Reversal of morphine-induced cell-type specific blocks ...Reversal of morphine-induced cell-type–specific synaptic plasticity in the nucleus accumbens shell blocks reinstatement

Reversal of morphine-induced cell-type–specificsynaptic plasticity in the nucleus accumbens shellblocks reinstatementMatthew C. Hearinga,b, Jakub Jedynaka,b, Stephanie R. Ebnera,b, Anna Ingebretsona,b, Anders J. Aspa,b,Rachel A. Fischera,b, Clare Schmidta,b, Erin B. Larsona,b, and Mark John Thomasa,b,c,1

aDepartment of Neuroscience, University of Minnesota, Minneapolis, MN 55455; bInstitute for Translational Neuroscience, University of Minnesota,Minneapolis, MN 55455; and cDepartment of Psychology, University of Minnesota, Minneapolis, MN 55455

Edited by Robert C. Malenka, Stanford University School of Medicine, Stanford, CA, and approved November 25, 2015 (received for review September28, 2015)

Drug-evoked plasticity at excitatory synapses on medium spinyneurons (MSNs) of the nucleus accumbens (NAc) drives behavioraladaptations in addiction. MSNs expressing dopamine D1 (D1R-MSN)vs. D2 receptors (D2R-MSN) can exert antagonistic effects in drug-related behaviors, and display distinct alterations in glutamatesignaling following repeated exposure to psychostimulants;however, little is known of cell-type–specific plasticity inducedby opiates. Here, we find that repeated morphine potentiates excit-atory transmission and increases GluA2-lacking AMPA receptorexpression in D1R-MSNs, while reducing signaling in D2-MSNsfollowing 10–14 d of forced abstinence. In vivo reversal of thispathophysiology with optogenetic stimulation of infralimbic cortex-accumbens shell (ILC-NAc shell) inputs or treatment with the an-tibiotic, ceftriaxone, blocked reinstatement of morphine-evokedconditioned place preference. These findings confirm the presenceof overlapping and distinct plasticity produced by classes of abuseddrugs within subpopulations of MSNs that may provide targetablemolecular mechanisms for future pharmacotherapies.

opiates | nucleus accumbens | plasticity | GluA2-lacking AMPARs |ceftriaxone

Opioid-based drugs are mainstays for pain management (1).However, side effects such as euphoria and the develop-

ment of tolerance and dependence contribute to an increasingdiversion of these readily available compounds for nonthera-peutic use (2). Opioid agonist-based treatments are known toreduce some aspects of opioid addiction. On the other hand,these therapies often lead to high relapse rates when discontinuedbecause they fail to eliminate key aspects of addiction such asconditioned associations that can trigger intense drug craving (2).Currently, development of alternative treatments for opioid ad-diction is hampered by a distinct lack of knowledge of the cellularplasticity that underlies persistent opioid-induced changes inbehavior.The nucleus accumbens (NAc) region of the ventral striatum is

involved in attribution of salience to drug-paired cues that can inturn motivate reward-related behavior (3, 4). Medium spinyneurons (MSNs), the principal cells of the NAc, are GABAergicprojection neurons that receive coordinated glutamatergic af-ferents arising from several cortical and limbic brain regions (5,6). MSNs are divided into two subpopulations based on expres-sion of the dopamine receptor 1 (D1R-MSN) or dopamine re-ceptor 2 (D2-MSN), with a small fraction (∼6–17%) expressingboth receptors (7). Importantly, these subpopulations have di-vergent projection targets and exert antagonistic effects in reward-related behaviors (8).Long-lasting alterations in excitatory synaptic strength and

glutamate release at MSNs produced by repeated exposure todrugs of abuse is a driving factor behind drug seeking and relapse(9–11). Numerous studies have examined effects of repeated psy-chostimulant exposure on synaptic strength and AMPA receptor(AMPAR)-mediated transmission in MSN subpopulations, with

a majority of adaptations observed in D1R-MSN (12–14). Althoughopiate-induced changes in extracellular glutamate and glutamatereceptor subunit expression have been reported, very little isknown about opiate-induced changes in the efficacy of synapticstrength and function in the NAc, the degree to which this plasticityis cell-type specific, and the potential role for this plasticity in opiate-induced changes in behavior (15). To address these questions, wemeasured effects of repeated morphine on glutamatergic synaptictransmission in the NAc MSN subpopulations and used optogeneticand pharmacological approaches to determine the role of thispathophysiology in reward-seeking behavior.

