Prefrontal cortex AMPA receptor plasticity is crucial for cue ... - Nature

© 2008 Nature Publishing Group http://www.nature.com/natureneuroscience

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Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking Michel C Van den Oever1, Natalia A Goriounova2, Ka Wan Li1, Roel C Van der Schors1, Rob Binnekade3, Anton N M Schoffelmeer3, Huibert D Mansvelder2, August B Smit1, Sabine Spijker1,4 & Taco J De Vries1,3,4 Associative learning processes have an important role in the initiation and persistence of heroin-seeking. Here we show in a rat self-administration model that reexposure to cues previously associated with heroin results in downregulation of AMPA receptor subunit GluR2 and concomitant upregulation of clathrin-coat assembly protein AP2m1 in synaptic membranes of the medial prefrontal cortex (mPFC). Reduced AMPA receptor expression in synaptic membranes was associated with a decreased AMPA/NMDA current ratio and increased rectification index in mPFC pyramidal neurons. Systemic or ventral (but not dorsal) mPFC injections of a peptide inhibiting GluR2 endocytosis attenuated both the rectification index and cue-induced relapse to heroin-seeking, without affecting sucrose-seeking. We conclude that GluR2 receptor endocytosis and the resulting synaptic depression in ventral mPFC are crucial for cue-induced relapse to heroin-seeking. As reexposure to conditioned stimuli is a major cause for heroin relapse, inhibition of GluR2 endocytosis may provide a new target for the treatment of heroin addiction.

In human heroin addicts, otherwise neutral environmental stimuli, such as paraphernalia associated with drug-taking, become associated over time with the drugs’ rewarding effects1. This associative learning process shares features with traditional learning models and is likely to involve mechanisms of synaptic plasticity2. For instance, exposure to drugs of abuse induces long-term depression or potentiation in neural circuits underlying natural reward learning3. Some of these neuroadaptations are long lasting4,5, which shows that drugs of abuse can persistently reconfigure neuronal circuitry function in the adult brain6. Similarly, reexposure to drug-associated cues can provoke drug craving and subsequent relapse to drug use even after prolonged abstinence periods1,7. However, the acute synaptic changes that take place immediately after reexposure to drug-conditioned stimuli, and that may lead to resumption of drug-seeking, have not yet been studied. In our earlier work, we showed that reexposure to cues previously associated with heroin self-administration leads to immediate-early gene expression in the mPFC8,9. This indicates neuronal activation in a brain area known to be crucially involved in cue-induced relapse to drug-seeking in animal models10–12. Here, we investigated acute changes in molecular composition and function of synapses in the mPFC upon reexposure to heroin cues after long-term abstinence from self-administration. Recent technological advances in identification of proteins through sensitive mass spectrometry and quantification by isotope labeling of proteins (iTRAQ, Applied Biosystems) allowed us to measure acute and subtle changes in the abundance of proteins in synaptic membrane samples of the rat mPFC13,14. In particular, we showed that synaptic membrane expression of AMPA receptor

(AMPAR) in the mPFC is reduced after reexposure to heroin cues. Accordingly, in glutamatergic synapses at mPFC pyramidal neurons, AMPA currents were reduced by almost 25%. Finally, rats in which endocytosis of AMPAR was specifically blocked in the ventral mPFC showed reduced cue-induced relapse to heroin-seeking. RESULTS Rats were trained in 3-h sessions to nose-poke for intravenous heroin infusions in the presence of audiovisual cues (Fig. 1a,b). Rats developed a stable preference for the active, heroin-paired hole. The number of nose-pokes in the active hole increased substantially when the fixed ratio (FR) was doubled from FR1 to FR2 and from FR2 to FR4, confirming the motivational drive to self-administer heroin (Fig. 1b). After a 3-week period of either abstinence in the rat’s home cage or extinction training in the operant cage in the absence of heroin and heroin-associated audiovisual cues, rats were allowed to nose-poke for response-contingent cue presentations during the relapse tests without receiving heroin. Rats that were not exposed to the cues served as controls. These conditions induced robust relapse to heroin-seeking in both the abstinence and extinction groups (Fig. 1c). Cue-induced AMPA receptor endocytosis in mPFC Immediately after the relapse test, we dissected the mPFC and isolated a synaptic membrane fraction containing membrane proteins and associated proteins using discontinuous sucrose gradients15. We then separated peptides from tryptic protein digests using two-dimensional liquid chromatography. Peptides were identified by tandem mass

