Synaptic depression via mglur1 positive allosteric modulation suppresses cue-induced cocaine craving

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1 Synptic depression vi mglur1 positive llosteric modultion suppresses cue-induced cocine crving Jessic A Loweth 1, Andrew F Scheyer 1,2, Mike Milovnovic 1, Amber L LCrosse 3, Eden Flores-Brrer 2, Crig T Werner 1, Xun Li 1, Kerstin A Ford 1,2, Tun Le 1, M Foster Olive 3, Kren K Szumlinski 4, Kuei Y Tseng 2,5 & Mrin E Wolf 1,5 npg 14 Nture Americ, Inc. All rights reserved. Cue-induced cocine crving is mjor cuse of relpse in bstinent ddicts. In rts, cue-induced crving progressively intensifies (incubtes) during withdrwl from extended-ccess cocine self-dministrtion. After ~1 month of withdrwl, incubted crving is medited by C 2+ -permeble AMPA receptors (CP-AMPARs) tht ccumulte in the nucleus ccumbens (NAc). We found tht decresed mglur1 surfce expression in the NAc preceded nd enbled CP-AMPAR ccumultion. Thus, restoring mglur1 trnsmission by dministering repeted injections of n mglur1 positive llosteric modultor (PAM) prevented CP-AMPAR ccumultion nd incubtion, wheres blocking mglur1 trnsmission t even erlier withdrwl times ccelerted CP-AMPAR ccumultion. In studies conducted fter prolonged withdrwl, when CP-AMPAR levels nd cue-induced crving re high, we found tht systemic dministrtion of n mglur1 PAM ttenuted the expression of incubted crving by reducing CP-AMPAR trnsmission in the NAc to control levels. These results suggest strtegy in which recovering ddicts could use systemiclly ctive compound to protect ginst cue-induced relpse. Relpse, which is often triggered by environmentl cues previously ssocited with cocine use, is mjor problem in treting cocine ddiction. Addicts remin vulnerble to relpse long fter the cute withdrwl phse. In rt model of this phenomenon, cueinduced cocine crving progressively intensifies (incubtes) during the first months of withdrwl from extended-ccess cocine selfdministrtion 1. Incubtion models humn scenrio in which hevy drug use is interrupted by hospitliztion or incrcertion 2 nd involves neurodpttions in the circuitry underlying motivtion nd ddiction 1. The NAc is brin region tht hs centrl role in this circuitry nd is comprised minly of medium spiny neurons (MSNs). These MSNs medite motivted behviors by serving s n interfce between corticl nd limbic regions nd the motor circuitry 3. AMPAR trnsmission onto NAc MSNs is criticl for drug-seeking in niml models of cocine ddiction 4. In drug-nive rts nd rts with limited cocine exposure, AMPAR trnsmission in the NAc is medited by GluA2-contining C 2+ -impermeble AMPARs (CI-AMPARs) 5. However, high-conductnce CP-AMPARs ccumulte in NAc synpses during withdrwl from extended-ccess cocine self-dministrtion 6,7. After elevtion of CP-AMPAR trnsmission hs occurred, intr-nac core injection of the selective CP-AMPAR ntgonist nspm mrkedly reduces cue-induced cocine-seeking 6. These results estblish tht CP-AMPAR trnsmission in the NAc medites the expression of incubtion fter prolonged withdrwl. Subsequently, we showed tht the synptic incorportion of CP-AMPARs enhnces the bseline responsiveness of NAc MSNs to glutmte trnsmission 8. Together, these findings suggest tht, when glutmte is relesed in the NAc in response to presenttion of cocine-ssocited cues, MSNs re ble to respond more robustly s result of the presence of CP-AMPARs. This in turn leds to enhnced cocine-seeking. The presence of CP-AMPARs in MSNs of the NAc cn be detected s n elevted rectifiction index for evoked AMPAR-medited excittory postsynptic currents (EPSCs) Using this mesure, we previously found tht CP-AMPAR medited trnsmission becomes elevted fter ~1 month of withdrwl nd endures through t lest withdrwl dy (WD), nd perhps much longer 5. Thus, once bstinence is chieved, the risk of relpse might be decresed if CP- AMPARs were removed from NAc synpses. Here we investigted whether this could be ccomplished by incresing mglur1 trnsmission. This ws inspired by work in other brin regions showing tht mglur1 produces postsynpticlly expressed form of long-term depression (LTD) tht relies on the removl of CP-AMPARs from synpses This form of mglur-ltd hd not been demonstrted in MSNs of drug-nive nimls; insted, synptic depression produced by the group I mglur gonist DHPG depends on mglur5 nd is expressed presynpticlly vi CB1R-medited inhibition of glutmte relese 17,18. However, in NAc slices prepred fter incubtion nd CP-AMPAR ccumultion, we found tht DHPG-induced synptic depression in MSN is ssocited with normliztion of the elevted rectifiction index through n mglur1 nd postsynptic PKC-dependent mechnism 10. This indictes mrked shift in group 1 Deprtment of Neuroscience, Roslind Frnklin University of Medicine nd Science, North Chicgo, Illinois, USA. 2 Deprtment of Cellulr nd Moleculr Phrmcology, Roslind Frnklin University of Medicine nd Science, North Chicgo, Illinois, USA. 3 Deprtment of Psychology, Arizon Stte University, Tempe, Arizon, USA. 4 Deprtment of Psychologicl nd Brin Sciences nd the Neuroscience Reserch Institute, University of Cliforni, Snt Brbr, Cliforni, USA. 5 These uthors contributed eqully to this work. Correspondence should be ddressed to M.E.W. (mrin.wolf@roslindfrnklin.edu). Received 23 August; ccepted 31 October; published online 24 November 13; doi: /nn.3590 nture NEUROSCIENCE VOLUME 17 NUMBER 1 JANUARY 14 73