ResultsAnatomic and Cell-Type Specificity of Morphine-Induced Adaptationsin NAc MSNs. To assess long-lasting effects of repeated morphineon glutamatergic synaptic transmission in NAc shell MSNs, weused BAC transgenic mice expressing tdTomato or enhancedgreen fluorescent protein (eGFP) in D1R- and D2R-MSNs,respectively. Electrophysiological recordings were primarily per-formed in the dorsal aspects of the rostral shell, a region consideredto be a hedonic “hot spot” for opioid reward (15). D1R-MSNswere identified using a crossover strategy in which we recordedfrom red cells in drd1a-tdtomato mice and nongreen cells in drd2a-eGFP mice (and vice versa for D2R-MSNs). This approachyielded very similar results; thus, data were pooled. Mice receivedfive daily injections of saline or morphine (10 mg/kg), as this

Significance

The recent rise in opioid addiction has made developmentof new treatments a public health priority. The effort has beenimpeded by a distinct lack of understanding how opioid-induced alterations in synaptic transmission and cellular plasticitywithin reward brain regions, such as the nucleus accumbens (NAc),drive addiction behavior. We examined whether repeated mor-phine induces differential alterations in synaptic strength andtransmission in subpopulations of NAc neurons, those expressingdopamine D1 or D2 receptors, that play opposing roles in addic-tion behavior. Morphine enhanced synaptic strength and trans-mission at D1 medium spiny neuron (MSN) synapses and reducedsignaling in D2-MSN. Reversal of this plasticity with in vivooptogenetics or the antibiotic ceftriaxone disrupted the rewardingproperties of morphine, providing a targetable molecular mech-anism for future pharmacotherapies.

Author contributions: M.C.H., J.J., E.B.L., and M.J.T. designed research; M.C.H., J.J., S.R.E.,A.I., A.J.A., R.A.F., and C.S. performed research; M.C.H., J.J., S.R.E., A.I., A.J.A., R.A.F.,and C.S. analyzed data; and M.C.H. and M.J.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1519248113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1519248113 PNAS | January 19, 2016 | vol. 113 | no. 3 | 757–762

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dose produced a maximal increase in AMPAR/NMDA receptor(NMDAR) (A/N) ratios (Fig. S1B) and transmitter releaseprobability (Fig. S1C) in wild-type mice following 10–14 d offorced abstinence. Baseline and morphine-induced motor activ-ity did not differ across wild-type and transgenic mice (Fig. S2).Morphine increased A/N ratios in NAc shell D1R-MSNs (Fig.1B) but not D2R-MSNs (Fig. 1C). To test for morphine-inducedsynaptic AMPAR-specific plasticity, analysis of miniature excit-atory postsynaptic current (mEPSCs) showed that amplitudeand frequency of these events were elevated in D1R-MSNscompared with saline controls (Fig. 1D), whereas frequency, butnot amplitude, was reduced in D2R-MSNs (Fig. 1E). Consistentwith previous work in the NAc core (16), D2R-MSNs exhibitedenhanced mEPSC frequency under basal conditions (saline)compared with D1R-MSNs [D1R-MSN, 4.8 ± 0.54 Hz, n(cells) =11, N(mice) = 6; D2R-MSN, 6.6 ± 0.42 Hz, n = 12, N = 6; t(21) =−2.501, P = 0.02]. These results indicate that morphine-inducedincreases in synaptic strength reflect, in large part, an up-regulationof postsynaptic AMPAR-signaling.

Alterations of synaptic AMPAR subunits can contribute to theexpression of long-term changes in synaptic strength. To test formorphine-induced changes in AMPAR subunit composition, weevaluated current–voltage relationships of evoked EPSCs. Mor-phine reduced the rectification index in D1R-MSNs (Fig. 2A),indicating an increased presence of synaptic GluA2-lackingAMPARs; however, no change was observed in D2R-MSNs (Fig.2B). In addition to postsynaptic receptor adaptations, fluctuationsin synaptic strength can be mediated presynaptically throughchanges in transmitter release. Given the morphine-inducedchanges in mEPSC frequency, paired-pulse (PPR) stimulation wasused to assess changes in glutamate release probability. Morphinereduced ratios in D1R-MSNs (Fig. 2C) and increased ratios inD2R-MSNs (Fig. 2D), suggesting significant increases and de-creases in release probability, respectively.To examine potential region-specific differences in morphine-

induced synaptic plasticity, we measured A/N ratios, mEPSCs,and PPR in D1R- and D2R-MSNs of the NAc core. No signif-icant differences were found in A/N in D1R- or D2R-MSNs (Fig.S3 B and E); however, mEPSC frequency, but not amplitude, wasselectively reduced in D2R-MSNs (Fig. S3 C and F), the latter ofwhich coincided with a trend toward reduced transmitter releaseselectively within D2R-MSNs (Fig. S3G). Because morphine’seffects on NAc synaptic function were more robust and diverse in