1Departments

of Molecular and Cellular Neurobiology and 2Integrative Neurophysiology, Center for Neurogenomics & Cognitive Research, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. 3Anatomy and Neurosciences, Center for Neurogenomics & Cognitive Research, VU Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. 4These authors contributed equally to this work. Correspondence should be addressed to S.S. ([email protected]). Received 19 May; accepted 12 June; published online 1 August 2008; doi:10.1038/nn.2165

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ARTICLES Figure 1 Heroin acquisition and relapse. Self-administration Extinction/abstinence Test (a) Overview of the paradigm used. (b) Rats 1h 22 d 21 d developed a stable preference for the active (heroin-paired) hole. Overall statistical analysis 60 140 Active hole Inactive hole Active A– Inactive A– FR4 # revealed an interaction for session nose-poke Active A+ Inactive A+ 120 50 hole (F14,1162 ¼ 170.6; P o 0.001). This shows Active E+ Inactive E+ 100 that there was no a priori difference between * 40 Active E– Inactive E– groups. A– , abstinence no cue; A+, abstinence 80 FR2 30 cue; E+, extinction cue; E–, extinction no cue. 60 (c) Response-contingent presentation of the 20 40 previously heroin-paired compound audiovisual # stimuli after 21 d of extinction training (Ext + 10 20 cue) or after abstinence in the home cage (Abst + 0 0 cue) induced robust resumption of response Abst + cue Ext + cue Ext no cue 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 in the formally heroin-associated (active) hole Session (F2,24 ¼ 64.12; P o 0.001). The Abst + cue group responded significantly more in the inactive hole compared to both extinction groups (F2,24 ¼ 16.57; P o 0.001), an effect often observed during a first extinction session. Control rats either underwent an additional extinction session (Ext no cue) or remained in the home cage (Abst no cue; no data). *P o 0.001 versus active Ext no cue; #P o 0.001 versus extinction groups. Bars represent mean ± s.e.m.

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to determine whether glutamatergic synaptic transmission was altered by previous heroin intake. We determined the contributions of AMPA and NMDA currents to extracellularly evoked excitatory postsynaptic currents by measuring current amplitudes at various membrane potentials19,20 (Fig. 3a) or from pharmacologically isolated AMPA and NMDA currents19,21 (Supplementary Fig. 1 online). Rats that were reexposed to heroin-conditioned cues had significantly lower AMPA/ NMDA current ratios than did rats that were not exposed, with no effect on other parameters (Fig. 3b and Supplementary Figs. 1 and 2 online). AMPA/NMDA current ratios in the saline control groups were identical to those observed in the heroin rats that were not reexposed to the heroin cues, indicating that heroin self-administration by itself did

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Figure 2 Protein changes after cue-induced relapse to heroin-seeking. After the relapse tests (60 min), the mPFC was dissected for synaptic membrane proteomics analysis. (a) Quantification of iTRAQ reagents revealed a cuespecific (two-factor ANOVA P o 0.01) regulation of AMPAR subunits GluR2 and GluR3, clathrin-coat assembly protein AP2m1, NR2B, b-catenin and calcium-transporting ATPase1 (Atp2b1). *P o 0.05 versus Abst no cue; #P o 0.05 versus Ext no cue. For details, see Supplementary Table 1. (b,c) Immunoblotting for GluR2 (b) and Ap2m1 (c) corroborated the observed downregulation of GluR2 and the upregulation of Ap2m1 resulting from cue exposure in heroin-trained animals (two-factor ANOVA P o 0.05). The Coomassie-stained lower (GluR2) or upper (AP2m1) portion of the same gel was used for correction of input. There was no long-term effect of heroin exposure on the synaptic expression of these proteins, as a saline (Sal) cueexposed group showed levels similar to those of the heroin no cue group. Expression levels are set to the saline cue group. *P o 0.05 versus heroin cue. Bars represent mean ± s.e.m.

