Donna M. Stone

The effects of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) on monoaminergic systems in the rat brain

The effects of two amphetamine-like designer drugs, 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA), on dopaminergic and serotonergic systems in the rat brain were investigated and compared to those of methamphetamine (METH). Like METH, single or multiple 10 mg/kg doses of either drug caused marked reductions in both tryptophan hydroxylase (TPH) activity and concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid, in several serotonergic nerve terminal regions. In all regions examined, the reduction in 5-HT content corresponded to the depression of TPH activity. Unlike multiple METH administrations, which induced pronounced deficits in dopaminergic neuronal markers, repeated doses of MDA or MDMA did not alter striatal tyrosine hydroxylase (TH) activities or reduce striatal dopamine concentrations. A single dose of MDA or MDMA significantly elevated striatal dopamine content; however, after repeated drug administrations dopamine concentrations were comparable to control values. At this time, striatal levels of homovanillic acid were significantly elevated suggesting that both drugs influence dopamine turnover. The effects of MDA or MDMA administration in the rat brain are reminiscent of those elicited by p-chloroamphetamine, a presumed serotonergic neurotoxin.

Immediate and long-term effects of 3,4-methylenedioxymethamphetamine on serotonin pathways in brain of rat.

In the rat, administration of the psychoactive analog of amphetamine 3,4-methylenedioxymethamphetamine (MDMA), causes selective, pronounced decreases in markers of central serotonergic function. The time course of these neurochemical changes was examined in several serotonergic nerve terminal regions of the brain. Fifteen min after subcutaneous injection of MDMA (10 mg/kg), the enzymatic activity of tryptophan hydroxylase (the rate-limiting enzyme for the biosynthesis of serotonin) was significantly decreased in the frontal cortex; by 1 hr after the injection, the activity of tryptophan hydroxylase had significantly declined in the neostriatum, hippocampus and hypothalamus as well. Although extensive recovery had occurred by 2 weeks, the activity of the enzyme remained significantly depressed in most regions. Decline of the regional content of 5-hydroxytryptamine (5-HT) closely paralleled, but was usually preceded by, that of the enzyme. Concentrations of the primary metabolite of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), were less responsive: in most regions levels of 5-HIAA had significantly decreased by 3 hr, but not by 1 hr, following treatment. Markers of dopamine function were altered transiently but had returned to control values by 24 hr. Administration of multiple doses of MDMA (5 doses over a 24-hr period) resulted in significant decreases in serotonergic parameters for up to 110 days after treatment. The rate and extent of recovery varied according to both the dose administered and the region examined. The persistence of these serotonergic deficits suggests that MDMA induced the destruction of serotonin-containing axon terminals.

Differences in the central serotonergic effects of Methylenedioxymethamphetamine (MDMA) in mice and rats

The effects of subcutaneous injection of 3,4-methylenedioxymethamphetamine (MDMA), a psychoactive amphetamine congener, on mouse central monoaminergic systems were assessed and compared to effects in rats. Whereas neostriatal concentrations of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in mouse were transiently decreased after a single moderately high dose of MDMA (15 mg/kg), mouse neostriatal or hippocampal tryptophan hydroxylase activity was not significantly affected, even after a dose of 60 mg/kg. These results are in contrast to effects in rats, in which a single 10 mg/kg dose of MDMA induced immediate and prolonged decreases in both central tryptophan hydroxylase activity and 5-hydroxyindole concentrations. Decreases in mouse central tryptophan hydroxylase activity, and prolonged decreases in mouse 5-hydroxyindole concentrations, were observed only after multiple doses of MDMA, suggesting that the duration of exposure may be an important determinant of toxic effects. These results show mice to be less susceptible than rats to MDMA-induced neurotoxicity, and are discussed in terms of possible interspecies differences in MDMA pharmacokinetics.

A comparison of the neurotoxic potential of methylenedioxyamphetamine (MDA) and its N-methylated and N-ethylated derivatives

Three psychoactive amphetamine congeners were evaluated for their ability to cause long-term changes in several neurochemical parameters indicative of central serotonergic function. Two weeks after multiple doses (10 mg/kg) of 3,4-methylenedioxyamphetamine (MDA) or its N-methylated derivative, 3,4-methylenedioxymethamphetamine (MDMA), selective and dramatic decreases were observed in regional brain tryptophan hydroxylase (TPH) activities, and in corresponding concentrations of 5-hydroxytryptamine (5-HT) and its primary metabolite, 5-hydroxyindoleacetic acid (5-HIAA). However, the N-ethylated derivative of MDA, N-ethyl-3,4-methylenedioxyamphetamine (MDE), was much less potent in its ability to lower brain hydroxyindoles, and in most regions examined did not significantly affect TPH activity. The neurotoxic implications of these results are discussed.

Acute inactivation of tryptophan hydroxylase by amphetamine analogs involves the oxidation of sulfhydryl sites

The activity of rat hippocampal tryptophan hydroxylase was reduced from 30-60% 3 h after the administration of a 10-15 mg/kg dose of either fenfluramine, methamphetamine or 3,4-methylenedioxymethamphetamine (MDMA). Tryptophan hydroxylase inactivated by these drug treatments could be reconstituted by a prolonged anaerobic incubation in the presence of 5 mM dithiothreitol and 50 microM Fe2+. Drug-inactivated enzyme obtained from rats killed 18 h after multiple doses of either D(+)- or L(-)-MDMA could not be similarly restored. These observations suggest that the rapid decrease in central tryptophan hydroxylase activity induced by amphetamine analogs results from the reversible oxidation of a sulfhydryl site(s) within the enzyme molecule, whereas the irreversible decrease in enzymatic activity measured 18 h after multiple-dose MDMA treatment may reflect serotonergic toxicity.