2 npg 14 Nture Americ, Inc. All rights reserved. I mglur regultion of NAc synptic trnsmission fter incubtion nd points to the potentil utility of mglur1-bsed strtegies for reducing incubted cocine crving. Given these findings, we exmined the effect of intr-nac nd systemic dministrtion of mglur1 PAMs on cue-induced crving fter withdrwl from the extended-ccess cocine self-dministrtion regimen leding to incubtion. We lso conducted mesures of CP-AMPAR trnsmission during withdrwl nd fter tretment with mglur1 PAMs or ntgonists, s well s biochemicl studies of group I mglurs nd Homer signling proteins. We focused on the NAc core becuse this subregion is criticl in cocine-seeking elicited by cues 19 nd, more specificlly, blockde of CP-AMPARs in the NAc core is sufficient to prevent expression of incubtion 6. We found tht mglur1 surfce expression ws decresed during withdrwl, just before the onset of incresed CP-AMPAR levels, suggesting tht mglur1 normlly exerts n inhibitory influence on CP-AMPAR levels, which is lost during incubtion. Supporting this hypothesis, we found tht enhncing mglur1 trnsmission during criticl period of withdrwl, using repeted mglur1 PAM injections, blocked the emergence of enhnced CP-AMPAR trnsmission nd incubtion. Conversely, decresing mglur1 tone in erly withdrwl ccelerted CP-AMPAR ccumultion. Notbly, stimulting mglur1 fter incubtion hd occurred ws sufficient to reduce CP-AMPAR trnsmission to control levels nd ttenute the expression of incubtion. The protective effects of systemiclly dministered mglur1 PAM lsted ~1 d fter single injection nd ~2 3 d fter repeted tretment, suggesting strong trnsltionl potentil. RESULTS mglur1 ctivtion in NAc core ttenutes incubted seeking All of the cocine self-dministrtion groups in this study underwent extended-ccess cocine self-dministrtion trining (6 h d 1 for 10 d; Fig. 1). Compred with limited-ccess cocine self-dministrtion, extended-ccess regimens produce different or enhnced behviors tht more closely model the compulsive drug-seeking nd drug-tking chrcteristic of ddiction. Furthermore, incubtion of cocine crving is most robust fter extended-ccess regimens, nd withdrwl-ssocited CP-AMPAR ccumultion occurs fter extended-ccess, but not limitedccess, cocine self-dministrtion 8. We first sked whether enhncing NAc mglur1 function in vivo could promote reduction in CP-AMPAR trnsmission sufficient to prevent the expression of incubted cocine crving. Using rts tht underwent prolonged withdrwl (>WD45) from the cocine regimen described bove, herefter referred to s incubted rts, we injected DHPG or vehicle into the NAc core just before test for cue-induced cocine-seeking (cnnule plcements shown in Supplementry Fig. 1). During the seeking test, nose poking in the ctive (previously ssocited with cocine nd cue delivery) only delivered the cue. Thus, responding under these conditions provides mesure of cue-induced crving. As predicted from DHPG s bility to ttenute CP-AMPAR medited trnsmission in vitro 10, incubted rts tht received intr-nac DHPG infusion exhibited significntly reduced cocine-seeking compred with vehicle-infused rts (t 25 = 2.35, P = 0.03; Fig. 1b). The sme effect ws produced by intr-nac infusion of the selective mglur1 PAM Ro (t 11 = 1.95, P = 0.04; Fig. 1c). Neither drug ltered inctive responding during the seeking test (Fig. 1b,c) or self-dministrtion of nturl rewrd (sucrose; Supplementry Fig. 2), indicting tht their effects on cocine-seeking were not ttributble to nonspecific motor depression or to generlized decrese in motivtion to seek rewrd. 0 Infusions (6 h) Trining dy b Nose pokes ( min) DHPG Active Inctive c Ro Systemic ttenutes seeking nd CP-AMPAR function If mglur1 PAMs re to be useful therpeutics, they must exert nticrving effects fter systemic dministrtion. Thus, we evluted, selective mglur1 PAM tht is behviorlly ctive fter systemic injection 21. We first confirmed tht reduced cueinduced cocine-seeking fter infusion into the NAc of incubted rts (t 14 = 2.17, P = 0.048; Fig. 1d). We then conducted MSN recordings in NAc brin slices obtined from incubted rts nd found tht bth ppliction of normlized the elevted rectifiction index of AMPAR trnsmission (exposure tretment interction: F 1,8 = 18.04, P = 0.003; Fig. 2) nd reduced EPSC 70 mv mplitude by ~25% (t 5 = 4.67, P = 0.01 versus bseline, n = 6 cells from 5 rts; dt not shown). This reduction ws result of the elimintion of CP-AMPAR medited trnsmission, s bth ppliction of the CP-AMPAR ntgonist nspm produced no further synptic depression (Fig. 2b). In the bsence of, nspm reduced EPSC 70 mv mplitude by ~30% in MSNs from incubted rts (Fig. 2b). Furthermore, nspm-induced ttenution of EPSC 70 mv mplitude ws not ltered by the presence of kinte receptor ntgonist (Supplementry Fig. 3). Next, we ssessed the effect of systemic on cocine crving by dministering single injection of or vehicle (intrperitonel) to incubted rts just before test for cue-induced cocineseeking. Reltive to the vehicle-injected group, cocine-seeking ws significntly blunted by systemic injection of (withdrwl dy tretment interction: F 1,32 = 6.21, P = 0.02; Fig. 2c). As with DHPG nd Ro , this ws not nonspecific effect ttributble to depression of motor ctivity, s there ws no group difference in inctive responding during the seeking test (Supplementry Fig. 4). Furthermore, s effect ws specific to cocine-seeking, s rts tested fter 10 d of sucrose self-dministrtion nd 30 d of withdrwl showed incubted cue-induced sucrose seeking tht ws not reduced by injection (Supplementry Fig. 5). To determine whether the reduction in cocine-seeking fter intrperitonel injection ws ttributble to reduced CP- AMPAR medited trnsmission, we performed recordings in slices prepred from subset of - nd vehicle-treted rts from the cocine experiment (Fig. 2d). As expected, incubted rts injected with vehicle before the seeking test exhibited n elevted rectifiction Active Inctive d Active Inctive Figure 1 Incubted cocine-seeking is ttenuted by intr-nac infusion of DHPG or mglur1 PAMs. () Trining. Nose pokes (men ± s.e.m.) in the ctive result in cocine infusion (0.5 mg per kg) pired with light cue. (b d) Seeking tests on >WD45. Shown re nose pokes (men ± s.e.m.) in the previously ctive ( mesure of cocine-seeking) nd inctive during -min test performed under extinction conditions (nose pokes deliver cue, but not cocine). Infusion of DHPG (500 µm; control, n = 16 rts; DHPG, n = 11 rts; b), Ro (10 µm; control, n = 7 rts; Ro , n = 6 rts; c) or (10 µm; control, n = 7 rts;, n = 9 rts; d) into the NAc core 10 min before the test decresed seeking compred with vehicle-infused controls (P < 0.05). Seeking ws lso significntly reduced by lower dose of DHPG (250 µm; 41% reduction, t 13 = 2.23, P = 0.04 versus vehicle; control, n = 8 rts; DHPG, n = 7 rts; dt not shown). 74 VOLUME 17 NUMBER 1 JANUARY 14 nture NEUROSCIENCE