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spectrometry and quantified by iTRAQ technology, enabling detection of subtle changes in protein abundance13,14. Overall statistical analysis of three independent experiments, including the factors cue (cue versus no cue) and heroin cessation condition (abstinence versus extinction), revealed that from a total of 417 proteins identified, the abundance of 6 proteins was significantly (P o 0.01) changed after cue exposure, independent of the experimental conditions after cessation of heroin self-administration (Fig. 2a and Supplementary Table 1 online). We observed a downregulation of AMPAR subunits GluR2 (10%) and GluR3 (15%) and the NMDA receptor (NMDAR) subtype 2B (10%); we also observed an increase in clathrin-coat assembly protein AP2 complex subunit mu-1 (Ap2m1, also known as AP50; 11%). Levels of the AMPAR subunit GluR1 and NMDAR subunit NR1 were unaffected by heroin-conditioned cue exposure (Supplementary Table 1). Immunoblotting of mPFC synaptic membranes of an independent group of rats that underwent extinction training confirmed the observed downregulation of GluR2 and upregulation of Ap2m1 caused by cue exposure (Fig. 2b,c). Collectively, these observations suggest that exposure to heroin cues results in AP2- and clathrin-mediated endocytosis of GluR2/3 AMPARs16–18, leading to synaptic depression in the mPFC. To test whether the changes in GluR2/3 AMPAR protein levels were paralleled by changes in synaptic input received by mPFC pyramidal neurons, we recorded glutamatergic synaptic transmission in mPFC pyramidal neurons in acute brain slices immediately after rats were reexposed to the heroin-conditioned cues (30-min test). Rats with a history of saline self-administration served as additional control groups

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Figure 3 Cue-induced heroin-seeking alters synaptic strength. After response-contingent cue presentations (30 min), acute mPFC slices were prepared, and AMPA and NMDA currents were recorded from infralimbic and prelimbic cortex pyramidal neurons in the presence of Gabazine. (a) Example traces of glutamatergic currents evoked by extracellular stimulation within layers II and III (at 50–100 mm) and recorded at holding potentials of –80 mV and +40 mV. Gray current traces represent responses to ten stimulations, with average waveform shown in black. Shaded rectangular areas indicate where measurements were taken to determine AMPA (at –80 mV) and NMDA (at +40 mV) current amplitudes. (b) In rats exposed to heroin-associated cues, AMPA/NMDA current ratios were significantly decreased (F3,103 ¼ 3.09; P ¼ 0.030) compared to rats unexposed to heroin-conditioned cues and compared to rats exposed or unexposed to saline-paired cues. Bars represent mean ± s.e.m., *P o 0.05.

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Preventing GluR2 endocytosis reduces heroin seeking Accumulating evidence suggests that regulated clathrin-dependent endocytosis of GluR2 subunits through interaction with AP2 underlies acute forms of synaptic plasticity17,18,22. We therefore examined whether blockade of clathrin-dependent GluR2 endocytosis using an HIV TAT protein–fused GluR2-derived C-terminal peptide (TAT-GluR23Y)23 could prevent the change in synaptic AMPA current properties induced by heroin-conditioned cue presentation. In hippo-

campal slices, this membrane-permeable peptide blocks the expression of homosynaptic long-term depression, but not long-term potentiation24, and the peptide has no effect on basal synaptic transmission in the nucleus accumbens (NAc)23. If GluR2/3 subunits were specifically internalized at mPFC glutamatergic synapses after reexposure to heroin-conditioned cues, the contribution of synaptic currents through AMPARs containing GluR2/3 subunits would be reduced at these synapses. As the presence of GluR2 subunits strongly determines the current-voltage behavior of AMPAR channels25–27, rectification of synaptic currents in the mPFC may be altered after cue exposure. To examine the contribution of GluR2 under various conditions, we assessed the rectification of pharmacologically isolated synaptic AMPA currents in rats that were presented with heroin-conditioned cues compared to rats that were not presented with these cues. We