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Fig. 1. Morphine-induced changes in NAc shell MSN synaptic strength andAMPAR-signaling. (A) Experimental timeline (Left) including two acclimationdays (H1–H2), 5 d of saline or morphine (10 mg/kg) injections, and 10–14 d offorced abstinence in the home cage. Electrophysiological recordings wereperformed in sagittal slices (Right) containing NAc shell D1-MSN (B and D)and D2-MSNs (C and E) following the 10- to 14-d abstinence period. (B andC) Representative AMPAR (red, green) and NMDAR (black) EPSCs at +40 mV(Left) and mean AMPAR/NMDAR (A/N) ratio values (Right) in D1R-MSN [B,t(14) = −5.072, P < 0.001, n = 8 per group, N = 6–7 per group] and D2R-MSN[C, t(14) = 1.618, P = 0.128, n = 8 per group, N = 5–7 per group] from saline(Sal, outlined) and morphine (Mor, filled) exposed mice. (Scale bar, 50 pA/50ms.) (D and E) Representative miniature EPSCs (Upper) and mean mEPSCamplitude (Left) and frequency (Right) in D1R-MSN [D, amplitude: t(30) =−3.773, P < 0.001; frequency: t(30) = −3.384, P = 0.002; n = 12–20 per group,N = 6–10 per group] and D2R-MSN [amplitude: t(13) = 1.072, P = 0.303; fre-quency: t(13) = 2.721, P = 0.017; n = 7–8 per group, N = 4–5 per group] fromsaline and morphine exposed mice. (Scale bar, 20 pA/50 ms.) *P < 0.05, **P <0.01, ***P < 0.001 vs. Sal.

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Fig. 2. Morphine-induced changes NAc shell MSN AMPAR subunit compo-sition and neurotransmitter release. (A and B) Examples of normalized EPSCamplitudes at +40, 0, and −80 mV (Upper), with mean I–V plots (Lower) andrectification index (RI) at +40 mV (Inset) in D1R-MSNs [A, t(11) = 3.460, P =0.005; n = 6–7 per group, N = 5 per group] and D2R-MSNs [B, t(8) = −0.579,P = 0.578; n = 4–6 per group, N = 4–6 per group] from saline- and morphine-exposed mice. (Scale bar, 50 pA/25 ms.) (C and D) Representative paired-pulseevoked EPSCs (50 ms; Upper) and mean ratios at 20- to 200-ms interstimulusintervals (Lower) in D1R- (C) and D2R-MSNs (D). A significant main effect oftreatment was observed in both D1R-MSNs [F(1,47) = 15.64, P = 0.003; n = 5–6 pergroup, N = 5 per group] and D2R-MSNs [D, F(1,55) = 5.89, P = 0.032; n = 6–8 pergroup, N = 5–8 per group]. (Scale bar, 60 pA/25 ms.) *P < 0.05, **P < 0.01 vs. Sal.

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the NAc shell, we focused our analysis and manipulation in sub-sequent experiments on this subdivision.

