Persistent Neuroadaptations in the Nucleus Accumbens Core Accompany Incubation of Methamphetamine Craving in Male and Female Rats.

Relapse is a major problem in treating methamphetamine use disorder. "Incubation of craving" during abstinence is a rat model for persistence of vulnerability to craving and relapse. While methamphetamine incubation has previously been demonstrated in male and female rats, it has not been demonstrated after withdrawal periods greater than 51 d and most mechanistic work used males. Here, we address both gaps. First, although methamphetamine intake was higher in males during self-administration training (6 h/d × 10 d), incubation was similar in males and females, with "incubated" craving persisting through withdrawal day (WD)100. Second, using whole-cell patch-clamp recordings in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) core, we assessed synaptic levels of calcium-permeable AMPA receptors (CP-AMPARs), as their elevation is required for expression of incubation in males. In both sexes, compared with saline-self-administering controls, CP-AMPAR levels were significantly higher in methamphetamine rats across withdrawal, although this was less pronounced in WD100-135 rats than WD15-35 or WD40-75 methamphetamine rats. We also examined membrane properties and NMDA receptor (NMDAR) transmission. In saline controls, MSNs from males exhibited lower excitability than females. This difference was eliminated after incubation because of increased excitability of MSNs from males. NMDAR transmission did not differ between sexes and was not altered after incubation. In conclusion, incubation persists for longer than previously described and equally persistent CP-AMPAR plasticity in NAc core occurs in both sexes. Thus, abstinence-related synaptic plasticity in NAc is similar in males and females although other methamphetamine-related behaviors and neuroadaptations show differences.

GluA2-lacking AMPA receptors in the nucleus accumbens core and shell contribute to the incubation of oxycodone craving in male rats.

One of the most challenging issues in the treatment of substance use disorder, including misuse of opioids such as oxycodone, is persistent vulnerability to relapse, often triggered by cues or contexts previously associated with drug use. In rats, cue-induced craving progressively intensifies ('incubates') during withdrawal from extended-access self-administration of several classes of misused drugs, including the psychostimulants cocaine and methamphetamine. For these psychostimulants, incubation is associated with strengthening of excitatory synapses in the nucleus accumbens (NAc) through incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors that lack the GluA2 subunit and are therefore Ca2+ -permeable (CP-AMPARs). Once CP-AMPAR upregulation occurs, their stimulation is required for expression of incubation. It is not known if a similar mechanism contributes to incubation of oxycodone craving. Using male rats, we established that incubation occurs by withdrawal day (WD) 15 and persists through WD30. Then, using cell-surface biotinylation, we found that surface levels of the AMPAR subunit GluA1 but not GluA2 are elevated in NAc core and shell of oxycodone rats on WD15, although this wanes by WD30. Next, using intra-NAc injection of the selective CP-AMPAR antagonist Naspm before a seeking test, we demonstrate that CP-AMPAR blockade in either subregion decreases oxycodone seeking on WD15 or WD30 (after incubation), but not WD1, and has no effect in saline self-administering animals. The Naspm results suggest CP-AMPARs persist in synapses through WD30 even if total cell surface levels wane. These results suggest that a common neurobiological mechanism contributes to expression of incubation of craving for oxycodone and psychostimulants.

CaMKII Modulates Diacylglycerol Lipase-α Activity in the Rat Nucleus Accumbens after Incubation of Cocaine Craving.

Relapse is a major challenge to the treatment of substance use disorders. A progressive increase in cue-induced drug craving, termed incubation of craving, is observed after withdrawal from multiple drugs of abuse in humans and rodents. Incubation of cocaine craving involves the strengthening of excitatory synapses onto nucleus accumbens (NAc) medium spiny neurons via postsynaptic accumulation of high-conductance Ca2+-permeable AMPA receptors. This enhances reactivity to drug-associated cues and is required for the expression of incubation. Additionally, incubation of cocaine craving is associated with loss of the synaptic depression normally triggered by stimulation of metabotropic glutamate receptor 5 (mGlu5), leading to endocannabinoid production, and expressed presynaptically via cannabinoid receptor 1 activation. Previous studies have found alterations in mGlu5 and Homer proteins associated with the loss of this synaptic depression. Here we conducted coimmunoprecipitation studies to investigate associations of diacylglycerol lipase-α (DGL), which catalyzes formation of the endocannabinoid 2-arachidonylglycerol (2-AG), with mGlu5 and Homer proteins. Although these interactions were unchanged in the NAc core at incubation-relevant withdrawal times, the association of DGL with total and phosphorylated Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) and CaMKIIß was increased. This would be predicted, based on other studies, to inhibit DGL activity and therefore 2-AG production. This was confirmed by measuring DGL enzymatic activity. However, the magnitude of DGL inhibition did not correlate with the magnitude of incubation of craving for individual rats. These results suggest that CaMKII contributes to the loss of mGlu5-dependent synaptic depression after incubation, but the functional significance of this loss remains unclear.

