Cue-elicited drug-craving is a cardinal feature of addiction that intensifies (incubates) during protracted withdrawal. In a rat model, these addiction-related behavioral pathologies are mediated, respectively, by time-dependent increases in PI3K/Akt1 signaling and reduced Group 1 metabotropic glutamate receptor (mGlu) expression, within the ventromedial prefrontal cortex (vmPFC). Herein, we examined the capacity of single oral dosing with everolimus, an FDA-approved inhibitor of the PI3K/Akt effector mTOR, to reduce incubated cocaine-craving and reverse incubation-associated changes in vmPFC kinase activity and mGlu expression. Rats were trained to lever-press for intravenous infusions of cocaine or delivery of sucrose pellets and then subjected to tests for cue-reinforced responding during early (3 days) or late (30–46 days) withdrawal. Rats were gavage-infused with everolimus (0–1.0 mg/kg), either prior to testing to examine for effects upon reinforcer-seeking behavior, or immediately following testing to probe effects upon the consolidation of extinction learning. Single oral dosing with everolimus dose-dependently blocked cocaine-seeking during late withdrawal and the effect lasted at least 24 h. No everolimus effects were observed for cue-elicited sucrose-seeking or cocaine-seeking in early withdrawal. In addition, everolimus treatment, following initial cue-testing, reduced subsequent cue hyper-responsivity exhibited observed during late withdrawal, arguing a facilitation of extinction memory consolidation. everolimus’ “anti-incubation” effect was associated with a reversal of withdrawal-induced changes in indices of PI3K/Akt1/mTOR activity, as well as Homer protein and mGlu1/5 expression, within the prelimbic (PL) subregion of the prefrontal cortex. Our results indicate mTOR inhibition as a viable strategy for interrupting heightened cocaine-craving and facilitating addiction recovery during protracted withdrawal.

IntroductionIncubation of drug-craving refers to a time-dependent increase in drug cue-elicited craving that occurs during protracted withdrawal. Historically, rat models of incubated cocaine craving employed extended-access (typically 6 h/day) intravenous drug self-administration (IV-SA) procedures, although incubated cocaine craving is reported to occur following shorter-access IV-SA paradigms. The notoriously low-throughput of extended-access IV-SA prompted us to determine whether two different short-access IV-SA procedures akin to those in the literature result in qualitatively similar changes in glutamate receptor expression and the activation of downstream signaling molecules within prefrontal cortex (PFC) subregions as those reported previously by our group under 6h-access conditions.MethodsFor this, adult, male Sprague-Dawley rats were trained to intravenously self-administer cocaine for 2 h/day for 10 consecutive days (2-h model) or for 6 h on day 1 and 2 h/day for the remaining 9 days of training (Mixed model). A sham control group was also included that did not self-administer cocaine.ResultsOn withdrawal day 3 or 30, rats were subjected to a 2-h test of cue-reinforced responding in the absence of cocaine and a time-dependent increase in drug-seeking was observed under both IV-SA procedures. Immunoblotting of brain tissue collected immediately following the cue test session indicated elevated phospho-Akt1, phospho-CaMKII and Homer2a/b expression within the prelimbic subregion of the PFC of cocaine-incubated rats. However, we failed to detect incubation-related changes in Group 1 metabotropic glutamate receptor or ionotropic glutamate receptor subunit expression in either subregion.DiscussionThese results highlight further a role for Akt1-related signaling within the prelimbic cortex in driving incubated cocaine craving, and provide novel evidence supporting a potential role also for CaMKII-dependent signaling through glutamate receptors in this behavioral phenomenon.

The glutamatergic system is purported to play an important role in the self‐administration of both drug and non‐drug reinforcers, such as sucrose. Sucrose‐experienced rats exhibit blunted glutamate release within the ventromedial prefrontal cortex (vmPFC) while cocaine‐experienced rats exhibit increased cue‐induced glutamate release within the vmPFC. While the behavioral phenomenon seems to be similar, there may be different mechanisms of glutamate in cocaine‐ and sucrose‐seeking. To address this question, this study investigated the role of vmPFC AMPA‐ and NMDA ‐type glutamate receptors in sucrose‐taking. For this, male and female Sprague‐Dawley rats were implanted with bilateral guide cannulae within the vmPFC and were trained to self‐administer sucrose pellets (6hr/day) for 10 days. Using a within‐subjects design, rats were microinjected (0.5 μl per side) with the GluA2‐lacking selective AMPA antagonist Naspm (40 μg), the non‐selective AMPA receptor antagonist NBQX (1 μg), the GluN2A‐selective NMDA antagonist PPPA (0.43 μg), the GluN2B‐selective NMDA antagonist TCN‐237 (0.002 μg) and the non‐selective NMDA receptor antagonist D‐AP5 (2.5 μg). Following each microinjection, rats underwent a 2‐hr test for sucrose self‐administration. In males, microinjection of D‐AP5 increased responding for sucrose reinforcement, while no significant effects were observed for the other compounds tested. While in females, there were no significant effects observed for any of the compounds tested. These results indicate that activation of NMDA receptors within the vmPFC suppress sucrose self‐administration in male rats, whereas AMPA receptors do not appear to be involved.

Cue-elicited drug-craving is a cardinal feature of addiction that intensifies (incubates) during protracted withdrawal. In a rat model, these addiction-related behavioral pathologies are mediated, respectively, by time-dependent increases in PI3K/Akt1 signaling and reduced Group 1 metabotropic glutamate receptor (mGlu) expression, within the ventromedial prefrontal cortex (vmPFC). Herein, we examined the capacity of single oral dosing with Everolimus, an FDA-approved inhibitor of the PI3K/Akt effector mTOR, to reduce incubated cocaine-craving and reverse incubation-associated changes in vmPFC kinase activity and mGlu expression. Rats were trained to lever-press for intravenous infusions of cocaine or delivery of sucrose pellets and then subjected to tests for cue-reinforced responding during early (3 days) or late (30-46 days) withdrawal. Rats were gavage-infused with Everolimus (0-1.0 mg/kg), either prior to testing to examine for effects upon reinforcer-seeking behavior, or immediately following testing to probe effects upon the consolidation of extinction learning. Single oral dosing with Everolimus dose-dependently blocked cocaine-seeking during late withdrawal and the effect lasted at least 24 h. No Everolimus effects were observed for cue-elicited sucrose-seeking or cocaine-seeking in early withdrawal. Additionally, Everolimus treatment, following initial cue-testing, reduced subsequent cue hyper-responsivity exhibited observed during late withdrawal, arguing a facilitation of extinction memory consolidation. Everolimus’ “anti-incubation” effect was associated with a reversal of withdrawal-induced changes in indices of PI3K/Akt1/mTOR activity, as well as Homer protein and mGlu1/5 expression, within the prelimbic (PL) subregion of the prefrontal cortex. Our results indicate mTOR inhibition as a viable strategy for interrupting heightened cocaine-craving and facilitating addiction recovery during protracted withdrawal.