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Figure 4 Blockade of GluR2 endocytosis attenuates cue-induced heroin-seeking. (a,b) In the presence of systemically injected control peptide (TAT-Glur23A) 90 min before the relapse test, cue exposure (30 min) increased the rectification index of synaptic AMPA currents. The GluR2 endocytosis blocking peptide (TAT-GluR23Y) prevented this cue-induced increase in rectification index (P ¼ 0.009; twofactor ANOVA P o 0.05; *P o 0.01 versus heroin cue with GluR23A). (c) Similarly, systemic injection of TAT-GluR23Y, but not TAT-Glur23A control peptide, attenuated cue-induced heroin-seeking (n ¼ 7–8 per treatment). Overall analysis revealed significant cue (F1,27 ¼ 60.67; P o 0.001), peptide (F1,27 ¼ 15.18; P o 0.001) and cue peptide (F1,27 ¼ 11.18; P ¼ 0.002) effects. Nose-poking in the active hole during extinction (no cue) conditions or in the inactive hole during both conditions was not altered by TAT-GluR23Y. #P o 0.01 versus active hole all groups, *P o 0.05 vs. active hole No cue groups. (d) Local infusion of TAT-GluR23Y into the ventral mPFC (n ¼ 10) mimicked the systemic injection and attenuated cue-induced heroin seeking. A significant difference in responding in the active hole (F1,18 ¼ 6.40; P ¼ 0.02) but not the inactive hole (P 4 0.05) was observed. *P o 0.05 versus heroin cue TAT-GluR23A. (e) In contrast, dorsal mPFC (n ¼ 7 per treatment) injection of TAT-GluR23Y was without effect (P 4 0.05). (f) Injection sites (black dots represent individual rats) were placed within an area that corresponds to the ventral mPFC (average depth, –4.9 mm) or dorsal mPFC (average depth, –2.8 mm) and ranged between bregma 2.7 and 3.7 anteroposterior. Bars represent mean ± s.e.m.


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Figure 5 TAT-GluR23Y does not impair relapse to sucrose-seeking. Systemic injection of TAT-GluR23Y (n ¼ 7–8 per treatment) did not affect responding on the active (previously sucrose-paired) hole or inactive hole (P 4 0.05). Bars represent mean ± s.e.m.

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had no effect on responding for sucrose reward under an FR4 schedule of reinforcement (Supplementary Fig. 3 online), confirming earlier observations23 that the peptide does not interfere with the rats’ ability to perform an operant response.