Role of the dopaminergic nigrostriatal pathway in methamphetamine-induced depression of the neostriatal serotonergic system

The prevention of the decrease in neostriatal tryptophan hydroxylase (TPH) activity with a single dose of methamphetamine (MA) was attempted by lesioning the nigrostriatal dopaminergic projections with bilateral nigral injections of 6-hydroxydopamine (6-OHDA). The rats were injected with MA (10 mg/kg) 11 days later, and killed 3 h after the injection. The 6-OHDA lesions prevented the decrease of TPH activity in the neostriatum, while the decrease of enzyme activity was slightly attenuated in the hippocampus and unaffected in the frontal cortex. This study demonstrates: that the attenuation of TPH activity can be prevented in a selected brain area by destroying its dopaminergic afferents, and implicates central dopamine (DA), or its metabolite, in the decrease in central TPH activity observed after a single injection of MA.

In Vitro Reactivation of Rat Cortical Tryptophan Hydroxylase Following In Vivo Inactivation by Methylenedioxymethamphetamine

Abstract: The activity of tryptophan hydroxylase (EC 1.14.16.4) from rat brain was significantly decreased 1 h following a single systemic injection of 3,4‐methylenedioxy‐methamphetamine (MDMA) when assessed ex vivo by ra‐dioenzymatic assay or in vivo by the quantitation of 5‐hy‐droxytryptophan accumulation following central L‐aromatic amino acid decarboxylase inhibition. Recovery of enzymatic activity in vivo, which occurred within 24 h of low‐dose MDMA treatment, appeared not to involve synthesis of new enzyme protein, because the return of enzymatic activity was not prevented by prior cycloheximide. Acutely MDMA‐depressed cortical tryptophan hydroxylase activity could be completely restored in vitro by a prolonged (20‐24 h) anaerobic incubation in the presence of dithiothreitol and Fe2+at 25°C; partial reconstitution occurred when 2‐mercapto‐ethanol was substituted for dithiothreitol. Cortical tryptophan hydroxylase acutely inactivated by methamphetamine or p‐chloroamphetamine could be similarly reactivated. MDMA‐inactivated cortical tryptophan hydroxylase derived from rats killed later than 3 days after drug treatment could not be significantly reactivated under the conditions described above, indicating the development of irreversible enzymatic damage. Kinetic analysis of enzyme reactivation revealed an approximate doubling of enzyme Vmax with no change in enzyme affinity for either substrate, tryptophan, or pterin cofactor. These studies suggest that MDMA and its congeners inactivate central tryptophan hydroxylase by inducing oxidation of key enzyme sulfhydryl groups. The reactivation capacity of drug‐inactivated enzyme at various times after MDMA treatment may provide a means of assessing the development of MDMA‐induced neurotoxicity.

Glucocorticoids and 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity.

The present study was carried out in order to explore the role of glucocorticoids in 3,4-methylenedio-xymethamphetamine (MDMA)-induced neurotoxicity of the central serotonergic system. The activity of tryptophan hydroxylase (TPH) was used as an index of this drug-induced neuronal degeneration. One week after a single high dose of MDMA (20 mg/kg), a significant decrease in the enzyme activity was measured in both the frontal cortex and hippocampus. Adrenalectomy (ADX) attenuated or blocked this decrease in TPH activity in the hippocampus but not in the frontal cortex. This protective effect of ADX on hippocampal serotonergic neurons disappeared with concurrent administration of corticosterone (CORT) and MDMA administration. The long-term MDMA-induced decreases in hippocampal serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were similarly affected by CORT replacement. However, ADX did not alter the short-term decline in hippocampal TPH activity and 5-HT concentrations measured 3 h after a single dose of MDMA (10 mg/kg s.c.). This study suggests that CORT play a role in the development of neurotoxicity induced by MDMA in the hippocampal serotonergic system, but may be less important in other brain structures.

Effects of amphetamine analogs on neurotensin concentrations in rat brain

The present study investigates the effects of amphetamine-like analogs on neurotensin systems and compares the same to those of methamphetamine. Like methamphetamine, multiple high doses of each of the analogs examined significantly increased the concentrations of neurotensin-like immunoreactivity in the striatum, substantia nigra and nucleus accumbens; these effects were reversible and specific. The changes in the neurotensin systems developed rapidly and were evident within 6 h following a single administration of two of the analogs studied.

Neurochemical Effects of MDMA

Since 1971 we have extensively investigated the neurochemical effects of amphetamine and related congeners. Early in those studies, we observed that methamphetamine, given in large repeated doses (10–15 mg/kg, s.c., every six hours for five doses), caused a dose-related decrease in tyrosine hydroxylase (TH) activity in the neostriatum [1, 2] and substantia nigra [3]. A parallel decline in concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) [4], accompanied the decrease in enzyme activity.