GluN3-Containing NMDA Receptors in the Rat Nucleus Accumbens Core Contribute to Incubation of Cocaine Craving.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.

mGlu5 function in the nucleus accumbens core during the incubation of methamphetamine craving.

Many studies have demonstrated that negative allosteric modulators (NAM) of metabotropic glutamate receptor 5 (mGlu5) reduce cocaine and methamphetamine seeking in extinction-reinstatement animal models of addiction. Less is known about effects of mGlu5 NAMs in abstinence models, particularly for methamphetamine. We used the incubation of drug craving model, in which cue-induced craving progressively intensifies after withdrawal from drug self-administration, to conduct the first studies of the following aspects of mGlu5 function in the rat nucleus accumbens (NAc) core during abstinence from methamphetamine self-administration: 1) functionality of the major form of synaptic depression in NAc medium spiny neurons, which is induced postsynaptically via mGlu5 and expressed presynaptically via cannabinoid type 1 receptors (CB1Rs), 2) mGlu5 surface expression and physical associations between mGlu5, Homer proteins, and diacylglycerol lipase-α, and 3) the effect of systemic and intra-NAc core administration of the mGlu5 NAM 3-((2-methyl-4-)ethynyl)pyridine (MTEP) on expression of incubated methamphetamine craving. We found that mGlu5/CB1R-dependent synaptic depression was lost during the rising phase of methamphetamine incubation but then recovered, in contrast to its persistent impairment during the plateau phase of incubation of cocaine craving. Furthermore, whereas the cocaine-induced impairment was accompanied by reduced mGlu5 levels and mGlu5-Homer associations, this was not the case for methamphetamine. Systemic MTEP reduced incubated methamphetamine seeking, but also reduced inactive hole nose-pokes and locomotion, while intra-NAc core MTEP had no significant effects. These findings provide the first insight into the role of mGlu5 in the incubation of methamphetamine craving and reveal differences from incubation of cocaine craving.

AMPA receptor and metabotropic glutamate receptor 1 adaptations in the nucleus accumbens core during incubation of methamphetamine craving.

Cue-induced drug craving progressively intensifies after withdrawal from self-administration of cocaine, methamphetamine, and other drugs of abuse, a phenomenon termed incubation of craving. For cocaine and methamphetamine, expression of incubated craving ultimately depends on strengthening of nucleus accumbens (NAc) synapses through an accumulation of high conductance Ca2+-permeable AMPA receptors (CP-AMPARs) that is detectable with electrophysiological approaches. This study sought to further characterize glutamate receptor adaptations in NAc core during methamphetamine incubation. Previous biochemical studies revealed that the CP-AMPARs accumulating after cocaine incubation are mainly homomeric GluA1 receptors and that their accumulation is reflected by increased cell surface GluA1. Here, for methamphetamine, we observed no significant change in surface or total GluA1 (GluA2 and GluA3 were also unchanged). Nonetheless, GluA1 translation was elevated after incubation of methamphetamine craving, as recently found for cocaine. Additionally, for cocaine, we previously observed a withdrawal-dependent decrease in mGlu1 surface expression that precedes and enables CP-AMPAR accumulation and incubation of craving, reflecting weakening of mGlu1-dependent mechanisms that normally limit synaptic CP-AMPAR levels in the NAc core. Here, we observed no change in surface or total mGlu1 protein or its coupling to Homer scaffolding proteins after methamphetamine withdrawal, nor did elevation of mGlu1 tone through repeated injections of an mGlu1-positive allosteric modulator delay incubation of craving. These findings suggest a common role for increased GluA1 translation, but not decreased mGlu1 function, in the incubation of methamphetamine and cocaine craving. We speculate that increased GluA1 translation near synapses may drive formation and synaptic insertion of homomeric GluA1 receptors in the absence of detectable changes in GluA1 protein levels.

