Cocaine produces a persistent reduction in cystine-glutamate exchange via system x c Ϫ in the nucleus accumbens that may contribute to pathological glutamate signaling linked to addiction. System x c Ϫ influences glutamate neurotransmission by maintaining basal, extracellular glutamate in the nucleus accumbens, which, in turn, shapes synaptic activity by stimulating group II metabotropic glutamate autoreceptors. In the present study, we tested the hypothesis that a long-term reduction in system x c Ϫ activity is part of the plasticity produced by repeated cocaine that results in the establishment of compulsive drug seeking. To test this, the cysteine prodrug N-acetylcysteine was administered before daily cocaine to determine the impact of increased cystine-glutamate exchange on the development of plasticity-dependent cocaine seeking. Although N-acetylcysteine administered before cocaine did not alter the acute effects of cocaine on self-administration or locomotor activity, it prevented behaviors produced by repeated cocaine including escalation of drug intake, behavioral sensitization, and cocaine-primed reinstatement. Because sensitization or reinstatement was not evident even 2-3 weeks after the last injection of N-acetylcysteine, we examined whether N-acetylcysteine administered before daily cocaine also prevented the persistent reduction in system x c Ϫ activity produced by repeated cocaine. Interestingly, N-acetylcysteine pretreatment prevented cocaine-induced changes in [35 S]cystine transport via system x c Ϫ, basal glutamate, and cocaine-evoked glutamate in the nucleus accumbens when assessed at least 3 weeks after the last N-acetylcysteine pretreatment. These findings indicate that N-acetylcysteine selectively alters plasticity-dependent behaviors and that normal system x c Ϫ activity prevents pathological changes in extracellular glutamate that may be necessary for compulsive drug seeking.

Repeated cocaine alters glutamate neurotransmission, in part, by reducing cystine-glutamate exchange via system x c -, which maintains glutamate levels and receptor stimulation in the extrasynaptic compartment. In the present study, we undertook two approaches to determine the significance of plasticity involving system x c -. First, we examined whether the cysteine prodrug Nacetylcysteine attenuates cocaine-primed reinstatement by targeting system x c -. Rats were trained to self-administer cocaine (1 mg/kg/200 µl, IV) under extended access conditions (6 hr/day). After extinction training, cocaine (10 mg/kg, IP) primed reinstatement was assessed in rats pretreated with N-acetylcysteine (0-60 mg/kg, IP) in the presence or absence of the system x c -inhibitor (S)-4-carboxyphenylglycine (CPG; 0.5 µM; infused into the nucleus accumbens). N-acetylcysteine attenuated cocaine-primed reinstatement, and this effect was reversed by co-administration of CPG. Secondly, we examined whether reduced system x c -activity is necessary for cocaine-primed reinstatement. To do this, we administered N-acetylcysteine (0 or 90 mg/kg, IP) prior to twelve daily self-administration sessions (1 mg/kg/200 µl, IV; 6 hr/day) since this procedure has previously been shown to prevent reduced activity of system x c -. On the reinstatement test day, we then acutely impaired system x c -in some of the rats by infusing CPG (0.5 µM) into the nucleus accumbens. Rats that had received N-acetylcysteine prior to daily self-administration sessions exhibited diminished cocaine-primed reinstatement; this effect was reversed by infusing the cystine-glutamate exchange inhibitor CPG into the nucleus accumbens. Collectively these data establish system x c -in the nucleus accumbens as a key mechanism contributing to cocaine-primed reinstatement. Keywords extrasynaptic; nonvesicular; glutamate; microdialysis; cystine-glutamate antiporter; reinstatement Long-term plasticity resulting in altered excitatory neurotransmission within corticostriatal pathways has been implicated in addiction. Human cocaine abusers exposed to cravinginducing stimuli exhibit increased activation of excitatory circuits originating in cortical regions, including orbital or prefrontal cortex, and projecting to the ventral striatum (Breiter et al., 1997;Dackis and O'Brien, 2005;Volkow et al., 2005). Preclinical data indicate that an injection of cocaine increases Fos protein expression throughout the corticostriatal pathway in