3 npg 14 Nture Americ, Inc. All rights reserved. Figure 2 A single systemic injection of the mglur1 PAM reduces cocine-seeking nd CP-AMPAR function. () Left, following prolonged withdrwl from cocine self-dministrtion (>WD45), MSNs exhibited n elevted rectifiction index (men ± s.e.m.) tht ws normlized by bth-pplied (SYN, 1 µm, 15 min). P < versus bseline; control, n = 4 cells from 4 rts;, n = 6 cells from 5 rts. Right, exmple trces illustrte the effects of in MSNs from controls nd incubted rts (clibrtion br = 50 pa, ms). The reversl potentils for both exmples re +10 mv. Overll, the reversl potentils did not differ significntly between sline nd cocine groups (P = 0.10). (b) Nspm ( µm) significntly reduced the rectifiction index in the bsence of (t 7 = 9.15, P < 0.001; bseline versus lst 5 min of nspm ppliction), but this ws not observed in the presence of, indicting tht s bility to normlize the rectifiction index ws result of the elimintion of CP-AMPAR trnsmission (control, n = 8 cells from 5 rts;, n = 5 cells from 4 rts). CSF, rtificil cerebrospinl fluid. (c) Nose pokes (men ± s.e.m.) in the previously ctive during seeking tests on WD1 nd >WD45. Injection of (10 mg per kg, intrperitonel) min before the second test significntly reduced seeking compred with vehicle-injected controls (P = 0.048; control, n = 13 rts;, n = 21 rts), lthough this reduction ws prtil, s WD1 nd WD45 differed significntly in the group (P = 0.02). (d) Left, recordings from subset of these rts reveled tht normlized the rectifiction index (men ± s.e.m.) for ~24 h. These recordings were performed immeditely fter the test (dy 0), 1 d lter (dy 1) or 2 d lter (dy 2). Right, representtive trces of EPSC 70 mv, EPSC + mv nd the reversl potentil (in gry, 0 mv) for cells from vehicle, dy 0 (D0) nd dy 2 (D2) groups (clibrtion brs = 50 pa, ms). Overll, the reversl potentils did not differ significntly between vehicle- nd -treted groups (P = 0.16). P < versus vehicle group; vehicle, n = 11 cells from 3 rts; D0, n = 12 cells from 3 rts; D1, n = 12 cells from 3 rts; D2, n = 4 cells from 2 rts. index regrdless of whether recordings were performed immeditely fter the test (dy 0), 1 d lter (dy 1) or 2 d lter (dy 2). In contrst, -injected rts recorded the dy of the seeking test (dy 0) exhibited normlized rectifiction index (Fig. 2d) resembling tht observed in sline-treted rts (Fig. 2). The prtil nture of s effect on seeking on dy 0 (Fig. 2c), in contrst with the complete reversl of the elevted rectifiction index, could indicte tht NAc synpses re strengthened not only by postsynptic CP- AMPAR ccumultion, but lso by presynptic dpttions 22 tht b Percentge sline Percentge sline Infusions (6 h) 50 Sline mglur1: surfce mglur5: surfce Trining dy mglur1: totl Sline mglur5: totl c Nose pokes (30 min) 1.8 Sline Bseline SYN WD1 WD45 Sline + mv 70 mv + mv 70 mv d Bseline 1.8 SYN Dys post-injection CSF 1.5 CSF or Nspm + mv 70 mv D0 D2 would not be ffected by. The rectifiction index mesured in -injected rts remined significntly ttenuted on dy 1, but returned to the elevted level chrcteristic of incubtion by dy 2 (group effect: F 3,35 = 15.44, P < 0.001; Fig. 2d). Thus, single systemic injection of inhibited CP-AMPAR medited trnsmission in NAc synpses for ~24 h. Decresed mglur1 precedes CP-AMPAR ccumultion Given tht mglur1 ctivtion inhibited CP-AMPAR function nd thereby reduced crving (Figs. 1 nd 2), we hypothesized tht mglur1 normlly exerts brking effect on CP-AMPAR ccumultion in the NAc. This would explin why CP-AMPARs ccount for only 5 10% of excittory trnsmission in NAc MSNs from dult, drug-nive rts 6,23. A similr negtive regultory role for bsl mglur1 trnsmission hs been described in other regions 7,15,24. We further hypothesized tht, during withdrwl from extended-ccess cocine self-dministrtion, decrese in NAc mglur1 trnsmission my occur tht removes this brke, permitting CP-AMPARs to ccumulte. One wy tht this could occur is through decrese in mglur1 surfce expression. To test this possibility, we used biotinyltion to mesure mglur1 surfce nd totl protein levels during withdrwl from extendedccess cocine or sline self-dministrtion. Given the time course of CP-AMPAR ccumultion during withdrwl 5, we selected three time points for study: WD14 (before ny chnge in AMPAR subunit b Normlized EPSC Minutes Figure 3 A withdrwl-dependent decrese in NAc mglur1 surfce expression just precedes CP-AMPAR ccumultion. () Trining dt for sline nd cocine groups (trining ws s described in Fig. 1). (b) Cell surfce nd totl protein levels of mglur1 nd mglur5 were mesured on three withdrwl dys: before (WD14), t the onset (WD25) nd fter (WD48) elevtion of CP-AMPAR levels. Dt re expressed s percent of sline group (±s.e.m.) t ech time point (n vlues re provided in ech br). P = 0.04 (surfce mglur1, WD25), P = 0.03 (surfce mglur1, WD48; surfce mglur5, WD48) nd P = 0.03 (totl mglur1, WD48; t 18 = 2.0) versus respective sline groups (P < 0.05). Differences in opticl density for sline groups on different withdrwl dys reflect differences in exposure time for different blots rther thn timedependent chnges in protein levels fter sline self-dministrtion. Full-length blots re presented in Supplementry Figure 9. nture NEUROSCIENCE VOLUME 17 NUMBER 1 JANUARY 14 75