Reversal of Morphine-Induced Plasticity by the Antibiotic Ceftriaxone.Opioid self-administration down-regulates expression of NAcglial excitatory amino acid transporter 2 (GLT-1) expression, atransporter responsible for 90% of glutamate uptake, and thiseffect increases vulnerability to relapse. Conversely, compoundsthat increase GLT-1 expression, such as the antibiotic ceftriax-one, reduce cocaine and heroin seeking (17, 18). Although theselatter effects have been attributed to normalization of extracel-lular glutamate levels following cocaine seeking, the effect ofceftriaxone on opioid-induced changes in NAc MSN synapticstrength and function remains unclear. To test the ability ofceftriaxone to normalize morphine-induced changes in excitatorysignaling, we treated mice with saline or ceftriaxone for 7–10d following five daily injections of saline or morphine exposure,and measured excitatory synaptic parameters in the NAc shell.Ceftriaxone restored morphine-induced increases in D1R-MSNA/N ratios (Fig. S4A) as well as mEPSC amplitude and fre-quency to control levels (Fig. 2A). Initial assessments of mEPSCsdemonstrated that amplitude and frequency were not altered inD1R- or D2R-MSNs from saline mice receiving ceftriaxone vs.vehicle (Fig. 2 B and C); thus, data were pooled. Unexpectedly,ceftriaxone treatment also normalized morphine-induced reduc-tions in D2R-MSN mEPSC frequency and potentiated mEPSCamplitude compared with vehicle-treated morphine mice andsaline controls (Fig. 3B). Evaluation of paired-pulse EPSCs inD1R- and D2R-MSNs showed that glutamate release probabilitywas restored to saline control levels in ceftriaxone-treated mor-phine mice, indicating that ceftriaxone normalizes morphine-induced alterations in glutamate release probability at both celltypes (Fig. 2 C and D).We next examined effects of ceftriaxone on the morphine-

induced increase in synaptic GluA2-lacking AMPARs in D1R-MSNs. Both the morphine-induced inward rectification of synapticAMPARs (Fig. 3 E and F) and the increased sensitivity of AMPAREPSCs to bath application of 1-naphthylacetylsperimine (Naspm),a selective antagonist of GluA2-lacking AMPARs, were blocked(Fig. 3 F–H), indicating that ceftriaxone treatment during ab-stinence restores expression of synaptic GluA2-lacking AMPARsto control levels. To test whether normalizing plasticity followingceftriaxone treatment alters reward behavior, mice underwentmorphine place preference conditioning (CPP) followed byvehicle or ceftriaxone treatment during extinction. In these ex-periments, all mice were conditioned with four daily injections ofmorphine (5 mg/kg). This treatment regimen produced robustplace preference and increased AMPAR plasticity in D1R-MSNsfollowing 10–14 d of abstinence only, similar to that previouslyobserved with five daily injections of 10 mg/kg morphine (Fig. 4 Aand B). Although ceftriaxone did not alter extinction of preference(Fig. S5), morphine-induced reinstatement of CPP was blocked inceftriaxone-treated morphine animals compared with vehicletreated controls (Fig. 4D).

Optically Induced LTD of ILC-NAc Shell Synapses Blocks Reinstatement.Ceftriaxone-dependent reversal of synaptic potentiation in D1R-MSNs and subsequent blockade of reinstatement identifies thisplasticity as a potential mechanism underlying the expression ofconditioned reward, a key component of opioid addiction. How-ever, an essential step in determining the functional role of opiateplasticity is elucidating the selectivity of these adaptations forspecific afferent populations. Pharmacological studies have im-plicated the infralimbic cortex-accumbens (ILC-NAc) shell path-way in the expression of opiate-induced reinstatement of drugseeking (19, 20). The extent to which this pathway is subject tomorphine-induced plasticity, and whether reversal of this plasticityprior to morphine reexposure prevents reinstatement behavior, animportant goal in addiction research, is unknown. Thus, to testwhether morphine-evoked potentiation in D1R-MSNs occurswithin ILC-NAc shell projections and whether this plasticity is

causally involved in addiction behavior, we used a CPPmodel wheremice were injected with a channelrhodopsin (ChR2)-expressingadeno-associated virus in the ILC and implanted with optical fibersinto the NAc shell, to allow selective stimulation of this major ex-citatory input (Fig. 5). Following extinction, mice were stimulatedwith light or received no stimulation 4 h before reinstatementtesting. In agreement with electrically evoked long-term depression(LTD) protocols (15), stimulation at 10 Hz for 10 min reducedoptically evoked A/N ratios in D1-MSN from morphine-injectedmice receiving saline during reinstatement testing vs. nonstimulatedmice (Fig. 5D). Although morphine increased motor activity com-pared with saline controls, no effect of optical stimulation in eithermorphine or saline reinstated mice was observed (Fig. S6A).