Cascades of Homeostatic Dysregulation Promote Incubation of Cocaine Craving.

In human drug users, cue-induced drug craving progressively intensifies after drug abstinence, promoting drug relapse. This time-dependent progression of drug craving is recapitulated in rodent models, in which rats exhibit progressive intensification of cue-induced drug seeking after withdrawal from drug self-administration, a phenomenon termed incubation of drug craving. Although recent results suggest that functional alterations of the nucleus accumbens (NAc) contribute to incubation of drug craving, it remains poorly understood how NAc function evolves after drug withdrawal to progressively intensify drug seeking. The functional output of NAc relies on how the membrane excitability of its principal medium spiny neurons (MSNs) translates excitatory synaptic inputs into action potential firing. Here, we report a synapse-membrane homeostatic crosstalk (SMHC) in male rats, through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic membrane excitability of NAc MSNs, and vice versa. After short-term withdrawal from cocaine self-administration, despite no actual change in the AMPA receptor-mediated excitatory synaptic strength, GluN2B NMDA receptors, the SMHC sensors of synaptic strength, are upregulated. This may create false SMHC signals, leading to a decrease in the membrane excitability of NAc MSNs. The decreased membrane excitability subsequently induces another round of SMHC, leading to synaptic accumulation of calcium-permeable AMPA receptors and upregulation of excitatory synaptic strength after long-term withdrawal from cocaine. Disrupting SMHC-based dysregulation cascades after cocaine exposure prevents incubation of cocaine craving. Thus, cocaine triggers cascades of SMHC-based dysregulation in NAc MSNs, promoting incubated cocaine seeking after drug withdrawal.SIGNIFICANCE STATEMENT Here, we report a bidirectional homeostatic plasticity between the excitatory synaptic input and membrane excitability of nucleus accumbens (NAc) medium spiny neurons (MSNs), through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the membrane excitability, and vice versa. Cocaine self-administration creates a false homeostatic signal that engages this synapse-membrane homeostatic crosstalk mechanism, and produces cascades of alterations in excitatory synapses and membrane properties of NAc MSNs after withdrawal from cocaine. Experimentally preventing this homeostatic dysregulation cascade prevents the progressive intensification of cocaine seeking after drug withdrawal. These results provide a novel mechanism through which drug-induced homeostatic dysregulation cascades progressively alter the functional output of NAc MSNs and promote drug relapse.

Withdrawal From Cocaine Self-administration Alters the Regulation of Protein Translation in the Nucleus Accumbens.

BACKGROUND: Cue-induced cocaine craving incubates during abstinence from cocaine self-administration. Expression of incubation ultimately depends on elevation of homomeric GluA1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in the nucleus accumbens (NAc). This adaptation requires ongoing protein translation for its maintenance. Aberrant translation is implicated in central nervous system diseases, but nothing is known about glutamatergic regulation of translation in the drug-naïve NAc or after incubation. METHODS: NAc tissue was obtained from drug-naïve rats and from rats after 1 or >40 days of abstinence from extended-access cocaine or saline self-administration. Newly translated proteins were labeled using 35S-Met/Cys or puromycin. We compared basal overall translation and its regulation by metabotropic glutamate receptor 1 (mGlu1), mGlu5, and N-methyl-D-aspartate receptors (NMDARs) in drug-naïve, saline control, and cocaine rats, and we compared GluA1 and GluA2 translation by immunoprecipitating puromycin-labeled proteins. RESULTS: In all groups, overall translation was unaltered by mGlu1 blockade (LY367385) but increased by mGlu5 blockade (MTEP). NMDAR blockade (AVP) increased overall translation in drug-naïve and saline control rats but not in cocaine/late withdrawal rats. Cocaine/late withdrawal rats exhibited greater translation of GluA1 (but not GluA2), which was not further affected by NMDAR blockade. CONCLUSIONS: Our results suggest that increased GluA1 translation contributes to the elevated homomeric GluA1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor levels in the NAc that mediate incubation. Additional contributions to incubation-related plasticity may result from loss of the braking influence on translation normally exerted by NMDARs. Apart from elucidating incubation-related adaptations, we found a suppressive effect of mGlu5 on NAc translation regardless of drug exposure, which is opposite to results obtained in the hippocampus and points to heterogeneity of translational regulation between brain regions.