4 Figure 4 Prolonged withdrwl from extendedccess cocine self-dministrtion does not lter mglur1-homer ssocitions, but does decrese mglur5-homer ssocitions in the NAc. () Co-immunoprecipittion experiments ssessing the physicl ssocitions between mglur1 nd Homer proteins on WD14, WD25 nd WD48 from extended-ccess cocine (coc) or sline (sl) self-dministrtion. No chnges in ssocition between mglur1 nd Homer proteins were observed t ny of the three withdrwl time points. IB, immunoblot; IP, immunoprecipittion. (b) In the cse of mglur5, no chnge in ssocition with Homer proteins ws observed t the two erlier withdrwl time points (WD14 nd WD25). However, significnt decrese in ssocition between mglur5 nd IP: mglur1 sl coc sl coc sl coc IB: mglur1 b IB: Homer1bc IB: Homer2 IP: mglur5 sl coc sl coc sl coc IB: mglur5 IB: Homer1bc IB: Homer2 both Homer isoforms ws found on WD48 in rts tht previously self-dministered cocine. Dt re expressed s percent of sline group (±s.e.m.) t ech time point (n vlues re provided in ech br). P = 0.01 (Homer1bc) nd P = 0.04 (Homer2) versus respective sline groups. Full-length blots re presented in Supplementry Figure 9. Percentge sline Percentge sline Sline IB: Homer1bc IB: Homer1bc IB: Homer IB: Homer npg 14 Nture Americ, Inc. All rights reserved. composition), WD25 (when the rectifiction index is just beginning to increse) nd WD48 (when CP-AMPARs re stbly expressed). Although the sline nd cocine groups did not differ on WD14, the cocine group exhibited decresed mglur1 surfce expression on WD25 (t 13 = 2.24, P = 0.04) nd WD48 (t 15 = 1.99, P = 0.03) (Fig. 3 nd Supplementry Fig. 6), suggesting tht persistently decresed mglur1 surfce expression during cocine withdrwl my contribute to CP-AMPAR ccumultion. We speculte tht the remining mglur1 receptors, lthough insufficient to mintin norml inhibitory tone on CP-AMPAR ccumultion, cn be stimulted phrmcologiclly when mglur1-ctivting drugs (such s ) re pplied to brin slice (Fig. 2) or injected directly into the NAc of n incubted rt (Fig. 1). We next exmined whether ltertions in Homer protein expression or function might further contribute to depressed mglur1 trnsmission during withdrwl. The Homer postsynptic scffolding proteins re criticl for group I mglur signling, regulting their ssocition with signling pthwys 25. Homer proteins re the product of three genes (Homer1, Homer2 nd Homer3) tht encode severl trnscriptionl vrints. Most re long forms tht re constitutively expressed (Homer1b d, Homer2b, Homer3), but two re truncted forms (Homer1, ni-3) tht re induced by synptic ctivity. In generl, the ssocition of long Homers with group I mglurs fvors receptor coupling to IP 3 receptor dependent pthwys, wheres short Homers disble this coupling, fvoring regultion of G protein dependent ion chnnels In the NAc, mglur1 ppers to elicit CP-AMPAR removl through the cnonicl pthwy linked to PLC ctivtion, IP 3 formtion, C 2+ relese nd PKC ctivtion 10. Thus, impired signling through the long Homers tht promote this pthwy could contribute, long with decresed mglur1 surfce expression, to reduced group I mglur signling during incubtion. Indeed, there is evidence for decresed long Homer protein levels in the NAc fter cocine exposure 25, including decreses in Homer1bc nd Homer 2 expression fter extended-ccess cocine self-dministrtion 30. However, when we ssessed Homer1bc nd Homer 2 expression during cocine withdrwl (WD14, WD25 nd WD48), we found no chnges in totl protein levels of these Homers (Supplementry Fig. 7) or their physicl ssocition with mglur1, s determined by coimmunoprecipittion (Fig. 4 nd Supplementry Fig. 8). Together, these results suggest tht CP-AMPAR ccumultion is likely result of the observed decreses in mglur1 surfce expression rther thn deficits in Homer signling. In light of our previous finding tht mglur5-medited synptic depression is disbled in the NAc of incubted rts 10, it ws lso of interest 50 b 250 Nose pokes (30 min) c WD1 WD Dys fter lst injection + mv 70 mv Percentge chnge (nose pokes) Dys fter lst injection D2 D4 5 Figure 5 Repeted injections during withdrwl interfere with CP-AMPAR ccumultion nd block incubtion of cocine crving. () Rts received vehicle or injections every other dy from WD15 through WD33. Repeted injections blocked incubtion of cocine crving. Dt re presented s the verge number of nose pokes (±s.e.m.) during seeking tests on WD1 nd WD35 (P = versus WD1; n = 13 rts per group). (b,c) To determine the durtion of -medited ttenution of incubtion nd its mechnism, seeking tests nd ptch-clmp recordings were conducted t vrible times fter the lst vehicle or injection. -medited reduction of cocine-seeking (dt expressed s percent chnge from WD1 test, men ± s.e.m.; b) nd the rectifiction index (men ± s.e.m., c, left) persisted for 2 3 d. Representtive trces re shown (c, right) of EPSC 70 mv, EPSC + mv nd the reversl potentil (in gry, +5 mv) for cells from repeted vehicle, dy 2 (D2) nd dy 4 5 (D4 5) groups (clibrtion brs = 50 pa, ms). Overll, the reversl potentils did not differ significntly between vehicle- nd SYN-treted groups (P = 0.49). P = 0.002, P < versus vehicle controls; repeted vehicle, n = 6 cells from 2 rts; D2, n = 4 cells from 2 rts; D3, n = 4 cells from 2 rts; D4 5, n = 6 cells from 3 rts. 76 VOLUME 17 NUMBER 1 JANUARY 14 nture neuroscience

