresses on the previously active lever are not reinforced.Thus, a reduction in responding on the previously rein-forced lever during that test could be viewed as facilitatedextinction learning and account for the reduction in lever



presses following MTEP infusion into the NA core(Fig. 1b). However, that is unlikely to have occurred fol-lowing MTEP infusion, as it is positive, not negative, allos-teric modulation of mGluR5 that has repeatedly beenshown to enhance multiple types of learning, includingextinction learning (Ayala et al. 2009; Gass & Olive2009a; Uslaner et al. 2009; Kufahl et al. 2012).

In conclusion, the present study suggests an impor-tant role for NA core mGluR5 in relapse followingboth abstinence and extinction training and for dSTRmGluR5 receptors in regulating extinction learning. Inrats, the administration of mGluR5 PAMs facilitate theextinction of a cocaine-associated contextual memoryand cocaine-seeking (Gass & Olive 2009a; Cleva et al.2011). Therefore, either pharmacological enhancementof mGluR5 function or increasing population of func-tional (surface) mGluR5 receptors may represent a novelapproach toward enhancing extinction learning in thecontext of drug addiction and reducing the risk ofrelapse.

Acknowledgements

The authors thank Rebecca Madell, Phong Do, IlanSondheimer and Stacey Sigmon for excellent technicalassistance. We also thank Dr. Stephen Lanier (MUSC,Charleston, SC) for kindly providing RasD1/AGS1 antise-rum and Dr. Jeffrey Conn (Vanderbilt University, Nashville,TN) for providing VU-29. This work was supported by theNational Institutes of Health Grant R21 DA025846(M.S.), R21 DA026010 (L.A.K), R21 MH080774 (H.L.T-D.), C06 RR015455 and P50 DA015369 (MUSC Neuro-biology of Addiction Research Center Pilot Project, M.S.).

Authors Contribution

MS and LAK were responsible for the study concept anddesign. MS and LAK (with the assistance of technicalpersonnel acknowledged above) conducted behavioralexperiments, as well as intracranial drug administrationand surface expression experiments. HLT-D performedelectrophysiological experiments. LAK, HLT-D and MSanalyzed the data. MS and LAK provided funds to supportanimal studies and wrote the manuscript. All authorshave critically reviewed content and approved finalversion submitted for publication.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:

Figure S1 Active lever pressing on Day 2 of extinctiontraining relative to Day 1 (context-induced relapse test).Mean number of active lever presses in Veh- and MTEP-treated groups expressed as a ratio of Day 2 to Day 1 inanimals with NAc or dSTR infusion. * = P < 0.05 in com-parison to dSTR vehicle. n = 4–12/group



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