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systemically injected (1.5 nmol per g body weight23 intravenously) either TAT-GluR23Y or the control peptide (TAT-GluR23A)23 90 min before the start of the cue-induced relapse test. Immediately after the 30-min relapse test, acute mPFC slices were prepared. In the presence of control peptide, synaptic AMPA currents in the mPFC of rats that were not presented with cues had a rectification index (absolute current at –80 mV, current at +40 mV) of 2.47 ± 0.25 (Fig. 4a,b). Rats that were exposed to heroin-conditioned cues had a significantly higher rectification index of 3.37 ± 0.20 (Fig. 4a,b), confirming a reduced contribution of GluR2 subunits to these synaptic currents. Thus, presentation of heroin-conditioned cues significantly altered the rectification of synaptic AMPA currents in the mPFC (Fig. 4a,b). In contrast, in the presence of the TAT-GluR23Y peptide, the rectification of synaptic AMPA currents in the mPFC of rats that were presented with heroin-conditioned cues was equal to that of rats that were not presented with these cues in the presence of control peptide (Fig. 4a,b). This indicates that clathrin-dependent GluR2 endocytosis alters the properties of synaptic AMPA currents in the mPFC after reexposure to heroin-conditioned cues. To determine whether clathrin-dependent GluR2 endocytosis is necessary for reinstatement of heroin-seeking after reexposure to heroinconditioned cues, we tested whether preventing GluR2 endocytosis with TAT-GluR23Y would attenuate cue-induced relapse to heroin-seeking. Statistical analysis that included the factors cue (cue versus no cue) and peptide (TAT-GluR23Y versus TAT-GluR23A) revealed that systemic blockade of GluR2 endocytosis attenuated cue-induced heroinseeking specifically for the previously heroin-paired hole (Fig. 4c). An interaction was observed for cue peptide on the active hole, but not the inactive hole, indicating that the peptide specifically interferes with cue-induced heroin-seeking but not with general motor output. To assess the role of the mPFC in the systemic effect of TAT-GluR23Y on cue-induced heroin-seeking, we injected TAT-GluR23Y or TATGluR23A (15 pmol bilaterally23) into either the ventral (ventral prelimbic and infralimbic cortex) or dorsal (anterior cingulate and dorsal prelimbic cortex) mPFC 60 min before the relapse tests. TAT-GluR23Y infusion into the ventral, but not the dorsal, mPFC attenuated cue-induced heroinseeking (Fig. 4d–f). Besides showing that the effect of TAT-GluR23Y is region specific, these results support the molecular and neurophysiological changes observed and indicate that cue-induced relapse to heroin-seeking depends on GluR2 endocytosis in the ventral mPFC. To verify whether this effect could be generalized to a natural reinforcer, we examined the effects of systemic infusion of TATGluR23Y during a relapse test in sucrose-trained animals under similar conditions and using the same compound audiovisual cue as in the heroin experiment. We found no effect of TAT-GluR23Y on responding in either the active or inactive hole (Fig. 5), indicating that the relapseattenuating effect of the peptide is specific for heroin-associated stimuli and that relapse to sucrose-seeking does not depend on clathrinmediated GluR2 endocytosis. Moreover, injection of TAT-GluR23Y

DISCUSSION To develop effective pharmacotherapies for the treatment of heroin addiction, it is crucial to understand the neurobiological underpinnings of the events that lead to relapse. Here, we showed that reexposure to cues that were previously associated with heroin self-administration elicit a rapid decrease in surface expression of AMPAR in the mPFC. Cue-induced heroin-seeking resulted in a concomitant decrease in synaptic strength in mPFC pyramidal neurons, caused by a reduction in AMPA, but not NMDA, currents. Finally, cue-induced relapse to heroin-seeking depended on clathrin-mediated AMPAR endocytosis in the ventral mPFC, as preventing GluR2 endocytosis in the ventral mPFC strongly reduced cue-induced heroin-seeking. Notably, this mechanism did not generalize to cue-induced seeking of a natural reward. Previous studies on drug-induced changes in neuronal connectivity and neuronal physiology have led to the concept that addiction might be viewed as a pathological process that involves plasticity mechanisms similar to those implicated in neuronal models of learning and memory28–31. However, the molecular mechanisms underlying these changes remain largely unsolved. The development of sensitive iTRAQbased proteomics13,14 enabled the quantification of acute cue-induced changes of protein levels at the synaptic membrane of the rat mPFC9. In the small set of differentially expressed proteins (1.4% of the identified proteome) upon cue exposure, we observed changes in the abundance of a group of proteins involved in a common pathway related to the process of internalization of AMPARs. Facilitated endocytosis of postsynaptic GluR2/3 AMPARs regulates synaptic strength at glutamatergic synapses16, a molecular and cellular substrate for learning and memory. Exposure to drugs of abuse changes the ratio of AMPA and NMDA currents in glutamatergic synapses in the ventral tegmental area (VTA) and NAc3,21,32. For instance, experimenter-administered cocaine increases AMPA/NMDA current ratios in the NAc shell after a 2-week drug-free period, whereas a single reexposure to cocaine abruptly reverses AMPA/NMDA current ratios32. Moreover, the induction of synaptic depression is abolished in the NAc core, but not the shell, after 21 d of abstinence from cocaine selfadministration5, indicating the importance of brain areas and drug administration paradigms in this synaptic effect. Until now, the effect of cues associated with drug self-administration on glutamatergic synaptic plasticity had never been tested. We found that exposure to cues previously associated with heroin self-administration induced an acute decrease in AMPA/NMDA current ratios in mPFC pyramidal neurons, resulting specifically from a reduction of the AMPA current amplitude. In addition to reduced levels of GluR2 and GluR3 AMPAR subunits, we observed a decrease in NR2B protein levels using iTRAQ proteomics (Fig. 2). This change was not associated with a change in NMDA currents at mPFC pyramidal neurons as measured by electrophysiology, suggesting that this NMDAR subunit is regulated in glutamatergic synapses received by other neuronal subtypes within the mPFC. We previously showed that cue-induced relapse to heroin-seeking results in enhanced expression of several immediate-early genes in the mPFC. For instance, reexposure to heroin cues—similar to conditions used in the present study—increases expression of Arc (activityregulated cytoskeleton-associated protein)8. Arc downregulates surface expression of AMPARs by increasing the basal rate of endocytosis33, thereby reducing the amplitude of synaptic GluR2/3-containing AMPA