5 npg 14 Nture Americ, Inc. All rights reserved. Figure 6 Correltions between effects of on rectifiction index, nspm sensitivity nd cue-induced cocine-seeking. () Anlysis of chnges in rectifiction index (from Fig. 5, men ± s.e.m.) following repeted tretment. (b) To further ssess the contribution of CP-AMPARs to the chnges in rectifiction index shown in, the CP-AMPAR ntgonist nspm ( µm) ws bth-pplied for 15 min nd EPSC mplitude (men ± s.e.m.) ws mesured t holding potentil of 70 mv (EPSC 70 mv, dt re normlized to pre-nspm bseline; left). Right, s expected, nspm decresed EPSC 70 mv by ~30% in cocine rts tht received repeted vehicle injections. In contrst, nspm sensitivity (men ± s.e.m.) ws significntly reduced in rts recorded 2 3 d (D2 or D3) fter the lst injection, time t which the rectifiction index ws lso reduced ( nd Fig. 5c) nd cue-induced cocine-seeking ws below incubted levels (Fig. 5b). By 4 5 d (D4 or D5) fter the lst injection, the rectifiction index nd nspm sensitivity hd returned to levels tht were more similr to those observed in cocine/vehicle (Coc/Veh) rts (,b), s hd cue-induced cocineseeking (Fig. 5b). P < 0.001, P = 0.003, P = 0.04 versus Coc/Veh; Coc/Veh, n = 5 cells from 2 rts; D2, n = 4 cells from 2 rts; D3, n = 4 cells from 2 rts; D4 5, n = 6 cells from 3 rts. (c) Anlyses of correltions between cocine-seeking (mesured s percent chnge from ctive responses on WD1), rectifiction index nd nspm sensitivity for ll rts shown in,b nd Figure 5b,c. Left, s expected, nspm sensitivity ws highly correlted with rectifiction index vlues, indicting tht n elevted rectifiction index is result of enhnced CP-AMPAR medited trnsmission. Right nd middle, in ddition, cocine-seeking correlted with both rectifiction index nd nspm sensitivity. to ssess mglur5 levels nd Homer coupling. We observed smll, but significnt, decrese in surfce mglur5 (t 17 = 1.96, P = 0.03; Fig. 3b) nd significnt decrese in the ssocition between mglur5 nd both Homer1bc (t 17 = 2.75, P = 0.01) nd Homer2 (t 17 = 1.91, P = 0.04) on WD48 (Fig. 4b). We note tht interprettion of the ltter result is complex becuse uncoupling of Homers nd group I mglurs cn both enhnce nd reduce signling 31. The level of mglur1 trnsmission regultes incubtion Given tht surfce expression of mglur1 decresed t pproximtely the sme withdrwl time s CP-AMPARs begn to ccumulte b Normlized EPSC Repeted vehicle +50 Repeted JNJ nspm MInutes Repeted vehicle Repeted JNJ + mv 70 mv 15 Percentge chnge 0 50 c Dys fter lst injection b Normlized EPSC r = 0.89 P = r = 8 P = Percentge nspm sensitivity Seeking (% chnge) JNJ Nspm Minutes 15 r = 0.71 P = Seeking (% chnge) 350 (Fig. 3), we wondered whether potentiting mglur1 trnsmission during this criticl period would block CP-AMPAR ccumultion nd inhibit incubtion of cocine crving. Thus, fter completion of cocine self-dministrtion trining, rts were rndomly ssigned to receive vehicle or injections every other dy from WD15 through WD33. These rts were tested for cue-induced cocineseeking on WD1, before vehicle or exposure, nd gin on WD35, 2 d fter the lst injection (Fig. 5). -treted rts exhibited incresed seeking on WD35 compred with WD1, tht is, incubtion occurred s expected (min effect of test dy: F 1,24 = 14.84, P < 0.001). In contrst, rts treted with during withdrwl did not exhibit incubtion of cocine-seeking between WD1 nd WD35, indicting tht enhncing mglur1 trnsmission during cocine withdrwl is sufficient to prevent incubtion (Fig. 5). To determine whether repeted tretment during withdrwl prevents incubtion by opposing CP-AMPAR ccumultion, Percentge chnge 0 Nspm sensitivity (%) Coc/Veh D0 1 Coc/ D2 Coc/ D3 Coc/ D4 5 Figure 7 The onset of CP-AMPAR ccumultion in the NAc is ccelerted by decresing mglur1 tone during erly withdrwl. () Rts selfdministered cocine nd then received vehicle or JNJ (JNJ) injections dily on WD10 15 (2 rts) or WD11 16 (1 rt). Left, in rts treted with vehicle during withdrwl, the rectifiction index on WD16 17 ws similr to sline controls, wheres it ws elevted fter repeted JNJ injections. Ech circle represents one cell. Lines re mens for ech group. Right, representtive trces of EPSC 70 mv, EPSC + mv nd the reversl potentil (in gry, 0 mv) for cells from cocine/vehicle (vehicle) nd cocine/jnj (JNJ) groups (clibrtion brs = 50 pa, ms). Overll, the reversl potentils did not differ significntly between the groups (P = 0.18). P = versus vehicle. (b) Repeted JNJ injections lso incresed nspm sensitivity on WD16 17 compred with the cocine/ vehicle group. Dt re shown s group verge EPSC 70 mv mplitude (±s.e.m.) before (5 min) nd during (15 min) nspm ppliction (left) nd s the verge percentge chnge from bseline during the lst 5 min of nspm ppliction (right; ech circle represents one cell; lines re mens for ech group) P < versus vehicle; vehicle, n = 8 cells from 3 rts; JNJ, n = 7 cells from 3 rts. nture NEUROSCIENCE VOLUME 17 NUMBER 1 JANUARY 14 77

6 Figure 8 Homer overexpression in the NAc core does not ffect incubtion of cue-induced cocine-seeking. () Stining for the hemgglutinin tg on cdna-homer1c verified loclized overexpression in the NAc core ( mgnifiction, imge tken ~3 months fter virus injection; c, nterior commissure). Scle br represents µm. (b) Following ~1 week of recovery from intr-nac AAV injection, rts self-dministered cocine (0.5 mg per kg) for 6 h d 1 for 10 d. No difference in cocine intke (verge infusions obtined ech dy c ±s.e.m.) ws observed between the three groups. (c) All rts showed n increse in cue-induced cocine-seeking (nose pokes, men ± s.e.m.) on WD48 compred with WD1 (tht is, incubtion), regrdless of which virus infusion they received. P < 0.001, P = 0.01, WD1 versus WD48 (ANOVA followed by lest significnt difference post hoc comprisons); GFP, n = 10 rts; Homer1c, n = 8 rts; Homer2, n = 10 rts. b 1 Infusions (6 h) 1 GFP Hom1c Hom Trining dy c 50 Nose pokes (30 min) GFP Hom1c Hom2 Active Test WD1 WD48 GFP Hom1c Hom2 Inctive npg 14 Nture Americ, Inc. All rights reserved. we subjected rts to the sme regimen of repeted or vehicle tretment (every other dy, WD15 33) nd then tested them for cue-induced cocine-seeking t vrible times fter the lst injection (0 5 d) (Fig. 5b). Immeditely fter ech test, slices were prepred for w-cell ptch-clmp recording. After repeted vehicle tretment during withdrwl, rts displyed incubted cocineseeking compred with WD1 (Fig. 5b) nd n elevted rectifiction index (Figs. 5c nd 6), indicting CP-AMPAR ccumultion. Notbly, repeted tretment on WD15 33 blocked incubtion nd reduced the rectifiction index when these endpoints were ssessed 2 3 d fter the lst injection (group effect: F 3,16 = 25.09, P < 0.001; Figs. 5b,c nd 6). This ttenuted rectifiction index ws result of reduced CP-AMPAR medited trnsmission, s