Protein Translation in the Nucleus Accumbens Is Dysregulated during Cocaine Withdrawal and Required for Expression of Incubation of Cocaine Craving.

Exposure to drug-associated cues can induce drug craving and relapse in abstinent addicts. Cue-induced craving that progressively intensifies ("incubates") during withdrawal from cocaine has been observed in both rats and humans. Building on recent evidence that aberrant protein translation underlies incubation-related adaptations in the NAc, we used male rats to test the hypothesis that translation is dysregulated during cocaine withdrawal and/or when rats express incubated cocaine craving. We found that intra-NAc infusion of anisomycin, a general protein translation inhibitor, or rapamycin, an inhibitor of mammalian target of rapamycin, reduced the expression of incubated cocaine craving, consistent with previous results showing that inhibition of translation in slices normalized the adaptations that maintain incubation. We then examined signaling pathways involved in protein translation using NAc synaptoneurosomes prepared after >47 d of withdrawal from cocaine or saline self-administration, or after withdrawal plus a cue-induced seeking test. The most robust changes were observed following seeking tests. Most notably, we found that eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 2α (eIF2α) are dephosphorylated when cocaine rats undergo a cue-induced seeking test; both effects are consistent with increased translation during the test. Blocking eIF2α dephosphorylation and thereby restoring its inhibitory influence on translation, via intra-NAc injection of Sal003 just before the test, substantially reduced cocaine seeking. These results are consistent with dysregulation of protein translation in the NAc during cocaine withdrawal, enabling cocaine cues to elicit an aberrant increase in translation that is required for the expression of incubated cocaine craving.SIGNIFICANCE STATEMENT Cue-induced cocaine craving progressively intensifies (incubates) during withdrawal in both humans and rats. This may contribute to persistent vulnerability to relapse. We previously demonstrated a role for protein translation in synaptic adaptations in the NAc closely linked to incubation. Here, we tested the hypothesis that translation is dysregulated during cocaine withdrawal, and this contributes to incubated craving. Analysis of signaling pathways regulating translation suggested that translation is enhanced when "incubated" rats undergo a cue-induced seeking test. Furthermore, intra-NAc infusions of drugs that inhibit protein translation through different mechanisms reduced expression of incubated cue-induced cocaine seeking. These results demonstrate that the expression of incubation depends on an acute increase in translation that may result from dysregulation of several pathways.

Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration.

It is well established that cocaine-induced changes in glutamate receptor expression in the nucleus accumbens (NAc) play a significant role in animal models of cocaine addiction. Far less is known about cocaine-induced changes in GABA transmission, despite its importance in regulating NAc output via local interneurons and medium spiny neuron (MSN) axon collaterals (GABA 'microcircuit'). Here we investigated whether GABAA receptor surface or total expression is altered following an extended-access cocaine self-administration regimen that produces a time-dependent intensification (incubation) of cue-induced cocaine craving in association with strengthening of AMPA receptor (AMPAR) transmission onto MSN. Rats self-administered cocaine or saline (control condition) 6h/day for 10 days. NAc tissue was obtained and surface proteins biotinylated on three withdrawal days (WD) chosen to span incubation of craving and associated AMPAR plasticity: WD2, WD25 and WD48. Immunoblotting was used to measure total and surface expression of three GABAA receptor subunits (α1, α2, and α4) that are strongly expressed in the NAc. We found a transient increase in surface, but not total, expression of the α2 subunit on WD2 from cocaine self-administration, an effect that was no longer observed by WD25. The expression of α1 and α4 subunits was not altered at these withdrawal times. On WD48, when AMPAR transmission is significantly potentiated, we did not find any alteration in GABAA receptor surface or total expression. Our findings suggest that the strengthening of AMPAR-mediated glutamate transmission in the NAc is not accompanied by compensatory strengthening of GABAergic transmission through insertion of additional GABAA receptors.

Response of the Ubiquitin-Proteasome System to Memory Retrieval After Extended-Access Cocaine or Saline Self-Administration.

