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

A key in understanding the neurobiology of addiction and developing effective pharmacotherapies is revealing drug-induced plasticity that results in heightened relapse susceptibility. Previous studies have demonstrated that increased extracellular glutamate, but not dopamine, in the nucleus accumbens core (NAcc) is necessary for cocaine-induced reinstatement. In this report, we examined whether drug-induced adaptations that are necessary to generate cocaine-induced reinstatement also determine relapse vulnerability. To do this, rats were assigned to self-administer cocaine under conditions resulting in low (2 h/d; 0.5 mg/kg/infusion, i.v.) or high (6 h/d; 1.0 mg/kg/infusion, i.v.) levels of drug intake since these manipulations produce groups of rats exhibiting differences in the magnitude of cocaine-induced reinstatement. Approximately 19 d after the last session, cocaine-induced drug seeking and extracellular levels of glutamate and dopamine in the NAcc were measured. Contrary to our hypothesis, high-intake rats exhibited a more robust cocaineinduced increase in extracellular levels of dopamine but not glutamate. Further, increased reinstatement in high-intake rats was no longer observed when the D 1 receptor antagonist SCH-23390 was infused into the NAcc. The sensitized dopamine response to cocaine in high-intake rats may involve blunted cystine-glutamate exchange by system x c Ϫ. Reduced 14 C-cystine uptake through system x c Ϫ was evident in NAcc tissue slices obtained from high-intake rats, and the augmented dopamine response in these rats was no longer observed when subjects received the cysteine prodrug N-acetyl cysteine. These data reveal a role for drug-induced NAcc dopamine in heightened relapse vulnerability observed in rats with a history of high levels of drug intake.

Factors that result in augmented reinstatement, including increased withdrawal period duration and high levels of cocaine consumption, may provide insight into relapse vulnerability. The neural basis of augmented reinstatement may arise from more pronounced changes in plasticity required for reinstatement and/or the emergence of plasticity expressed only during a specific withdrawal period or under specific intake conditions. In this study, we examined the impact of withdrawal period duration and cocaine intake on the magnitude of cocaine-primed reinstatement and extracellular glutamate in the nucleus accumbens, which has been shown to be required for cocaine-primed reinstatement. Rats were assigned to self-administer under conditions resulting in low (2 hr/day; 0.5 mg/kg/infusion, IV) or high (6 hr/day; 1.0 mg/kg/infusion, IV) levels of cocaine intake. After 1, 21, or 60 days of withdrawal, drug seeking and extracellular glutamate levels in the nucleus accumbens were measured before and after a cocaine injection. Cocaine reinstated lever pressing and elevated extracellular glutamate at every withdrawal time point tested, which is consistent with the conclusion that increased glutamatergic signaling in the nucleus accumbens is required for cocaine-induced reinstatement. Interestingly, high-intake rats exhibited augmented reinstatement at every time point tested, yet failed to exhibit higher levels of cocaine-induced increases in extracellular glutamate relative to low-intake rats. Our current data indicate that augmented reinstatement in high-intake rats is not due to relative differences in extracellular levels of glutamate in the nucleus accumbens, but rather may stem from intake-dependent plasticity.