3,4-Methylenedioxy-N-methamphetamine (ecstasy) promotes the survival of fetal dopamine neurons in culture
Introduction
The widely abused club-drug 3,4-methylenedioxymethamphetamine (MDMA or ‘ecstasy’) is a substituted amphetamine that binds with high affinity to the dopamine and serotonin transporters (DAT and SERT, respectively; for a review see Green et al., 2003). MDMA has been shown to reduce the phenotypic expression of 5-HT throughout the adult brain (Colado and Green, 1995). Some consider this loss of phenotype evidence for MDMA-induced serotonin neurotoxicity in adults (McCann and Ricaurte, 1991, Ricaurte et al., 1988a, Ricaurte et al., 1988b) while others are more skeptical (for reviews see Baumann et al., 2007; Helton et al., 1993).
In contrast, rats prenatally exposed to MDMA exhibit few discernible changes in serotonin (5-HT) neurochemistry when examined postnatally at various ages (Galineau et al., 2005, Koprich et al., 2003, Srivastava and Crippen, 1993, St Omer et al., 1991). However, prenatal MDMA increased the mesocortical dopaminergic innervation of prefrontal cortex by more than 4-fold (Koprich et al., 2003). Significant increases in TH+ neuropil density in the striatum and the nucleus accumbens were also evident. These changes were accompanied by reduced dopamine (DA) turnover which resulted in significantly lower levels of DA metabolites. These findings implied that MDMA acts on the developing fetus to increase DA neuron differentiation, survival and/or promote neurite outgrowth into target structures (Koprich et al., 2003).
The mechanisms underlying MDMA's ability to increase fetal dopaminergic neurite density are currently unknown. The effect could be a direct result of MDMA acting on developing DA neurons via its actions at DAT, or an indirect result of maternal factors impinging upon fetal development, such as peripheral alterations in hormones, growth factors or vascular perfusion in response to MDMA administration. In the present series of experiments we utilized primary mesencephalic cell culture to determine whether direct application of MDMA could specifically promote DA neuron survival and neurite outgrowth. Use of this in vitro approach allowed us to evaluate MDMA's effect on midbrain DA neurons, independent of maternal, placental or fetal factors.
Section snippets
Experiment 1: determination of fetal brain MDMA levels
We have previously determined that 4 days in vitro (DIV, equivalent to E14–E18) is an optimal cell culture interval for determining the effectiveness of trophic compounds in primary mesencephalic cultures (Collier et al., 2003). Therefore, to add external validity to this in vitro MDMA model, it was imperative to determine the levels of MDMA achieved in fetal brains as a result of repeated MDMA administration from E14 to E18. By incorporating these fetal brain MDMA concentrations into our in
Experiment 1: fetal brain levels of MDMA
At time zero (tzero) on E18, MDMA concentration in brain was at 6.88 ± 0.648 μM as a result of the carryover from repeated exposure on prior days. By 30 min fetal brain MDMA levels had risen to 66.1% of the maximum recorded concentration (28.04 ± 2.92 μM). Through visual inspection of the concentration-time curve, MDMA levels reached an apparent maximum concentration (Cmax) of 42.41 ± 3.93 μM at 120 min post-injection (tmax). The rate of elimination (kelim) was determined to be 0.099 min−1 and the
Discussion
These results are the first to demonstrate that concentrations of the substituted amphetamine MDMA achieved in vivo can increase the survival of ventral mesencephalic DA neurons in culture in a dose-dependent, and DA-specific manner. The results also demonstrate that MDMA is effective not only against the early wave of cell death in vitro, but also at later intervals, since delayed application of MDMA still significantly increased DA neuron survival. MDMA exposure significantly increased the
Acknowledgments
The authors would like to thank Prof. Dan Peterson for his expertise and guidance in quantifying neurons and neurites in culture using Stereo Investigator. We would also like to thank Prof. Kathy Steece-Collier for permitting the use of her embryonic tissue dissection images. This research was supported by the NIH: 1R21 DA019261 (JWL), 1R01DA017399 (JWL), and the George and Elizabeth Wile Fund. MDMA and methylphenidate were provided by the NIDA Research Drug Supply System.
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