Esther O’Shea

Acute and long-term effects of MDMA on cerebral dopamine biochemistry and function

Rationale and objectivesThe majority of experimental and clinical studies on the pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) tend to focus on its action on 5-HT biochemistry and function. However, there is considerable evidence for MDMA having marked acute effects on dopamine release. Furthermore, while MDMA produces long-term effects on 5-HT neurones in most species examined, in mice its long-term effects appear to be restricted to the dopamine system. The objective of this review is to examine the actions of MDMA on dopamine biochemistry and function in mice, rats, guinea pigs, monkeys and humans.Results and discussionMDMA appears to produce a major release of dopamine from its nerve endings in all species investigated. This release plays a significant role in the expression of many of the behaviours that occur, including behavioural changes, alterations of the mental state in humans and the potentially life-threatening hyperthermia that can occur. While MDMA appears to be a selective 5-HT neurotoxin in most species examined (rats, guinea pigs and primates), it is a selective dopamine neurotoxin in mice. Selectivity may be a consequence of what neurotoxic metabolites are produced (which may depend on dosing schedules), their selectivity for monoamine nerve endings, or the endogenous free radical trapping ability of specific nerve endings, or both. We suggest more focus be made on the actions of MDMA on dopamine neurochemistry and function to provide a better understanding of the acute and long-term consequences of using this popular recreational drug.

Dopamine D2-receptor knockout mice are protected against dopaminergic neurotoxicity induced by methamphetamine or MDMA

Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), amphetamine derivatives widely used as recreational drugs, induce similar neurotoxic effects in mice, including a marked loss of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum. Although the role of dopamine in these neurotoxic effects is well established and pharmacological studies suggest involvement of a dopamine D2-like receptor, the specific dopamine receptor subtype involved has not been determined. In this study, we used dopamine D2 receptor knock-out mice (D2R(-/-)) to determine whether D2R is involved in METH- and MDMA-induced hyperthermia and neurotoxicity. In wild type animals, both drugs induced marked hyperthermia, decreased striatal dopamine content and TH- and DAT-immunoreactivity and increased striatal GFAP and Mac-1 expression as well as iNOS and interleukin 15 at 1 and 7days after drug exposure. They also caused dopaminergic cell loss in the SNpc. Inactivation of D2R blocked all these effects. Remarkably, D2R inactivation prevented METH-induced loss of dopaminergic neurons in the SNpc. In addition, striatal dopamine overflow, measured by fast scan cyclic voltammetry in the presence of METH, was significantly reduced in D2R(-/-) mice. Pre-treatment with reserpine indicated that the neuroprotective effect of D2R inactivation cannot be explained solely by its ability to prevent METH-induced hyperthermia: reserpine lowered body temperature in both genotypes, and potentiated METH toxicity in WT, but not D2R(-/-) mice. Our results demonstrate that the D2R is necessary for METH and MDMA neurotoxicity and that the neuroprotective effect of D2R inactivation is independent of its effect on body temperature.

Effect of repeated ('binge') dosing of MDMA to rats housed at normal and high temperature on neurotoxic damage to cerebral 5-HT and dopamine neurones.

The technique of ‘binge’ dosing (several doses in one session) by recreational users of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) requires evaluation in terms of its consequences on the acute hyperthermic response and long-term neurotoxicity. We examined the neurotoxic effects of this dosing schedule on 5-HT and dopamineneurones in the rat brain. When repeated (three) doses of MDMA (2, 4 and 6 mg/kg i.p.) were given 3 h apart to rats housed at 19 °C, a dose-dependent acute hyperthermia and long-term loss of 5-HT was observed in several brain regions (hippocampus, cortex and striatum), with an approximate 50% loss following 3 × 4mg/kg and 65% decrease following 3 × 6mg/kg. No decrease in striatal dopamine content was detected. When MDMA (4 mg/kg i.p.) was given repeatedly to rats housed at 30 °C, a larger acute hyperthermic response than that observed in rats treated at 19 °C environment was seen (maximum response 2.6 ± 0.1 °C versus 1.3 ± 0.2 °C). A long-term cerebral 5-HT loss of approximately 65% was also detected in both the cortex and hippocampus, but no loss in striatal dopamine content occurred. These data emphasize the increased acute hyperthermic response and neurotoxicity which occurs when MDMA is administered in a hot room environment compared to normal room temperature conditions, and support the view that MDMA is a selective 5-HT neurotoxin, even when a binge dosing schedule is employed and the rats are present in a hot environment.