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ARTICLES currents34. Cue-induced Arc expression may therefore provide a mechanism for increased rates of GluR2/3 endocytosis after cue exposure. We showed here that relapse to heroin-seeking depends on AMPAR endocytosis in the ventral mPFC, but not the dorsal mPFC. Previous studies on relapse to cocaine-seeking, in which pharmacological intervention or lesions were used to silence various areas of the mPFC, underscore the importance of the dorsal mPFC in initiating cocaineseeking11,35,36. However, more recent studies confirm a crucial role of the ventral part of the mPFC in reinstatement of both cocaine-seeking37,38 and heroin-seeking39. The ventral mPFC (particularly the infralimbic region) exerts an inhibitory control over cocaine-seeking through its projection to the shell region of the ventral striatum37, whereas the projection of the more dorsal prelimbic region to the core region of the ventral striatum actually drives cocaine-seeking. Thus, reinstatement of drug-seeking may depend on interplay between the dorsal and ventral parts of the mPFC; glutamatergic output from the dorsal mPFC may regulate executive control over response to drug-conditioned stimuli40, whereas diminished output from the ventral mPFC may contribute to drug-seeking by impairing inhibitory response control37. Our observations fit within this conceptual framework by showing that acute synaptic depression of the ventral mPFC upon heroin-cue exposure results in a loss of inhibitory control over heroin-seeking. Apart from projections of the ventral mPFC to the NAc, the entire mPFC has reciprocal connections with the VTA41,42 and amygdala41,43, with a predominance of interconnections with the more ventral areas of the mPFC44. Both the VTA and amygdala have a crucial role in cueinduced reinstatement of heroin-seeking39,45–47, and both these brain regions are heavily interconnected with the ventral striatum48. Together, these studies suggest that the ventral mPFC, ventral striatum, VTA and amygdala function as a concerted neuronal system that controls cue-induced relapse to heroin-seeking. Our findings are in agreement with previous work showing an important role of glutamate signaling in cue-induced heroin-seeking and a more general role of glutamate systems in the motivational processes related to drug addiction12,37,40,46,49. We have now shown that acute cue-induced alterations in AMPAR plasticity and physiology at the synaptic level are crucial for relapse to heroin-seeking. Cueinduced changes in AMPAR subunit expression did not result from extinction learning processes, as GluR2 and GluR3 protein levels changed after extinction and abstinence of heroin self-administration. Moreover, there was no long-term effect of heroin self-administration, as GluR2 or Ap2m1 synaptic protein levels were not affected 3 weeks after the last heroin exposure, nor did it affect AMPA/NMDA current ratios in mPFC pyramidal neurons. Rather, our data suggest that cueinduced retrieval of memories related to heroin use, and the translation of those memories into goal-directed behavior, depends on acute decreases in AMPAR surface expression in the ventral mPFC through a reduced contribution of GluR2, and possibly GluR3, subunits. These findings are potentially relevant for human heroin addiction, as pharmacotherapeutic intervention aimed at inhibiting regulated AMPAR endocytosis may provide a new avenue for the treatment of cue-induced relapse to heroin use. METHODS Animals. Male Wistar rats (280–300 g; Harlan) were housed on a reversed 12-h light-dark cycle with food (Teklad Global 2016, Harlan) and water ad libitum throughout the experiments. The Animal Users Care Committee of the Vrije Universiteit approved all experiments. Heroin self-administration, extinction and relapse tests. Rats were trained to self-administer heroin (100 mg kg–1, diacetylmorphine-HCL; OPG) in 15 daily 3-h sessions9. Rats were then divided into two groups and underwent either


