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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 (CP-AMPARs) that accumulate in synapses onto medium spiny neurons (MSN) in the nucleus accumbens (NAc) core. However, the delay in their accumulation (approximately 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 days after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 days of cocaine withdrawal and this persisted for at least 68 days 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 (MSN) of the nucleus accumbens 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.

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 Ca

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 STATEMENTHere, 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.

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 STATEMENTCue-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.

Background The incubation of cue-induced drug craving in rodents provides a model of persistent vulnerability to craving and relapse in human addicts. After prolonged withdrawal, incubated cocaine craving depends on strengthening of nucleus accumbens (NAc) core synapses through incorporation of Ca 2+ -permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (CP-AMPARs). Through metabotropic glutamate receptor 1 (mGluR1)–mediated synaptic depression, mGluR1 positive allosteric modulators remove CP-AMPARs from these synapses and thereby reduce cocaine craving. This study aimed to determine if similar plasticity accompanies incubation of methamphetamine craving. Methods Rats self-administered saline or methamphetamine under extended-access conditions. Cue-induced seeking tests demonstrated incubation of methamphetamine craving. After withdrawal periods ranging from 1 to >40 days, rats underwent one of the following procedures: 1) whole-cell patch clamp recordings to characterize AMPAR transmission, 2) intra–NAc core injection of the CP-AMPAR antagonist 1-naphthyl acetyl spermine followed by a seeking test, or 3) systemic administration of a mGluR1 positive allosteric modulator followed by a seeking test. Results Incubation of methamphetamine craving was associated with CP-AMPAR accumulation in NAc core, and both effects were maximal after ~1 week of withdrawal. Expression of incubated craving was decreased by intra–NAc core 1-naphthyl acetyl spermine injection or systemic mGluR1 positive allosteric modulator administration. Conclusions These results are the first to demonstrate a role for the NAc in the incubation of methamphetamine craving and describe adaptations in synaptic transmission associated with this model. They establish that incubation of craving and associated CP-AMPAR plasticity occur much more rapidly during withdrawal from methamphetamine compared with cocaine. However, a common mGluR1-based therapeutic strategy may be helpful for recovering cocaine and methamphetamine addicts.

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 Ca(2+)-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.

Animal models of relapse reveal that the motivation to seek drug is regulated by enduring morphological and physiological changes in the nucleus accumbens, as well as transient synaptic potentiation in the accumbens core (NAcore) that parallels drug-seeking behavior. The current study sought to examine the link between the behavioral and synaptic consequences of cue-induced cocaine seeking by optically silencing glutamatergic afferents to the NAcore from the prelimbic cortex (PL). Adeno-associated virus coding for the inhibitory opsin archaerhodopsin was microinjected into PL, and optical fibers were targeted to NAcore. Animals were trained to self-administer cocaine followed by extinction training, and then underwent cue-induced reinstatement in the presence or absence of 15 min of optically-induced inhibition of PL fibers in NAcore. Inhibiting the PL-to-NAcore projection blocked reinstated behavior and was paralleled by decreased dendritic spine head diameter and AMPA/NMDA ratio relative to sham-laser control rats. Interestingly, while spine density was elevated after extinction training, no further effects were observed by cued reinstatement or optical inhibition. These findings validate the critical role for PL afferents to the NAcore in simultaneously regulating both reinstated behavior and the associated transient synaptic potentiation.

The regulation of protein translation by glutamate receptors and its role in plasticity have been extensively studied in the hippocampus. In contrast, very little is known about glutamatergic regulation of translation in nucleus accumbens (NAc) medium spiny neurons (MSN), despite their critical role in addiction-related plasticity and recent evidence that protein translation contributes to this plasticity. We used a co-culture system, containing NAc MSNs and prefrontal cortex (PFC) neurons, and fluorescent non-canonical amino acid tagging (FUNCAT) to visualize newly synthesized proteins in neuronal processes of NAc MSNs and PFC pyramidal neurons. First, we verified that the FUNCAT signal reflects new protein translation. Next, we examined the regulation of translation by group I metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors by incubating co-cultures with agonists or antagonists during the 2-h period of non-canonical amino acid labeling. In NAc MSNs, basal translation was modestly reduced by blocking Ca(2+)-permeable AMPARs whereas blocking all AMPARs or suppressing constitutive mGluR5 signaling enhanced translation. Activating group I mGluRs with dihydroxyphenylglycine increased translation in an mGluR1-dependent manner in NAc MSNs and PFC pyramidal neurons. Disinhibiting excitatory transmission with bicuculline also increased translation. In MSNs, this was reversed by antagonists of mGluR1, mGluR5, AMPARs or NMDARs. In PFC neurons, AMPAR or NMDAR antagonists blocked bicuculline-stimulated translation. Our study, the first to examine glutamatergic regulation of translation in MSNs, demonstrates regulatory mechanisms specific to MSNs that depend on the level of neuronal activation. This sets the stage for understanding how translation may be altered in addiction.