nspm sensitivity ws significntly reduced in these rts compred with incubted rts treted with vehicle (group effect: F 3,15 = 11.17, P < 0.001; Fig. 6b). Significnt correltions (ll P vlues < 0.05) were lso observed between levels of cocine-seeking, nspm sensitivity nd CP-AMPAR trnsmission (Fig. 6c). However, 4 5 d fter discontinuing repeted injections, both seeking nd CP-AMPAR levels recovered towrd incubted levels (Figs. 5b,c nd 6,b). Thus, potentition of mglur1 trnsmission during withdrwl, vi repeted systemic injections of n mglur1 PAM, reduced cocine crving by blocking CP-AMPAR ccumultion in the NAc, n effect tht lsted for ~2 3 d. If mglur1 normlly exerts inhibitory tone on CP-AMPAR ccumultion, then decresing mglur1 trnsmission very erly in withdrwl should ccelerte CP-AMPAR ccumultion. To test this, we injected rts tht hd self-dministered cocine with n mglur1 ntgonist (JNJ , 5 mg per kg, intrperitonel) or vehicle dily on WD10 15 (2 rts) or WD11 16 (1 rt) nd crried out ptch-clmp recordings 1 d fter the lst injection (WD16 or WD17). Normlly, this is too erly in withdrwl for detection of incresed CP-AMPAR levels 5. However, fter repeted JNJ injections, recordings on WD16 17 reveled elevtion of both the rectifiction index (F 1,13 = 15., P = 0.002) nd nspm sensitivity (F 1,13 = 52.12, P < 0.001) (tht is, profile identicl to tht of incubted rts recorded fter >WD45; Fig. 7). Prior studies in niml models of cocine nd lcohol ddiction hve shown tht geneticlly ltering long Homer expression in the NAc cn mimic or disrupt drug-induced ltertions in group I mglur medited signling pthwys nd modify behviorl responding to nd/or intke of the drug 25,32,33. Given tht long Homer isoforms cn potentite certin group I mglur medited signling pthwys (see bove), we ssessed whether we could mimic the effect of repeted mglur1 PAM tretment (Fig. 5) by overexpressing long Homers in the NAc. We used previously chrcterized deno-ssocited viruses (AAVs) to overexpress Homer1c, Homer2 or GFP (control) (Fig. 8). We found tht rts in ll three groups demonstrted equivlent cocine selfdministrtion (Fig. 8b) nd incubtion of cocine crving (min effect of test dy, F 1,25 = 47.47, P < 0.001; Fig. 8c). These results indicte tht Homer overexpression does not mimic the effects of repeted mglur1 potentition during withdrwl nd tht enhncing mglur1 trnsmission using PAM is more promising therpeutic strtegy. DISCUSSION We found tht mglur1 receptors in the NAc negtively regulted CP-AMPAR levels nd thereby reduced cue-induced cocine crving. First, we showed tht intr-nac infusions of the group I gonist DHPG or n mglur1 PAM (Ro or ) ttenuted the expression of incubted cue-induced cocine-seeking. Next, in behviorl studies followed by ptch-clmp recordings, we found tht cute systemic dministrtion of reduced the expression of incubted cocine-seeking nd removed CP-AMPAR trnsmission from NAc synpses. This protective effect persisted for ~1 d. Control experiments reveled tht the sme tretment tht reduced incubtion of cocine-seeking hd no effect on incubtion of sucrose seeking. This is consistent with the findings of prior study 34, indicting selectivity of GluA1-relted dpttions for cocine versus sucrose withdrwl. On the bsis of these findings, we hypothesized tht mglur1 normlly exerts inhibitory tone on CP-AMPAR levels in NAc synpses nd tht loss of mglur1 tone during cocine withdrwl enbles CP-AMPAR ccumultion. Indeed, we found tht NAc mglur1 surfce expression ws norml on WD14, but ws decresed by WD25 (tht is, just preceding CP-AMPAR ccumultion), lthough ssocitions with Homer proteins were pprently not ltered. Restoring mglur1 tone during this period by dministering repeted, intermittent injections of blocked CP-AMPAR ccumultion nd the incubtion of cue-induced cocine-seeking, effects tht persisted 2 3 d fter the lst PAM injection. Conversely, phrmcologicl reduction of mglur1 trnsmission during n erly withdrwl period before the decrese in mglur1 surfce expression ccelerted CP-AMPAR ccumultion. Together, these results suggest tht recovering ddict could tke n mglur1 PAM before n nticipted encounter with cocine-ssocited cues nd be fforded resonble period of protection. Although more persistent effect would be desirble, it is reveling tht both CP-AMPARs nd crving recover to incubted levels within dys of discontinuing PAM exposure. This indictes tht mglur1 tone dicttes whether CP-AMPARs nd incubted crving cn emerge, but n s yet unknown underlying mechnism must drive both phenomen. Although suppressed during repeted mglur1 PAM exposure, this mechnism re-engges within dys of discontinuing tretment. In the sme rts tht we used for nlysis of mglur1 expression during withdrwl, mglur5 surfce expression ws unchnged 78 VOLUME 17 NUMBER 1 JANUARY 14 nture neuroscience

7 npg 14 Nture Americ, Inc. All rights reserved. on WD14 nd WD25; however, modest decrese ws observed on WD48, long with decresed ssocition between mglur5 nd long Homer proteins, s mesured by co-immunoprecipittion. These results provide possible explntion for the impirment of mglur5-dependent synptic depression tht we observed in the NAc of incubted rts t these lte withdrwl times 10. There re other reports of ttenuted mglur-ltd in the NAc fter cocine exposure 35 38, but those studies used different cocine regimens nd withdrwl times; thus, our biotinyltion nd co-immunoprecipittion dt re not necessrily pplicble. A lrge number of prior studies hve suggested tht nti-ddictive effects in niml models cn be chieved by blocking group I mglurs (primrily mglur5) 39 rther thn by enhncing mglur1 trnsmission, s we observed here. However, none of these prior studies were conducted in rts tht hd undergone extended-ccess cocine selfdministrtion nd prolonged withdrwl. Both conditions must be met for CP-AMPARs to ccumulte in the NAc of dult rts. Thus, CP-AMPAR ccumultion is not observed, regrdless of withdrwl time, fter experimenter-dministered