Differential effect of dietary selenium on the long-term neurotoxicity induced by MDMA in mice and rats

We examined the effect of dietary selenium (Se) on the long-term effect of 3,4-methylenedioxymethamphetamine (MDMA) on dopamine (DA) and 5-hydroxytryptamine (5-HT) containing neurons in the brain of mice and rats. Animals were fed either a Se-deficient (<0.02 ppm) or Se-replete (0.2 ppm) diet for 8 weeks. On the seventh week mice received three injections of MDMA (15 mg/kg, i.p. 3 h apart) or saline and rats a single dose of MDMA (12.5 mg/kg i.p.) or saline. All animals were sacrificed 7 days later. MDMA administration to mice depleted striatal DA concentration in both dietary groups, although depletion was considerably larger in the Se-deficient mice (64%) than Se-replete mice (30%). In addition, a decrease in 5-HT (17-32%) occurred in brain regions of Se-deficient but not Se-replete mice. In rats, MDMA decreased cortical [(3)H]-paroxetine binding (62%) and 5-HT content, the depletion being similar in the Se-deficient and Se-replete groups. No DA loss occurred in either group. There was no difference in the hyperthermic response induced by MDMA in Se-deficient or Se-replete animals. The Se-deficient diet decreased glutathione peroxidase (GPx) activity by 30% in mouse striatum and cortex and increased the degree of lipid peroxidation in cortical synaptosomes. Se-deficient rats also showed a decrease in brain GPx activity compared with the Se-replete group, but the degree of lipid peroxidation in synaptosomes was similar in both dietary groups. These results suggest that the antioxidant capacity of rats and mice differ leading to a differential susceptibility to the oxidative stress caused by MDMA in situations of low dietary Se.

Evidence that MDMA ('ecstasy') increases cannabinoid CB2 receptor expression in microglial cells: role in the neuroinflammatory response in rat brain.

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06578.x

The role of 5-HT in the impairment of thermoregulation observed in rats administered MDMA ('ecstasy') when housed at high ambient temperature.

RationaleAdministration to rats of a neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) produces an impairment in thermoregulation which is reflected in a prolonged hyperthermic response to a subsequent dose of MDMA given to rats housed at high ambient temperature.ObjectiveWe wished to examine whether the impaired thermoregulation was associated with decreased cerebral 5-HT content produced by the prior neurotoxic dose of MDMA.MethodsRats were injected with drugs decreasing 5-HT function [the tryptophan hydroxlase inhibitor p-chlorophenylalanine (PCPA), and 5-HT receptor antagonists] and rectal temperature was measured after administering MDMA to rats housed at 30°C.ResultsPCPA pretreatment decreased 5-HT and 5-HIAA concentrations in cortex, hippocampus and striatum by >80% and prolonged the hyperthermia induced in rats housed at 30°C by administering MDMA (5 mg/kg i.p.). A similar prolongation of the hyperthermic response to MDMA was seen when rats were pretreated with methysergide (10 mg/kg i.p.) or the 5-HT1A antagonist WAY100635 (0.5 mg/kg s.c.).ConclusionsDecreasing 5-HT function in diverse ways enhanced the hyperthermic response to MDMA given to rats housed at high ambient temperature. This suggests that loss of 5-HT acting on 5-HT1A receptors leads to impaired thermoregulation in rats and suggests that the impairment seen in MDMA pretreated rats housed at high ambient temperature is due to a loss in 5-HT function. These data could have implications for recreational users of MDMA, who may have damaged serotoninergic neurons because of prior heavy or frequent use of the drug, when taking further doses of MDMA in hot environments such as dance clubs.

3,4-Methylenedioxymethamphetamine increases pro-interleukin-1β production and caspase-1 protease activity in frontal cortex, but not in hypothalamus, of Dark Agouti rats: Role of interleukin-1β in neurotoxicity

3,4-Methylenedioxymethamphetamine (ecstasy) increases mature interleukin-1beta production in rat brain shortly after injection. This effect is a consequence of the 3,4-methylenedioxymethamphetamine-induced hyperthermia and is reduced when rats are maintained at low ambient room temperature. Since interleukin-1beta is generated as an inactive 31-kDa precursor protein and processed into mature form by caspase-1, we have now examined the effect of 3,4-methylenedioxymethamphetamine on pro-interleukin-1beta production and caspase-1-like protease activity in the hypothalamus and frontal cortex of Dark Agouti rats. 3,4-Methylenedioxymethamphetamine increased the immunoreactivity of pro-interleukin-1beta in frontal cortex, not in hypothalamus, 3 h and 6 h after administration. Caspase-1-like protease activity was increased in frontal cortex 3 h after 3,4-methylenedioxymethamphetamine injection compared with saline-treated animals. 3,4-Methylenedioxymethamphetamine did not modify the expression of pro-caspase-1 but increased the immunoreactivity for the caspase-1 active cleavage product (p20) in frontal cortex 3 h after dosing. No change on caspase-1-like protease activity was observed in hypothalamus. The basal immunoreactivity of pro-interleukin-1beta and caspase-1-like protease activity was higher in the hypothalamus than in frontal cortex of control (saline-treated) animals. These data indicate that 3,4-methylenedioxymethamphetamine alters, in a region-specific manner, the mechanisms which regulate interleukin-1beta production in the brain of Dark Agouti rats and suggest that the release of interleukin-1beta in hypothalamus may be regulated independently of caspase-1 activation. Administration (i.c.v.) of interleukin-1beta enhanced the 3,4-methylenedioxymethamphetamine-induced long-term loss of brain 5-HT parameters and immediate hyperthermia. Neither of these effects was observed when interleukin-1beta was given into hippocampus. These results indicate that exogenous interleukin-1beta potentiates 3,4-methylenedioxymethamphetamine neurotoxicity as a consequence of its effect on body temperature and suggest that the 3,4-methylenedioxymethamphetamine-induced rise in interleukin-1beta levels could in turn contribute to the maintenance of 3,4-methylenedioxymethamphetamine-induced hyperthermia and subsequent neurotoxicity.