The ubiquitin-proteasome system (UPS) has been implicated in the retrieval-induced destabilization of cocaine- and fear-related memories in Pavlovian paradigms. However, nothing is known about its role in memory retrieval after self-administration of cocaine, an operant paradigm, or how the length of withdrawal from cocaine may influence retrieval mechanisms. Here, we examined UPS activity after an extended-access cocaine self-administration regimen that leads to withdrawal-dependent incubation of cue-induced cocaine craving. Controls self-administered saline. In initial experiments, memory retrieval was elicited via a cue-induced seeking/retrieval test on withdrawal day (WD) 50-60, when craving has incubated. We found that retrieval of cocaine- and saline-associated memories produced similar increases in polyubiquitinated proteins in the nucleus accumbens (NAc), compared with rats that did not undergo a seeking/retrieval test. Measures of proteasome catalytic activity confirmed similar activation of the UPS after retrieval of saline and cocaine memories. However, in a subsequent experiment in which testing was conducted on WD1, proteasome activity in the NAc was greater after retrieval of cocaine memory than saline memory. Analysis of other brain regions confirmed that effects of cocaine memory retrieval on proteasome activity, relative to saline memory retrieval, depend on withdrawal time. These results, combined with prior studies, suggest that the relationship between UPS activity and memory retrieval depends on training paradigm, brain region, and time elapsed between training and retrieval. The observation that mechanisms underlying cocaine memory retrieval change depending on the age of the memory has implications for development of memory destabilization therapies for cue-induced relapse in cocaine addicts.

Synaptic depression via mGluR1 positive allosteric modulation suppresses cue-induced cocaine craving.

Cue-induced cocaine craving is a major cause of relapse in abstinent addicts. In rats, cue-induced craving progressively intensifies (incubates) during withdrawal from extended-access cocaine self-administration. After ~1 month of withdrawal, incubated craving is mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs) that accumulate in the nucleus accumbens (NAc). We found that decreased mGluR1 surface expression in the NAc preceded and enabled CP-AMPAR accumulation. Thus, restoring mGluR1 transmission by administering repeated injections of an mGluR1 positive allosteric modulator (PAM) prevented CP-AMPAR accumulation and incubation, whereas blocking mGluR1 transmission at even earlier withdrawal times accelerated CP-AMPAR accumulation. In studies conducted after prolonged withdrawal, when CP-AMPAR levels and cue-induced craving are high, we found that systemic administration of an mGluR1 PAM attenuated the expression of incubated craving by reducing CP-AMPAR transmission in the NAc to control levels. These results suggest a strategy in which recovering addicts could use a systemically active compound to protect against cue-induced relapse.

AMPA receptor upregulation in the nucleus accumbens shell of cocaine-sensitized rats depends upon S-nitrosylation of stargazin.

Behavioral sensitization to cocaine is associated with increased AMPA receptor (AMPAR) surface expression in the nucleus accumbens (NAc). This upregulation is withdrawal-dependent, as it is not detected on withdrawal day (WD) 1, but is observed on WD7-21. Its underlying mechanisms have not been clearly established. Nitric oxide (NO) regulates AMPAR trafficking in the brain by S-nitrosylation of the AMPAR auxiliary subunit, stargazin, leading to increased AMPAR surface expression. Our goal was to determine if stargazin S-nitrosylation contributes to AMPAR upregulation during sensitization. First, we measured stargazin S-nitrosylation in NAc core and shell subregions on WD14 after 8 daily injections of saline or 15 mg/kg cocaine. Stargazin S-nitrosylation was markedly increased in NAc shell but not core. To determine if this is associated with AMPAR upregulation, rats received 8 cocaine or saline injections followed by twice-daily treatments with vehicle or the nitric oxide synthase inhibitor l-NAME (50 mg/kg) on WD1-6, the time when AMPAR upregulation is developing in cocaine-exposed rats. Cocaine/vehicle rats showed elevated stargazin and GluA1 surface expression on WD7 compared to saline/vehicle rats; the GluA1 increase was more robust in core, while stargazin increased more robustly in shell. These effects of cocaine were attenuated in shell but not core when cocaine injections were followed by l-NAME treatment on WD1-6. Together, these results indicate that elevated S-nitrosylation of stargazin contributes to AMPAR upregulation during sensitization selectively in the NAc shell. It is possible that AMPAR upregulation in core involves a different TARP, γ4, which also upregulates in the NAc of sensitized rats.

An investigation of interactions between hypocretin/orexin signaling and glutamate receptor surface expression in the rat nucleus accumbens under basal conditions and after cocaine exposure.