cocine or limited-ccess cocine self-dministrtion 8,11, or even fter short withdrwls from extended-ccess cocine self-dministrtion 5,6. In the bsence of CP-AMPARs, no nti-ddictive effects of mglur1 PAMs would be nticipted. Insted, mglur1 blockde my be beneficil (for exmple, see ref. ) by tpping into the sme mechnisms chieved with mglur5 blockde. In young nimls, CP-AMPAR plsticity ppers to be induced more redily 7. However, bsed on our results in dult rodents, mglur1 PAM bsed therpies would selectively benefit ddicts who hve chieved bstinence nd seek to sustin it, wheres different pproches would be preferred in other situtions, such s when n ddict is still using or just ttempting to bstin 39,41. We recognize libilities nd mixed clinicl results obtined with previous glutmte-bsed ddiction tretments 41, but mglur1 PAMs my represent n improvement s result of their selectivity for single trget (vis à vis nonselective ion chnnel blockers such s gbpentin) nd durtion of effect (for exmple, cmproste must be tken three times per dy). Although we focused on the effects of mglur1 PAMs medited in the NAc, their effects in the ventrl tegmentl re (VTA) would lso be expected to benefit cocine users. After mny types of cocine exposure (even single injection), CP-AMPARs re inserted into synpses onto VTA dopmine neurons in exchnge for lower conductnce CI-AMPARs, resulting in synptic potentition 13,14,42. This is reversed by cute mglur1 stimultion; tht is, CP-AMPAR trnsmission is decresed nd CI-AMPAR trnsmission is enhnced, resulting in LTD 7, Furthermore, the level of mglur1 tone in the VTA determines the durtion of CP-AMPAR elevtion in the VTA; for exmple, decresing mglur1 tone permits CP-AMPARs to persist for longer time 7. Notbly, mglur1 receptors in the VTA lso regulte CP-AMPAR plsticity downstrem in the NAc. Thus, lthough single cocine injection is not sufficient to increse CP-AMPAR levels in the NAc shell of young mice, it becomes sufficient fter interruption of coupling between VTA group I mglurs nd Homer1bc 7. Combined with our findings, these results predict tht mglur1 PAMs would oppose ddiction-promoting plsticity in the VTA nd NAc of humn cocine users. They would lso hve potentil benefits with regrd to cognitive deficits ssocited with cocine use 43. It is intriguing tht, when CP-AMPARs re present, mglur1 receptors exhibit unique bility to remove them from synpses. This is true not only in the VTA nd NAc (bove), but lso in other brin regions 44. For exmple, mglur1 stimultion results in LTD by incresing CI-AMPAR nd reducing CP-AMPAR synptic trnsmission in cerebellr stellte cells 15. In the lterl mygdl, mglur1-ltd medited by CP-AMPAR removl hs been implicted in the extinction of fer memories 16. The cellulr mechnisms underlying these effects of mglur1 stimultion re best understood in the VTA, where strong evidence supports internliztion of CP-AMPARs nd trnsloction of CI-AMPARs from intrcellulr comprtments to the synpse 14. It is likely tht similr exchnge occurs in the NAc 10, lthough it my not be driven by incresed GluA2 synthesis, s seen in the VTA 5. In stellte cells, lterl diffusion of CI-AMPARs into the synpse my contribute to enhnced CI-AMPAR trnsmission 45. In light of evidence tht D1 receptor nd D2 receptor expressing MSNs in the NAc my contribute differently to cocine-relted behviors 46,47, it my seem surprising tht we observed CP-AMPAR ccumultion in nerly ll MSN in the NAc core of incubted rts (see lso ref. 11). An importnt considertion is tht CP-AMPAR ccumultion in the NAc is first detected ~1 month fter the lst cocine self-dministrtion session. Bsed on this dely, we propose tht CP- AMPAR ccumultion is more likely to reflect withdrwl-dependent chnges in glutmte trnsmission onto MSNs (for exmple, decrese in trnsmission tht triggers homeosttic plsticity) s opposed to differentil ctivtion of D1 nd D2 receptors during the period of cocine self-dministrtion 5. Nevertheless, lthough our recordings estblish tht nerly ll of the MSNs in the NAc core exhibited CP-AMPAR plsticity, it remins to be determined whether CP-AMPARs ccumulte t most spines or whether they re selectively positioned to respond to prticulr fferents. Recently, it ws shown tht CP-AMPAR insertion during incubtion leds to the un-silencing of cocine-generted silent synpses in the mygdl-ccumbens projection 48. It will be importnt to determine whether this occurs in other pthwys nd how mglur1 nd silent synpse bsed reorgniztion interct to shpe excittory synptic trnsmission in the NAc. In conclusion, dministrtion of mglur1 PAMs to incubted rts reduced cue-induced cocine crving by inhibiting CP-AMPAR medited synptic trnsmission in the NAc. Furthermore, repeted mglur1 PAM injections during criticl period of withdrwl opposed the decrese in surfce mglur1 tht normlly occurs, thereby mintining mglur1-medited inhibitory control over CP-AMPAR ccumultion nd delying incubtion. These results suggest tht mglur1 PAMs could be used by recovering ddicts to control cue-induced crving nd prolong bstinence. This hs mjor trnsltionl relevnce, s no tretment is presently vilble tht provides such protection. Methods Methods nd ny ssocited references re vilble in the online version of the pper. Note: Any Supplementry Informtion nd Source Dt files re vilble in the online version of the pper. Acknowledgments This work ws supported by US Public Helth Service grnts DA (M.E.W. nd K.Y.T.), DA (M.E.W.), DA (M.E.W.), DA (M.F.O.), Roslind Frnklin University of Medicine nd Science (K.Y.T.), nd postdoctorl Ntionl Reserch Service Awrd DA (J.A.L.). AUTHOR CONTRIBUTIONS J.A.L., K.Y.T. nd M.E.W. were responsible for overll study design. J.A.L. nd M.M. conducted the biochemicl experiments. A.F.S. conducted the electrophysiologicl experiments, with help from E.F.-B. Surgeries, self-dministrtion trining nd behviorl testing were performed by J.A.L., with help from C.T.W., X.L., K.A.F. nd T.L., except for the incubtion of sucrose crving study, which ws performed by A.L.L. K.K.S. provided virl vectors nd dvice on virl vector experiments. M.F.O. provided, dvised on in vivo experiments, nd designed nture NEUROSCIENCE VOLUME 17 NUMBER 1 JANUARY 14 79