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abstinence for 21 d (home cage) or 15 once-daily extinction sessions (1 h) spanning a drug abstinence time frame of 21 d9. We reexposed half of the rats of each group to heroin-conditioned stimuli (60 min; see Supplementary Methods online). Immediately after the test, we decapitated the rats and froze the brains rapidly using ice-cold (–50 1C) isopentane and stored them at –80 1C until further use. For all other experiments (immunoblotting, electrophysiology and behavior), a 30-min test session (relapse) was used after 21 d of 1-h extinction sessions. For immunoblotting and electrophysiological experiments, we also included saline self-administering rats that received similar relapse tests. Sucrose self-administration, extinction and relapse tests. We trained rats (n ¼ 20) to self-administer a 10% sucrose solution (0.19 ml) with a maximum of 100 rewards per session in daily 3-h sessions9, under conditions similar to those for heroin self-administration except that a liquid receptacle was placed between the nose-poke holes. Tissue preparation, iTRAQ labeling, two-dimensional liquid-chromatography tandem mass spectrometry and immunoblotting. We dissected the mPFC freehand at –20 1C from 1-mm-thick brain slices according to bregma coordinates 3.7–2.7 mm. We isolated synaptic membranes15 (pool of three rats), and we either digested proteins (100 mg per sample) for iTRAQ (Applied Biosystems) or dissolved them in loading buffer (0.32 mg per lane) for immunoblotting. In triplicate experiments, we iTRAQ-labeled the digested peptides and processed them for two-dimensional liquid-chromatography tandem mass spectrometry (4800 Proteomics Analyzer; Applied Biosystems). Details on synaptic membrane isolation, iTRAQ labeling and protein identification are provided in Supplementary Methods. Samples for immunoblotting were prepared according to the manufacturer (NuPage and iBlot, Invitrogen) and incubated with antibodies to mouse GluR2 (NeuroMab clone L21/32, UC Davis/NIH NeuroMab Facility; 1:5,000) or goat AP2m1 (BD Biosciences PharMingen; 1:20,000). For correction of input, we stained the lower (GluR2) or upper (Ap2m1) half of the same gel with Coomassie. Electrophysiology. Immediately after reinstatement testing (30-min session), independent batches of rats (n ¼ 7–11 per treatment) were decapitated and their brains rapidly removed. Coronal mPFC slices of 350-mm thickness were prepared in sucrose-containing artificial cerebrospinal fluid (ACSF) consisting of 3.5 mM KCl, 2.4 mM CaCl2, 1.3 mM MgSO4*7H2O, 1.2 mM KH2PO4, 215.5 mM sucrose, 26 mM NaHCO3 and 10 mM D-glucose. Slices were stored in holding chambers containing normal ACSF consisting of 125 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 1 mM CaCl2, 26 mM NaHCO3 and 10 mM glucose, bubbled with carbogen gas (95% O2 and 5% CO2). Whole-cell recordings of AMPA and NMDA currents (Figs. 3 and 4 and Supplementary Figs. 1 and 2) from pyramidal neurons were made using standard electrophysiological techniques19,21,50. The pipette medium contained 120 mM cesium gluconate, 10 mM CsCl, 8 mM NaCl, 2 mM MgATP, 10 mM phosphocreatine, 0.2 mM EGTA, 10 mM HEPES, 0.3 mM Tris-GTP and 1 mM QX-314Cl. All recordings were made at 32 1C. Systemic and intra-mPFC injection of synthetic GluR2-derived peptide. To block clathrin-dependent GluR2 endocytosis, we injected a synthetic peptide derived from the rat GluR2 C terminus (GluR23Y; 869YKEGYNVYG877) fused to the cell membrane transduction domain of the HIV-1 TAT protein23, or an HIV-TAT–fused GluR2 negative control peptide (GluR23A; AKEGANVAG)23 (Netherlands Cancer Institute). For systemic injections, peptides were injected intravenously (1.5 nmol per g body weight) 90 min23 before the start of the reinstatement test. For intra-mPFC injections, rats (n ¼ 10 per treatment) were implanted with bilateral guide cannulas (26-gauge; C235G-1.5/2.5, PlasticsOne), as described previously9, aimed at the dorsal mPFC (anterior cingulate and dorsal prelimbic area) or the ventral mPFC (ventral prelimbic and infralimbic area). Coordinates relative to bregma were +3.2 mm anteroposterior and 0.75 mm mediolateral. All injections were given in a volume of 0.25 ml ACSF in 120 s, with an additional 60 s to allow diffusion. Statistics. Data from the relapse tests were analyzed separately for active and inactive nose-pokes and subjected to analysis of variance (ANOVA) with a post hoc two-tailed Tukey-Kramer test. iTRAQ-based proteomics analysis was done in triplicate with independent biological samples. Experiments were