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Fig. 3. Ceftriaxone normalizes plasticity in NAc shell D1R- and D2R-MSNs.Electrophysiological recordings were performed in NAc shell D1R- and D2R-MSN 10–14 d following abstinence from five daily injections of saline ormorphine (10 mg/kg), during which they received 7–10 daily injections ofvehicle or ceftriaxone (400 mg/kg) beginning 72 h following the final drugexposure. (A and B) Mean mEPSC amplitude (Left) and frequency (Right) inD1R-MSN [A, amplitude: F(2,32) = 11.368, P < 0.001; frequency: F(2,32) =11.747, P < 0.001; n = 10–15 per group, N = 5–9 per group] and D2R-MSN[B, amplitude: F(2,36) = 10.325, P < 0.001); frequency: F(2,36) = 6.997, P = 0.003;n = 8–17 per group, N = 6–9 per group] from saline mice treated with vehicleor ceftriaxone (Sal, data are pooled) and morphine mice treated with vehicle(Mor) or ceftriaxone (Cef). (C and D) Mean EPSC paired-pulse ratios in D1R-(Left) and D2R- (Right) MSNs at 20–200 ms interstimulus intervals from micetreated with saline + vehicle, morphine + vehicle, or morphine + ceftriax-one. A significant interaction of drug-treatment and time was observed inD1R-MSN [C, F(6,91) = 2.881, P = 0.016; n = 6–8 per group, N = 4–6], whereas asignificant effect of treatment [D, F(2,63) = 6.037, P = 0.014; n = 6–7 pergroup, N = 3–4 per group] and time [F(3,63) = 8.974, P < 0.001] was observedin D2R-MSNs. (E and F) I–V plots of mean normalized AMPAR-EPSCs at +40,0, and −80 mV (E) and mean rectification indices at +40 mV (F) in D1R-MSNfrom mice treated with saline + vehicle, morphine + vehicle, or morphine +ceftriaxone [F(2,18) = 6.058, P = 0.011; n = 5–8, N = 5–6]. (G) Time course ofEPSCs (−80 mV) before and during perfusion of Naspm (100 μM). (Scale bar,100 pA/25 ms.) (H) Relative amplitudes of individual EPSCs in D1R-MSN be-fore (black outlined) and after Naspm (gray filled) bath application frommice treated with saline + vehicle, morphine + vehicle, and morphine +ceftriaxone [F(2,15) = 6.641, P = 0.009; n = 4–7 per group, N = 4–6 per group].*P < 0.05, **P < 0.01, ***P < 0.001 vs. Sal; #P < 0.05, ##P < 0.01, ###P < 0.001vs. Cef; ^̂ ^P < 0.001 vs. Mor.

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Furthermore, reductions in this synaptic strength correlated withblockade of morphine-induced reinstatement of place preference(Fig. 5E). Taken together, these data indicate that morphine po-tentiates synaptic strength at D1R-MSNs within the ILC-NAcshell pathway and that this plasticity is a central factor in drivingreinstatement of reward behavior.

DiscussionTo our knowledge, our study is the first to identify region-specificbimodal changes in synaptic plasticity within discrete subpopu-lations of NAc MSNs that are induced by repeated morphineexposure and demonstrate a causal link between this plasticityand behavior that models a key aspect of opiate addiction. As dataon experience-dependent synaptic plasticity induced by cocaineare much more abundant, they provide a useful basis for compar-ison. Similar to cocaine, repeated morphine augmented synapticstrength and AMPAR-mediated transmission exlcusively in D1R-MSNs of the NAc shell (14). On the other hand, morphine-dependent increases in mEPSC frequency and reductions inpaired-pulse ratios suggest an increase in release probabilityat D1R-MSNs, contrasting with cocaine studies, which havedirectly observed increases in glutamate release probability inthe NAc core, but not shell (21–24).In addition to enhanced synaptic strength and transmission in

D1R-MSNs, morphine also weakens excitatory input at D2R-MSNs, which has not been observed following cocaine exposure(25). Indeed, increased release probability has been reportedat D1R-MSNs 24 h following remifentanil self-administration;however, no alterations in release probability were found at D2R-MSNs, indicating that, unlike D1R-MSNs, alterations in gluta-mate release at D2R-MSNs requires more prolonged abstinence(26). This divergent regulation of synaptic transmission in D1R-vs. D2R-MSNs is congruent with their antagonistic roles in ad-diction behavior and the notion that MSN subpopulationsreceive differential afferent innervation (27). The exact mecha-nism behind cell-type–specific plasticity in the current study isunclear; however, increases in transmitter release at D1R-MSNsmay reflect a reduction in presynaptic mu opioid receptor (MOR)inhibition (26). Alternatively, it is possible that reductionsin transmitter release at D2R-MSNs may be due in part to

endocannabinoid-mediated LTD (eCB-LTD), which selectivelyoccurs in striatal D2R-MSNs (16).