Evidence for a role of Hsp70 in the neuroprotection induced by heat shock pre‐treatment against 3,4‐methylenedioxymethamphetamine toxicity in rat brain

3,4‐Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22°C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4°C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30°C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22°C. Fluoxetine‐induced inhibition of 5‐HT release and hydroxyl radical formation did not modify MDMA‐induced Hsp70 expression 3 h later. Four‐ or 8‐day heat shock (elevation of basal rectal temperature by 1.5°C for 1 h) or geldanamycin pre‐treatment induced Hsp70 expression and protected against MDMA‐induced serotoninergic neurotoxicity without affecting drug‐induced hyperthermia. Thus, MDMA‐induced Hsp70 expression depends on the drug‐induced hyperthermic response and not on 5‐HT release or hydroxyl radical formation and pre‐induction of Hsp70 protects against the long‐term serotoninergic damage produced by MDMA.

Factors contributing to the enhancement of MDMA-induced 5-HT depletion by ethanol: a reply to Byron and Cassel

This letter is in response to the comments by Byron and Cassel on our study entitled ‘Binge ethanol administration enhances the MDMA-induced long-term neurotoxicity in rat brain’. Byron and Cassel have published several studies in which they analyzed some of the behavioral and neurochemical effects induced by the concurrent administration of ethanol and MDMA to rats. As most of the results differed from those reported in our paper, they speculated in their comments about the possible reasons for this discrepancy. Nevertheless, doses of ethanol and MDMA, experimental design, ambient temperature, and strain are so different that what is really difficult is to find some common point between the studies carried out by the two groups. In contrast to the studies by Cassel’s group, in our study, the hyperthermia induced by MDMA was similar in rats exposed and not exposed to ethanol; in other words, ethanol did not prevent the rise in rectal temperature induced by MDMA. From our point of view, the key factor is that the last exposure to ethanol was carried out approximately 24 h before MDMA injection—that is, ethanol was not injected simultaneously with MDMA as in the studies by Cassel et al. We chose this experimental protocol precisely in order to avoid the possible interferences of the hypothermia induced by ethanol with the long-term neurotoxicity caused by MDMA. Numerous studies have shown that the prevention of MDMA-induced hyperthermia attenuates or abolishes the long-term neurotoxicity by the drug (Colado et al. 1999; O’Shea et al. 2006). Had ethanol been given together with MDMA, the result would have probably been quite different as the rats developed a significant hypothermia at least at the end of the first day of ethanol vapor exposure (Fig. 4; Izco et al. 2007). It is interesting to note that, in a similar protocol to ours, Cassel’s group also described a lack of effect of the 4-day ethanol preexposure (1.5 g/kg, i.p.) on MDMA-induced (6.6 mg/kg, i.p.) hyperthermia 24 h after the last ethanol dose (Hamida et al. 2006). Another crucial difference between our study and that of Cassel’s group is that in our study, MDMA was given at a high ambient temperature, which is often the environment in which MDMA is consumed recreationally. At a high ambient temperature, not only was the body temperature response to MDMA different to that observed at standard temperature, but rats preexposed to ethanol also showed a body temperature significantly higher than that of salinetreated rats. This could be related to alterations of the compensatory physiological responses that mediate temperature control and are triggered in response to a thermal challenge (Myers 1981). In the study where no enhancement of MDMA neurotoxicity by ethanol is observed (Cassel et al. 2005), the authors claimed that the dose of ethanol used (1.5 g/kg, i.p.) typically results in blood ethanol levels of 175 mg/dl. This appears to be below the level necessary to cause neurodegeneration (reflected by silver staining) in areas such as the hippocampus and the cortex of adults (250– 400 mg/dl; Obernier et al. 2002; Hamelink et al. 2005; Crews et al. 2006) and neonatal rats (379–432 mg/dl; West et al. 1986; Miki et al. 2000). In addition, the blood ethanol level and dose used by Cassel et al. (2005) were below those reported to produce oxidative stress in rat brains (Dahchour et al. 2005). An increase in the production of hydroxyl radicals is important in mediating the neurotoxic Psychopharmacology (2007) 190:581–582 DOI 10.1007/s00213-007-0700-8