MDMA (ecstasy) effects on cultured serotonergic neurons: evidence for Ca²⁺-dependent toxicity linked to release

Animal studies have established a correlation between release of 5-hydroxytryptamine (5-HT) and the long-term reduction of 5-HT (toxicity) by 3,4-methylenedioxymethamphetamine (MDMA) with the S(+) enantiomer being more active than the R(-). Using a microculture system of fetal raphe neurons, the enantiomers of MDMA were tested to determine if a similar difference in potency existed. The results showed that the development of the uptake capacity of [³H]5-HT in 4-day cultures was half-maximally inhibited by a single application at time of plating of 5 × 10^-6 M S(+)-MDMA and 5 × 10^-5 M R(-)-MDMA. In order to determine if the Ca²⁺-independent release (chemically induced through the transporter protein and inhibited by reuptake blockers) or the Ca²⁺-dependent release (K⁺-induced and inhibited by presynaptic receptors) contributed to the toxicity, fluoxetine and D₁ and α₂ agonists were studied. The results showed that both forms of release were involved in the loss of [³H]5-HT uptake capacity, with the direct MDMA-induced Ca²⁺-independent (fluoxetine-sensitive) release being the first step. Evidence from binding studies indicates that MDMA has a micromolar affinity for the 5-HT₂ receptor, and our studies in culture showed that ketanserin, a specific 5-HT₂ antagonist, was effective at attenuating the effects of S(+)-MDMA on the development of the [³H]5-HT uptake capacity by the cultured raphe neurons. The 5-HT₂ receptor is linked to increased intracellular Ca²⁺ through a second messenger phosphatidylinositol (PI)-hydrolysis mechanism. Since increases in Ca²⁺ intracellular levels have been shown to be a key step in neuronal death, the interaction between MDMA and caffeine (stimulates release of Ca²⁺ from intracellular stores) and nimodipine (a Ca²⁺ channel blocker) were studied. The results showed that caffeine (10^-6 M) potentiated aand nimodipine (10^-9 M) attenuated the inhibitory effects of MDMA. Our results are consistent with both types of 5-HT release being involved in producing a Ca²⁺-dependent toxicity induced by MDMA on cultured serotonergic fetal neurons.