Morphine Increases Expression of Ca2+-Permeable AMPARs. In co-caine studies, increases in Ca2+-permeable, GluA2-lackingAMPARs within NAc MSNs have emerged as a key mediatorof increased drug seeking and “incubation of craving” duringprolonged (∼30–45 d) abstinence (28). Whether other classesof abused drugs produce this neuroadaptation is unknown. Toour knowledge, our data provide the first evidence that repeatedopiate exposure increases synaptic GluA2-lacking AMPARs inNAc, and that this adaptation occurs exclusively in D1R-MSNs.Interestingly, incubation of drug seeking appears to manifest morerapidly for opiates (within 14 d) (29) than for cocaine, consistentwith the time frame for the appearance of GluA2-lacking AMPARsshown here. In addition, only a brief regimen of experimenter-administered morphine was sufficient for this plasticity, in contrast

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Fig. 4. Ceftriaxone and reinstatement of morphine place preference.(A) Experimental time line of CPP to assess AMPAR-transmission followingCPP. Electrophysiological recordings were performed following 10–14 d of ab-stinence from the final conditioning session. (B) Mean mEPSC amplitude (Left)and frequency (Right) in NAc shell D1R-MSN from mice conditioned withmorphine (5 mg/kg × 4) or saline [amplitude: t(11) = −3.527, P = 0.005; fre-quency: t(11) = −3.132, P = 0.010; n = 8–9 per group, N = 6 per group].(C) Experimental time line of place preference to assess ceftriaxone’s ability todisrupt reinstatement of place preference. (D) Preference scores during 20-minsession during pretesting, preference testing, the final extinction test, and re-instatement testing in morphine conditioned mice treated with vehicle (Veh,outlined) or ceftriaxone (Cef, filled). A significant interaction of treatment andtest day was observed [F(6,79) = 4.126, P = 0.002; n = 6–8 per group]; **P < 0.01,***P < 0.001 vs. Sal; @@@P < 0.001 vs. Mor/Cef/Sal; +++P < 0.001 vs. Mor/Cef/Mor.

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Fig. 5. Optical stimulation of ILC-NAc shell afferents depotentiatesD1R-MSN synaptic strength and blocks reinstatement of morphine placepreference. (A) Schematic depicting intra-ILC infusion of channelrhodopsin-expressing adeno-associated viral infusion (ChR2-AAV), optical stimulationof ILC terminals in the NAc shell, and recording of optically evoked EPSCsfollowing reinstatement. (B) Experimental timeline of CPP. All mice wereconditioned with morphine. Following optical (or mock) stimulation, micewere reinstated with an injection of morphine or saline. Electrophysiologyrecordings were performed 30–45 min following testing in a subset of micereceiving saline injections during reinstatement. (C) Representative opticfiber track placement (yellow outline) targeting the NAc shell (Left) and ILCChR2-AAV infusion (Right). (Inset) High-magnification image of drd1a-tomato transgene (red) and ChR2-AAV terminal expression in NAc shell.(Scale bars, 20 μm.) (D) Optically evoked EPSCs in D1-MSNs from mice re-ceiving stimulation (black) or mock stimulation (gray) and reinstated withsaline [t(6) = 3.858, P = 0.008; n = 4 per group, N = 4 per group]. (Scale bar,50 pA/50 ms.) (E) Preference scores across test days in stimulated (gray) andnonstimulated (black) mice reinstated with saline (outlined) or morphine(filled). All groups displayed statistically significant preference and extinc-tion across test days with no differences observed across groups within testday. A significant interaction of test day and treatment group was observed[F(9,103) = 4.305, P < 0.001; n = 6–8]. **P < 0.01; ***P < 0.001 vs. Mor/No stim/Sal;###P < 0.001 vs. Mor/Stim/Sal; ^̂ ^P < 0.001 vs. Mor/Stim/Mor. PrLC, prelimbiccortex; cc, corpus callosum.

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to the extended-access self-administration procedure needed forGluA2-lacking AMPAR accumulation by cocaine (28). Thus, al-though we do not yet know the mechanisms by which (i) drugexposure increases GluA2-lacking AMPARs and (ii) the syn-aptic incorporation of these receptors drives the incubation ef-fect, use of opiate exposure models should facilitate the study ofthese important questions.