Altered glutamate signaling contributes to a myriad of neural disorders, including schizophrenia. While synaptic levels are intensely studied, nonvesicular release mechanisms, including cystine-glutamate exchange, maintain high steady-state glutamate levels in the extrasynaptic space. The existence of extrasynaptic receptors, including metabotropic group II glutamate receptors (mGluR), pose nonvesicular release mechanisms as unrecognized targets capable of contributing to pathological glutamate signaling. We tested the hypothesis that activation of cystine-glutamate antiporters using the cysteine prodrug N-acetylcysteine would blunt psychotomimetic effects in the rodent phencyclidine (PCP) model of schizophrenia. First, we demonstrate that PCP elevates extracellular glutamate in the prefrontal cortex, an effect that is blocked by N-acetylcysteine pretreatment. To determine the relevance of the above finding, we assessed social interaction and found that N-acetylcysteine reverses social withdrawal produced by repeated PCP. In a separate paradigm, acute PCP resulted in working memory deficits assessed using a discrete trial t-maze task, and this effect was also reversed by Nacetylcysteine pretreatment. The capacity of N-acetylcysteine to restore working memory was blocked by infusion of the cystineglutamate antiporter inhibitor (S)-4-carboxyphenylglycine into the prefrontal cortex or systemic administration of the group II mGluR antagonist LY341495 indicating that the effects of N-acetylcysteine requires cystine-glutamate exchange and group II mGluR activation. Finally, protein levels from postmortem tissue obtained from schizophrenic patients revealed significant changes in the level of xCT, the active subunit for cystine-glutamate exchange, in the dorsolateral prefrontal cortex. These data advance cystine-glutamate antiporters as novel targets capable of reversing the psychotomimetic effects of PCP.

BACKGROUND Alcohol use among adolescents is widespread and a growing concern due to long-term behavioral deficits, including altered Pavlovian behavior, that potentially contribute to addiction vulnerability. We tested the hypothesis that adolescent intermittent ethanol (AIE) exposure alters Pavlovian behavior in males and females as measured by a shift from goal-tracking to sign-tracking. Additionally, we investigated GLT-1, an astrocytic glutamate transporter, as a potential contributor to a sign-tracking phenotype. METHODS Male and female Sprague-Dawley rats were exposed to AIE (5g/Kg, intragastric) or water intermittently 2 days on, 2 days off from postnatal day (P) 25 to 54. Around P70, animals began 20 daily sessions of Pavlovian conditioned approach, where they learned that a cue predicted non-contingent reward delivery. Lever pressing indicated interaction with the cue, or sign-tracking, and receptacle entries indicated approach to the reward delivery location, or goal-tracking. To test for effects of AIE on nucleus accumbens excitatory signaling, we isolated membrane subfractions and measured protein levels of the glutamate transporter GLT-1 after animals completed behavior as a measure of glutamate homeostasis. RESULTS Females exhibited elevated sign-tracking compared to males with significantly more lever presses, faster latency to first lever press, and greater probability to lever press in a trial. AIE significantly increased lever pressing while blunting goal tracking, as indicated by fewer cue-evoked receptacle entries, slower latency to receptacle entry, and lower probability to enter the receptacle in a trial. No significant Sex-by-Exposure interactions were observed in sign- or goal-tracking metrics. Moreover, we found no significant effects of Sex or Exposure on membrane GLT-1 expression in the nucleus accumbens. CONCLUSIONS Females exhibited enhanced sign-tracking compared to males, while AIE decreased goal-tracking compared to control exposure. Our findings support the hypothesis that adolescent binge ethanol can shift conditioned behavior from goal- to cue-directed in Pavlovian conditioned approach, especially in females.

The research in the current study used mice single-or group-housed on a 12 h light/dark cycle with food and water available ad libitum with experiments run during the light portion. All experiments were approved by the University of Minnesota and Marquette University Institutional Animal Care and Use Committee. The following funding sources made the study possible: National Institute for Neurological Disorders and Stroke (P30 NS062158); National Institute on Drug Abuse grant K99 DA038706 (to M.H.), R00DA038706 (M.H.), R00DA038706-04S1 (A.C.M), R01DA019666 (M.J.T.), K02DA035459 (M.J.T.) and T32 DA007234 (A.E.I.)..