Elsevier

Neuroscience

Volume 139, Issue 3, 2006, Pages 1069-1081
Neuroscience

Neuropharmacology
Ecstasy-induced cell death in cortical neuronal cultures is serotonin 2A-receptor-dependent and potentiated under hyperthermia

https://doi.org/10.1016/j.neuroscience.2006.01.007Get rights and content

Abstract

Studies on 3,4-methylenedioxymethamphetamine (“ecstasy”)-induced neurotoxicity mainly focus on damage of serotonergic terminals. Less attention has been given to neuronal cell death produced by 3,4-methylenedioxymethamphetamine and other amphetamines in areas including the cortex, striatum and thalamus. In the present study we investigated 3,4-methylenedioxymethamphetamine-induced neurotoxicity in neuronal serum free cultures from rat cortex. Since 3,4-methylenedioxymethamphetamine intake induces hyperthermia in both animals and humans, the experiments were performed under normal (36.5 °C) and hyperthermic conditions (40 °C). Our findings showed a dose-, time- and temperature-dependent apoptotic cell death induced by 3,4-methylenedioxymethamphetamine in cortical neurons. 3,4-Methylenedioxymethamphetamine-induced damage was potentiated under hyperthermia. The neurotoxicity was reduced by the serotonin 2A-receptor antagonists, ketanserin and (2R,4R)-5-[2-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]ethyl]-1-methyl-3-pyrrolidinol hydrochloride, in both normothermic and hyperthermic conditions. (±)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride, a model agonist for the serotonin 2A-receptor, also induced a dose- and time-dependent apoptotic cell death. Again, protection was provided by ketanserin and (2R,4R)-5-[2-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]ethyl]-1-methyl-3-pyrrolidinol hydrochloride against (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride-induced neurotoxicity, thereby indicating that the 3,4-methylenedioxymethamphetamine stimulation of the serotonin 2A-receptor leads to neurotoxicity. This study provides for the first time evidence that direct 3,4-methylenedioxymethamphetamine serotonin 2A-receptor stimulation leads to neuronal cortical death. α-Phenyl-N-tert-butyl nitrone a free radical scavenger and the nitric oxide synthase inhibitor Nω-nitro-l-arginine as well as the NMDA-receptor antagonist MK-801 provided protection under normothermia and hyperthermia, thereby suggesting the participation of free radicals in 3,4-methylenedioxymethamphetamine-induced cell death. Since 3,4-methylenedioxymethamphetamine serotonin 2A-receptor agonistic properties lead to neuronal death, clinically available atypical antipsychotic drugs with serotonin 2A-antagonistic properties could be a valuable therapeutic tool against 3,4-methylenedioxymethamphetamine-induced neurodegeneration.

Section snippets

Materials

Materials for cell cultures were obtained from the following sources: neurobasal medium and supplement B27 from Invitrogen (Paisley, UK); modified Eagle’s medium, phosphate-buffered saline (PBS), HEPES buffer, trypsin/EDTA, penicillin/streptomycin, l-glutamine, collagen-G and poly-l-lysine from Biochrom (Berlin, Germany); multiwell plates from NUNC A/S (Roskilde, Denmark); 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), 3,3-diaminobenzidine (DAB) and enzyme-standard for

Hyperthermia induced cell death in cortical neuronal cultures

Hyperthermia induced moderate temperature- and time-dependent neurotoxicity at 39 °C and 40 °C, as revealed by LDH measurements at different time-points (24 h, 48 h and 72 h after incubation under hyperthermic conditions) and MTT assay (data not shown). In contrast the temperature of 41 °C proved to be highly toxic already at early time points. This comes in agreement with in vivo experiments which show that hyperthermia in rats with a core temperature above 41 °C can be lethal (Malberg and

Discussion

The key findings of our study are the following: (1) MDMA-induced cortical neurotoxicity in cortical cultures is time- and concentration-dependent, (2) and further potentiated under hyperthermia conditions. (3) MDMA-induced neuronal death follows an apoptotic pattern, (4) which is at least partially mediated by the direct MDMA stimulation of the 5-HT2A-receptor, (5) involves glutamate excitotoxicity, (6) as well as free oxygen radicals. (7) DOI induced a concentration-dependent apoptotic

Conclusion

In conclusion, using a cortical neuronal serum free culture we showed that MDMA neurotoxicity occurs in a dose-, time- and temperature-dependent manner. As the misuse of MDMA as a recreational drug (particularly in crowded and hot environments) can induce hyperthermia, the results presented here corroborate the serious concern previously reported in the literature. This study provides for the first time evidence that direct 5-HT2A-receptor stimulation by MDMA leads to neuronal cortical death.

Acknowledgments

This work was supported by the Hermann & Lilly Schilling Foundation and the Althoff Program of the Charité. J.P.C. was the recipient of a PhD grant from “Fundação para a Ciência e a Tecnologia” (FCT) Portugal (Ref. SFRD/BD/10908/2002). A.R.G. was supported by the “Centro de Matemática da Universidade do Porto” (CMUP), financed by “Fundação para a Ciência e a Tecnologia” (FCT), Portugal, through the programs POCTI and POSI.

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