Morphine-Induced Synaptic Plasticity in the NAc Core. The two ma-jor subdivisions of the NAc, the shell and core, are distinguishedbased on anatomic connectivity and their role in reward-relatedbehavior (6, 30–33). In the present study, no significant adap-tations in synaptic strength (A/N ratios) or glutamate releaseprobability were observed in either D1R- or D2R-MSNs of theNAc core. Although repeated exposure to cocaine producesenduring increases in synaptic strength in both NAc shell andcore, we recently demonstrated NAc synaptic plasticity followingrepeated amphetamine (22, 24, 32–34), which raises a questionof whether cocaine, rather than morphine, may be unusual inregards to plasticity in the NAc core. That said, given that thebehavioral and neurochemical effects of opiates are dependenton activation of mu opioid receptors (2, 35), the regional dif-ferences in morphine-induced plasticity demonstrated here maybe related to the higher prevalence of mu opioid receptors in theNAc shell compared with the core (36, 37).Although a specific role glutamate signaling in the shell vs.

core in opiate seeking remains debatable, the shell-specific plas-ticity shown here is consistent with previous reports implicating arole for this region in opiate reward and relapse to drug seeking(19, 38–43). It is worth noting, however, that reductions in NAccore A/N ratios due to up-regulation of NR2B-containing NMDARcurrents have been reported following heroin self-administrationin rats, and that this adaptation drives reinstatement of heroinseeking (24). The mechanisms underlying this apparent discrep-ancy in plasticity are unclear; however, morphine and heroin havebeen shown to differentially modulate synaptic strength (44).Furthermore, distinctions in learning and motivational processesassociated with operant responding tasks vs. CPP, as well as in-corporation of extinction vs. abstinence following drug exposuremay be contributing factors to these differences.

Reversal of NAc Morphine-Induced Plasticity Blocks Reinstatement.Many studies on the neurobiology of drug relapse have focusedon measuring glutamate release and cellular adaptations at ex-citatory synapses in the NAc (45). A common finding across drugclasses is that reinstated drug seeking in animal models is asso-ciated with increased extracellular glutamate in the NAc (17).These increases in extrasynaptic glutamate have been ascribed inpart, to reduced uptake of synaptically released glutamate viaGLT-1 in the NAc, as compounds that increase expression ofGLT-1 (i.e., ceftriaxone) can inhibit reinstatement of drug seeking(17). Although repeated cocaine reduces GLT-1 expression in theNAc core and shell, the antirelapse effects of ceftriaxone havebeen attributed to restoration of GLT-1 function selectively inthe core (46). Similarly, heroin self-administration increasesextrasynaptic glutamate spillover and impairs GLT-1 function inthe NAc core (40). Reversal of this adaptation with ceftriaxonewas associated with attenuated reinstatement of cue-inducedheroin-seeking behavior. Although these findings support thenotion that ceftriaxone normalizes reuptake in the NAc core,they do not address potential alterations in NAc shell glutamateor synaptic plasticity in either core or shell. Here, we find thatrepeated treatment with ceftriaxone during abstinence normalizesmorphine-induced synaptic plasticity in D1R- and D2R-MSNs,providing, to our knowledge, the first evidence that ceftriaxonenormalizes opiate-induced synaptic strength and transmission, andthat these effects are distinctly different across MSN subpopula-tions in the NAc shell. In congruence with previous opiate extra-cellular glutamate work, ceftriaxone-dependent normalization ofsynaptic plasticity also blocks reinstatement reward-related be-havior (41, 47); however, future studies are needed to determine

whether this blockade is due to ceftriaxone’s effects on D1R- orD2R-MSN plasticity or a combination of the two.The ability of ceftriaxone to normalize synaptic strength and