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ARTICLES combined, and protein abundance per treatment was subjected to two-factor ANOVA (significance set at P o 0.01 to reduce the chance of false positives that can result from multiple testing) with the factors of heroin cessation type (abstinence versus extinction) and cue (cue versus no cue), followed by a post hoc two-tailed Fisher’s least significant difference test. Immunoblotting data were analyzed by two-factor ANOVA with the factors of drug administration type (saline versus heroin) and cue (cue versus no cue), followed by a post hoc one-tailed Fisher’s least significant difference test. Statistical significance of AMPA/NMDA current ratios and rectification of AMPA currents were evaluated by two-factor ANOVA followed by post hoc Fisher’s least significant difference test or Tukey-Kramer multiple comparisons test.

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Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTS The authors thank W. de Vries, H. Raasø, M. Stegeman and Y. Gouwenberg for technical assistance and Y. Shaham for valuable comments on the manuscript. This work was supported by grants from the Center for Medical Systems Biology (to M.C.V.d.O., S.S., R.C.V.d.S., K.W.L. and A.B.S.) and the Netherlands Organization for Scientific Research (to N.A.G. and H.D.M.). AUTHOR CONTRIBUTIONS M.C.V.d.O. and N.A.G. contributed equally to this work. M.C.V.d.O., A.N.M.S., H.D.M., A.B.S., S.S. and T.J.D.V. designed the experiments. M.C.V.d.O. and R.B. executed the behavior experiments. M.C.V.d.O. and T.J.D.V. analyzed the behavioral data. M.C.V.d.O., K.W.L., R.C.V.d.S., R.B. and S.S. executed the molecular experiments. M.C.V.d.O. and S.S. analyzed the molecular data. N.A.G. executed the electrophysiology experiments. N.A.G. and H.D.M. analyzed the electrophysiology data. M.C.V.d.O., A.B.S., H.D.M., T.J.D.V. and S.S. wrote the manuscript. Published online at http://www.nature.com/natureneuroscience/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/

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