The selective serotonergic neurotoxicity of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) depends on their systemic metabolism. We have recently shown that inhibition of brain endothelial cell ␥-glutamyl transpeptidase (␥-GT) potentiates the neurotoxicity of both MDMA and MDA, indicating that metabolites that are substrates for this enzyme contribute to the neurotoxicity. Consistent with this view, glutathione (GSH) and N-acetylcysteine conjugates of ␣-methyl dopamine (␣-MeDA) are selective neurotoxicants. However, neurotoxic metabolites of MDMA or MDA have yet to be identified in brain. Using in vivo microdialysis coupled to liquid chromatography-tandem mass spectroscopy and a high-performance liquid chromatography-coulometric electrode array system, we now show that GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA are present in the striatum of rats administered MDMA by subcutaneous injection. Moreover, inhibition of ␥-GT with acivicin increases the concentration of GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA in brain dialysate, and there is a direct correlation between the concentrations of metabolites in dialysate and the extent of neurotoxicity, measured by decreases in serotonin (5-HT) and 5-hydroxyindole acetic (5-HIAA) levels. Importantly, the effects of acivicin are independent of MDMA-induced hyperthermia, since acivicin-mediated potentiation of MDMA neurotoxicity occurs in the context of acivicin-mediated decreases in body temperature. Finally, we have synthesized 5-(N-acetylcystein-S-yl)-N-methyl-␣-MeDA and established that it is a relatively potent serotonergic neurotoxicant. Together, the data support the contention that MDMA-mediated serotonergic neurotoxicity is mediated by the systemic formation of GSH and N-acetylcysteine conjugates of N-methyl-␣-MeDA (and ␣-MeDA). The mechanisms by which such metabolites access the brain and produce selective serotonergic neurotoxicity remain to be determined.Although the selectivity of (Ϯ)-3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and (Ϯ)-3,4-methylenedioxyamphetamine (MDA) for the serotonergic system in rats and humans is firmly established, the mechanism(s) involved are not fully understood. In rats, MDMA is cleared mainly by hepatic metabolism by N-demethylation to form MDA. MDMA and MDA are further O-demethylenated to 3,4-dihydroxymethamphetamine (N-methyl-␣-methyldopamine; N-Me-␣-MeDA) and 3,4-dihydroxyamphetamine (␣-methyldopamine; ␣-MeDA), respectively. N-Me-␣-MeDA and ␣-MeDA are highly redox-unstable catechols and are conjugated with sulfate and glucuronic acid. Both catechols can also be rapidly oxidized to their corresponding orthoquinones and form adducts with glutathione (GSH) and other thiol-containing compounds (Lim and Foltz, 1988;Hiramatsu et al., 1990). Alternatively, N-Me-␣-MeDA and ␣-MeDA can be O-methylated to form 4-hydroxy-3-methoxymethamphetamine (3-O-Me-N-Me-␣-MeDA) or 4-hydroxy-3-methoxyamphetamine (3-O-Me-␣-MeDA), respectively.

Short 50-kilohertz (kHz) range frequency-modulated ultrasonic vocalizations (USVs) produced by rats and mice are unconditionally elicited by drugs of abuse or electrical stimulation that increase dopamine activity in the nucleus accumbens, and it has been suggested that they reflect “positive affect” or incentive motivational states associated with appetitive behavior. The repeated administration of amphetamine is known to not only produce “psychomotor” sensitization, but also to facilitate a number of appetitive behaviors, including conditioned drug pursuit behavior. We were interested, therefore, in whether amphetamine-induced 50-kHz USVs would also increase with repeated drug exposure. USV recordings were made during 5-min sessions immediately after a saline infusion, and again 4-5 hours later, after 1.0 mg/kg intravenous amphetamine exposure. These sessions took place every other day over a 5-day period. A challenge dose of 1.0 mg/kg amphetamine was administered 2 weeks later to determine whether sensitization would persist. The initial amphetamine infusion increased 50-kHz USVs relative to the saline infusion. This effect was enhanced over trials and during the amphetamine challenge two weeks later. Classification of 50-kHz range call types revealed that complex frequency-modulated trill calls were sensitized by amphetamine, but not flat 50-kHz calls. It is possible that 50-kHz USV recordings could provide a potentially valuable behavioral measure of sensitization linked to enhanced incentive salience and increased tendency to self-administer drugs of abuse.

Ultrasonic vocalization (USV) in the 50-kHz range occurs in rats immediately upon first-time exposure to cocaine or amphetamine, and rapidly increases with repetitive drug exposure at the same dose. This sensitized positive-affect response to these drugs of abuse is persistent in that the peak level of USVs again appears when the drug is reintroduced after several weeks of drug discontinuation. The present study explored whether with enough experience USVs might be elicited, and gradually escalate, in anticipation of impending drug delivery. Rats were trained to selfadminister (SA) cocaine intravenously by lever pressing 5 days per week for 4 weeks. Yoked rats received experimenter-delivered cocaine matching that of SA rats. USVs and locomotor activity were recorded during each 10-min period prior to 60-min drug access sessions. Extinction trials in which drug access was denied were then carried out over an additional 4-week period. After about a week of cocaine experience, both the SA and Yoked groups began to progressively increase USVs when placed in an environment that predicted forthcoming drug exposure. Extinction of anticipatory calls and locomotion occurred over days after drug access ended. USVs may be a useful model for specifically investigating the neural basis of drug anticipation and aid in developing and assessing new addiction treatment strategies for reducing craving and relapse.

Dopamine increases in the nucleus accumbens after ethanol administration in rats, but the contributions of the core and shell subregions to this response are unclear. The goal of this study was to determine the effect of various doses of i.v. ethanol infusions on dopamine in these two subregions of the nucleus accumbens. Male Long-Evans rats were infused with either acute i.v. ethanol (0.5, 1.0, 1.5 g/kg), repeated i.v. ethanol (four 1.0 g/kg infusions resulting in a cumulative dose of 4.0 g/kg), or saline as a control for each condition. Dopamine and ethanol were measured in dialysate samples from each experiment. The in vivo extraction fraction for ethanol of probes was determined using i.v. 4-methylpyrazole, and was used to estimate peak brain ethanol concentrations after the infusions. The peak brain ethanol concentrations after the 0.5, 1.0 and 1.5 g/kg ethanol infusions were estimated to be 20, 49 and 57 mM, respectively. A significant dopamine increase was observed for the 0.5 g/kg ethanol group when collapsed across subregions. However, both the 1.0 g/kg and 1.5 g/kg ethanol infusions produced significant increases in dopamine levels in the shell that were significantly higher than those in the core. An ethanol dose-response effect on dopamine in the shell was observed when saline controls, 0.5, 1.0, and 1.5 g/kg groups were compared. For the cumulative-dosing study, the first, second, and fourth infusions resulted in significant increases in dopamine in the shell. However, these responses were not significantly different from one another. The results of this study show that the shell has a stronger response than the core to i.v. ethanol, that dopamine in the shell increases in a dose-dependent manner between 0.5-1.0 g/kg doses, but that the response to higher ethanol doses reaches a plateau.

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