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-naive NAc or after incubation. Methods NAc tissue was obtained from drug-naive 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-naive, 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-naive 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.

Inhibitory optogenetics was used to examine the roles of the prelimbic cortex (PL), the nucleus accumbens core (NAcore) and the PL projections to the NAcore in the reinstatement of cocaine seeking. Rats were microinjected into the PL or NAcore with an adeno-associated virus containing halorhodopsin or archaerhodopsin. After 12 days of cocaine self-administration, followed by extinction training, animals underwent reinstatement testing along with the presence/absence of optically induced inhibition via laser light. Bilateral optical inhibition of the PL, NAcore or the PL fibers in the NAcore inhibited the reinstatement of cocaine seeking.

The core subcompartment of the nucleus accumbens (NAcore) contributes significantly to behavioral responses following motivationally relevant stimuli, including drug-induced, stress-induced, and cue-induced reinstatement of cocaine seeking. Projections from NAcore that could carry information necessary to initiate reinstated cocaine seeking include outputs via the indirect pathway to the dorsolateral subcompartment of the ventral pallidum (dlVP) and through the direct pathway to the medial substantia nigra (SN). Here we used an optogenetic strategy to determine whether the dlVP or nigral projections from the NAcore are necessary for cocaine seeking initiated by a cocaine and conditioned cue combination in rats extinguished from cocaine self-administration. Rats were pretreated in the NAcore with an adeno-associated virus expressing the inhibitory opsin archaerhodopsin, and fiber-optic cannulae were implanted above the indirect pathway axon terminal field in the dlVP, or the direct pathway terminal field in the SN. Inhibiting the indirect pathway to the dlVP, but not the direct pathway to the SN, prevented cocaine-plus-cue-induced reinstatement. We also examined projections back to the NAcore from the ventral tegmental area (VTA) and dlVP. Inhibiting the dlVP to NAcore projection did not alter, while inhibiting VTA afferents abolished reinstated cocaine seeking. Localization of green fluorescent protein reporter expression and whole-cell patch electrophysiology were used to verify opsin expression. These data reveal a circuit involving activation of VTA inputs to the NAcore and NAcore projections through the indirect pathway to the dlVP as critical for cocaine-plus-cue-induced reinstatement of cocaine seeking.

Stimuli previously associated with drugs of abuse can become triggers that elicit craving and lead to drug-seeking behavior. The basolateral amygdala (BLA) is a key neural structure involved in cue-induced reinstatement of cocaine seeking. Previous studies have also implicated projections from the BLA directly to the nucleus accumbens (NAc) in these behaviors. However, other structures critically involved in cocaine seeking are targets of BLA innervation, including the prelimbic prefrontal cortex (PL). It has been shown that BLA or PL innervation direct to the NAc can modulate reward-related behaviors but the BLA also projects to the PL, and given the importance of the PL projection to the NAc for reinstated drug seeking, we hypothesized the BLA to PL projection may indirectly influence behavior via PL innervation to the NAc. We delivered a virus expressing the inhibitory optogenetic construct ArchT into the BLA and implanted fiber optics above the injection site or axon terminal fields in either the NAc or PL. Rats then went through 12 days of cocaine self-administration followed by extinction training. Following extinction, animals underwent cue-induced reinstatement sessions in the presence or absence of optical inhibition. Inactivation of the BLA and either the BLA core subcompartment of the NAc (BLA-to-NAcore) BLA-to-PL projections inhibited cue-induced reinstatement. These data demonstrate that the BLA projection either directly into the NAc, or indirectly via the PL, is a necessary regulator of drug-seeking behavior.