transmitter release probability in D1R-MSNs indicated thatmorphine-induced potentiation in D1R-MSN synapses is a keyfactor for morphine reward-related behavior. However, thesecells receive prominent glutamate input from multiple brain re-gions, and pathway-specific activation of these fibers has beendemonstrated to elicit distinct behavioral responses (5, 48–50).Here we show that prior optogenetic stimulation of ILC-NAcshell terminals using a protocol known to produce LTD in NAcMSNs produces a depotentiation of morphine synaptic strengthin D1R-MSNs and prevents morphine-induced reinstatement ofplace preference. Previous work has shown that reversible in-activation of the NAc shell during testing can block heroin-primed reinstatement (18). Furthermore, inactivation of the ILCin conjunction with D1R antagonism in the NAc shell duringtesting attenuates context-induced reinstatement of heroin seeking(19). These studies indicate a role for the ILC and NAc shell inthe expression of drug-seeking behavior, but do not directly assessthe role of the ILC-NAc shell pathway, as these brain regionsretain numerous efferent and afferent connections, nor do theyaddress any role for plasticity in this pathway. Thus, to ourknowledge, these data are the first to directly demonstrate thatmorphine potentiates ILC-NAc shell synaptic strength, and thatthis pathway-specific plasticity is a primary mechanism by whichopiates exert their rewarding effects. The possibility of anti-dromic activation of cell bodies within the ILC and subsequentengagement of collaterals projecting to other brain regionsshould be considered when interpreting these results (51). Im-portantly, these data are consistent with recent data suggestingthat the ventromedial prefrontal cortex acts as a neural “off”switch for cocaine seeking, but an “on” switch for opiates (52).Future experiments will be necessary to determine whether plas-ticity at ILC-NAc shell synapses is important for reinstatement byother modalities (i.e., cues, context).The need for developing nonopioid based treatments that

target neural processes corrupted by chronic opioid use is clear.However, a better understanding of molecular and cellularmechanisms underlying effects of opioids on synaptic transmissionand plasticity within reward-circuits is required. Our study iden-tifies cell-type and pathway-specific alterations in synaptic strengthand transmission within the NAc and implicates the NAc shell asa key site for morphine plasticity involved in the conditionedrewarding effects of morphine. Furthermore, our data highlightthe complexities of NAc microcircuits, and confirm the presenceof distinct and overlapping plasticity produced by classes ofabused drugs that may provide targetable molecular mechanismsfor future pharmacotherapies.

Materials and MethodsMice. C57BL/J6, drd1a-tdtomato, or drd2-eGFP male mice (P48-60) were usedand have been described (refs. 14 and 21; SI Materials and Methods). Allexperiments were approved by the University of Minnesota InstitutionalAnimal Care and Use Committee.

Locomotor Sensitization. Initial studies involving repeated morphine andabstinence (Figs. 1–3 and Figs. S1–S4) included habituation, five once-dailyinjections of saline or morphine (10 mg/kg, i.p.), and 10–14 d of home cageabstinence. For ceftriaxone experiments (Fig. 2 A–G and Fig. S2 A–C), micereceived 7–10 daily injections of vehicle or ceftriaxone (400 mg/kg, i.p) be-ginning 72 h following the final drug exposure (SI Materials and Methods).

Conditioned Place Preference. All conditioned place preference experimentsused a two-chamber apparatus, and included eight daily alternating sessionsof saline or morphine (5 mg/kg, s.c.) and six daily extinction sessions as de-scribed (ref. 53; SI Materials and Methods).

Virus Injection of ChR2-AAV, Optic Fiber Implantation, and in Vivo StimulationProtocols. AAV2-CaMKII-ChR2-eYFP or AAV2-CaMKII-ChR2-mcherry (Univer-sity of North Carolina Vector Core Facility) was bilaterally injected into theinfralimbic cortex. Approximately 4–6 wk following virus surgeries, mice

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were implanted with indwelling optic fibers targeting the NAc shell. Blue LEDs(Plexon, 465 nm, 15 mW) were used to deliver a 10-min train of 5-ms pulses at10 Hz in the home cage 4 h before receiving an injection of saline or morphinefor testing reinstatement of place preference (SI Materials and Methods).

Slice Electrophysiology. Whole-cell patch clamp recordings from MSNs inacute NAc sagittal slices (250 μm) were performed as described (22, 54).

Data Analysis. All data are expressed as a mean ± SEM. Data obtained fromindividual cells is represented alongside bar graphs. Sample size in experimentsis presented as n and N, where n is the number of cells and N is the number of

mice. Statistical significance was assessed using Student’s t tests, one-way,two-way, and repeated measures ANOVAs (Sigma Plot) with further com-parisons made using a Student–Newman–Keuls post hoc test. Traces in figureshave had stimulus artifacts removed and are averages of 20–25 consec-utive responses.

ACKNOWLEDGMENTS. The MnDRIVE Optogenetics Core at the University ofMinnesota provided invaluable technical support. This work was supportedby funding from the National Institute on Drug Abuse Grants R01 DA019666,K02 DA035459 (to M.J.T.), K99 DA038706 (to M.C.H.), and T32 DA007234 (toA.I. and S.R.E.); the MnDRIVE Initiative on Brain Conditions; and the Breyer-Longden Family Research Fund.

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