8 npg 14 Nture Americ, Inc. All rights reserved. nd supervised sucrose incubtion studies. J.A.L., A.F.S., K.Y.T. nd M.E.W. nlyzed the dt. J.A.L., K.Y.T. nd M.E.W. wrote the pper. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is vilble online t reprints/index.html. 1. Pickens, C.L. et l. Neurobiology of the incubtion of drug crving. Trends Neurosci. 34, (11). 2. Reichel, C.M. & Bevins, R.A. Forced bstinence model of relpse to study phrmcologicl tretments of substnce use disorder. Curr. Drug Abuse Rev. 2, (09). 3. Sesck, S.R. & Grce, A.A. Cortico-bsl gngli rewrd network: microcircuitry. Neuropsychophrmcology 35, (10). 4. Klivs, P.W. & Volkow, N.D. The neurl bsis of ddiction: pthology of motivtion nd choice. Am. J. Psychitry 162, (05). 5. Wolf, M.E. & Tseng, K.Y. Clcium-permeble AMPA receptors in the VTA nd nucleus ccumbens fter cocine exposure: when, how nd why? Front. Mol. Neurosci. 5, 72 (12). 6. Conrd, K.L. et l. Formtion of ccumbens GluR2-lcking AMPA receptors medites incubtion of cocine crving. Nture 454, (08). 7. Mmeli, M. et l. -evoked synptic plsticity: persistence in the VTA triggers dpttions in the NAc. Nt. Neurosci. 12, (09). 8. Purginto, A. et l. Different dpttions in AMPA receptor trnsmission in the nucleus ccumbens fter short versus long ccess cocine self-dministrtion regimens. Neuropsychophrmcology 38, (13). 9. Ferrrio, C.R. et l. Altertions in AMPA receptor subunits nd TARPs in the rt nucleus ccumbens relted to the formtion of C 2+ -permeble AMPA receptors during the incubtion of cocine crving. Neurophrmcology 61, (11). 10. McCutcheon, J.E. et l. Group I mglur ctivtion reverses cocine-induced ccumultion of clcium-permeble AMPA receptors in nucleus ccumbens synpses vi protein kinse C-dependent mechnism. J. Neurosci. 31, (11). 11. McCutcheon, J.E., Wng, X., Tseng, K.Y., Wolf, M.E. & Mrinelli, M. Clciumpermeble AMPA receptors re present in nucleus ccumbens synpses fter prolonged withdrwl from cocine self-dministrtion but not experimenterdministered cocine. J. Neurosci. 31, (11). 12. Bellone, C. & Lüscher, C. mglurs induce long-term depression in the ventrl tegmentl re tht involves switch of the subunit composition of AMPA receptors. Eur. J. Neurosci. 21, (05). 13. Bellone, C. & Lüscher, C. triggered AMPA receptor redistribution is reversed in vivo by mglur-dependent long-term depression. Nt. Neurosci. 9, (06). 14. Mmeli, M., Bllnd, B., Luján, R. & Lüscher, C. Rpid synthesis nd synptic insertion of GluR2 for mglur-ltd in the ventrl tegmentl re. Science 317, (07). 15. Kelly, L., Frrnt, M. & Cull-Cndy, S.G. Synptic mglur ctivtion drives plsticity of clcium-permeble AMPA receptors. Nt. Neurosci. 12, (09). 16. Clem, R.L. & Hugnir, R.L. Clcium-permeble AMPA receptor dynmics medite fer memory ersure. Science 330, (10). 17. Robbe, D., Kopf, M., Remury, A., Bockert, J. & Mnzoni, O.J. Endogenous cnnbinoids medite long-term synptic depression in the nucleus ccumbens. Proc. Ntl. Acd. Sci. USA 99, (02). 18. Lovinger, D.M. Presynptic modultion by endocnnbinoids. Hndb. Exp. Phrmcol. 184, (08). 19. Milton, A.L. & Everitt, B.J. The persistence of mldptive memory: ddiction, drug memories nd nti-relpse tretments. Neurosci. Biobehv. Rev. 36, (12).. Vnderschuren, L.J. & Ahmed, S.H. Animl studies of ddictive behvior. Cold Spring Hrb. Perspect. Med. 3, (13). 21. Ngomb, R.T. et l. Protective role for type-1 metbotropic glutmte receptors ginst spike nd wve dischrges in the WAG/Rij rt model of bsence epilepsy. Neurophrmcology, (11). 22. Klivs, P.W. The glutmte homeostsis hypothesis of ddiction. Nt. Rev. Neurosci. 10, (09). 23. Reimers, J.M., Milovnovic, M. & Wolf, M.E. Quntittive nlysis of AMPA receptor subunit composition in ddiction-relted brin regions. Brin Res. 1367, (11). 24. Bellone, C., Mmeli, M. & Lüscher, C. In utero exposure to cocine delys postntl synptic mturtion of glutmtergic trnsmission in the VTA. Nt. Neurosci. 14, (11). 25. Szumlinski, K.K., Ary, A.W. & Lominc, K.D. Homers regulte drug-induced neuroplsticity: implictions for ddiction. Biochem. Phrmcol. 75, (08). 26. Xio, B. et l. Homer regultes the ssocition of group 1 metbotropic glutmte receptors with multivlent complexes of homer-relted, synptic proteins. Neuron 21, (1998). 27. Kmmermeier, P.J., Xio, B., Tu, J.C., Worley, P.F. & Iked, S.R. Homer proteins regulte coupling of group I metbotropic glutmte receptors to N-type clcium nd M-type potssium chnnels. J. Neurosci., (00). 28. Kmmermeier, P.J. & Worley, P.F. Homer 1 uncouples metbotropic glutmte receptor 5 from postsynptic effects. Proc. Ntl. Acd. Sci. USA 104, 55 (07). 29. Kmmermeier, P.J. Endogenous Homer proteins regulte metbotropic glutmte receptor signling in neurons. J. Neurosci. 28, (08). 30. Ben-Shhr, O. et l. 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Cognitive effects of Group I metbotropic glutmte receptor lignds in the context of drug ddiction. Eur. J. Phrmcol. 639, (10). 44. Loweth, J.A., Tseng, K.Y. & Wolf, M.E. Using metbotropic glutmte receptors to modulte cocine s synptic nd behviorl effects: mglur1 finds niche. Curr. Opin. Neurobiol. 23, (13). 45. Grdner, S.M. et l. Clcium-permeble AMPA receptor plsticity is medited by subunit-specific interctions with PICK1 nd NSF. Neuron 45, (05). 46. Lobo, M.K. et l. Cell type specific loss of BDNF signling mimics optogenetic control of cocine rewrd. Science 330, (10). 47. Bock, R. et l. Strengthening the ccumbl indirect pthwy promotes resilience to compulsive cocine use. Nt. Neurosci. 16, (13). 48. Lee, B.R. et l. Mturtion of silent synpses in mygdl-ccumbens projection contributes to incubtion of cocine crving. Nt. Neurosci. 16, (13). VOLUME 17 NUMBER 1 JANUARY 14 nture neuroscience