Hypocretin peptides are critical for the effects of cocaine on excitatory synaptic strength in the ventral tegmental area (VTA). However, little is known about their role in cocaine-induced synaptic plasticity in the nucleus accumbens (NAc). First, we tested whether hypocretin-1 by itself could acutely modulate glutamate receptor surface expression in the NAc, given that hypocretin-1 in the VTA reproduces cocaine's effects on glutamate transmission. We found no effect of hypocretin-1 infusion on AMPA or NMDA receptor surface expression in the NAc, measured by biotinylation, either 30 min or 3h after the infusion. Second, we were interested in whether changes in hypocretin receptor-2 (Hcrtr-2) expression contribute to cocaine-induced plasticity in the NAc. As a first step towards addressing this question, Hcrtr-2 surface expression was compared in the NAc after withdrawal from extended-access self-administration of saline (control) versus cocaine. We found that surface Hcrtr-2 levels remain unchanged following 14, 25 or 48 days of withdrawal from cocaine, a time period in which high conductance GluA2-lacking AMPA receptors progressively emerge in the NAc. Overall, our results fail to support a role for hypocretins in acute modulation of glutamate receptor levels in the NAc or a role for altered Hcrtr-2 expression in withdrawal-dependent synaptic adaptations in the NAc following cocaine self-administration.

A protein cross-linking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments.

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface-expressed proteins are covalently cross-linked using the membrane-impermeable, bifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS(3)). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross-linking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants.

Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca²âº-permeable AMPA receptors during the incubation of cocaine craving.

Cue-induced cocaine seeking intensifies or incubates after withdrawal from extended access cocaine self-administration, a phenomenon termed incubation of cocaine craving. The expression of incubated craving is mediated by Ca²âº-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Thus, CP-AMPARs are a potential target for therapeutic intervention, making it important to understand mechanisms that govern their accumulation. Here we used subcellular fractionation and biotinylation of NAc tissue to examine the abundance and distribution of AMPAR subunits, and GluA1 phosphorylation, in the incubation model. We also studied two transmembrane AMPA receptor regulatory proteins (TARPs), γ-2 and γ-4. Our results, together with earlier findings, suggest that some of the new CP-AMPARs are synaptic. These are probably associated with γ-2, but they are loosely tethered to the PSD. Levels of GluA1 phosphorylated at serine 845 (pS845 GluA1) were significantly increased in biotinylated tissue and in an extrasynaptic membrane-enriched fraction. These results suggest that increased synaptic levels of CP-AMPARs may result in part from an increase in pS845 GluA1 in extrasynaptic membranes, given that S845 phosphorylation primes GluA1-containing AMPARs for synaptic insertion and extrasynaptic AMPARs supply the synapse. Some of the new extrasynaptic CP-AMPARs are likely associated with γ-4, rather than γ-2. The maintenance of CP-AMPARs in NAc synapses during withdrawal is accompanied by activation of CaMKII and ERK2 but not CaMKI. Overall, AMPAR plasticity in the incubation model shares some features with better described forms of synaptic plasticity, although the timing of the phenomenon and the persistence of related neuroadaptations are significantly different.

Distribution of AMPA receptor subunits and TARPs in synaptic and extrasynaptic membranes of the adult rat nucleus accumbens.

We characterized the distribution of AMPA receptor (AMPAR) subunits and the transmembrane AMPA receptor regulatory proteins (TARPs) γ-2 and γ-4 in adult rat nucleus accumbens (NAc) using a method that separates plasma membranes into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions. We also measured GluA1 phosphorylated at serine 845 (pS845 GluA1) and serine 831 (pS831 GluA1). GluA1-3 protein levels and pS831 GluA1/total GluA1 were higher in synaptic membranes. However, pS845 GluA1/total GluA1 was higher in extrasynaptic membranes, consistent with a role for S845 phosphorylation in GluA1 insertion at extrasynaptic sites. Homeric GluA1 receptors were detected in extrasynaptic membranes, consistent with evidence for extrasynaptic Ca(2+)-permeable AMPARs in other systems. The TARP γ-2 was enriched in synaptic membranes, whereas γ-4 was mainly found in extrasynaptic membranes, suggesting distinct roles for these proteins in the NAc. These experiments provide fundamental information that will aid in the interpretation of studies on AMPAR-related plasticity in the NAc.