| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Neuropharmacology Research Centre, School of Pharmacy, De Montfort University, Leicester, United Kingdom (A.R.G., A.O.M., J.M.E.); AstraZeneca R&D Charnwood, Loughborough, United Kingdom (A.R.G.); and Departamento de Farmacologia, Facultad de Medicina, Universidad Complutense, Madrid, Spain (E.O., M.I.C.)
 |
Abstract |
|---|
 Top Next |
|---|
|
I. Introduction |
|---|
|
TopPreviousNext |
|---|
) reported that it was actually first patented in Germany in 1914 as a precursor agent for therapeutically active compounds and was never intended for use as an anorectic drug. The toxicology of MDMA was first examined in the 1950s, together with other mescaline analogs, by the U.S. military, presumably as part of a chemical warfare program (Hardman et al., 1973).
|
The first report that MDMA was psychoactive in humans appears to be the report of Shulgin and Nichols (1978), although this paper does not describe the effects encountered. In the 1980s, MDMA started to be used in psychotherapy and was said to increase patient self-esteem and facilitate therapeutic communication. In such settings it was administered orally (75-175 mg) and noted to produce acute sympathomimetic effects, such as increased heart rate and blood pressure, and transient anxiety (Greer and Strassman, 1985; Grinspoon and Bakalar, 1986).
In 1985, the U.S. Drug Enforcement Administration classified MDMA as a Schedule 1 drug due to its high abuse potential, lack of clinical application, lack of accepted safety for use under medical supervision (www.usdoj.gov/dea) and evidence that 3,4-methylenedioxyamphetamine (MDA), a related compound and major MDMA metabolite, induced serotonergic nerve terminal degeneration in rat brain (Ricaurte et al., 1985). Possession of MDMA is also illegal in the United Kingdom, it being controlled as a Class A drug under the Misuse of Drugs Act (1971). Nevertheless, since the mid 1980s it has become popular as a recreational drug, often being taken at "rave" or "techno" parties, particularly in large dance clubs. "Raves" comprise heavily mixed, electronically generated sound and computer-generated video and laser light shows, where individuals are able to dance all night.
Ecstasy comes in a variety of colors, shapes, and sizes of tablet, which are decorated with a wide variety of designs or logos and may also be available in capsule form (see www.drugscope.org.uk; www.ecstasy.org; www.erowid. org; www.thesite.org). As with any illicitly prepared and obtained recreational drug, both doses and purity vary greatly (Ziporyn, 1986), but tablets have regularly been found to contain between 80 and 150 mg of MDMA.
The onset of effects can take 20 to 60 min to occur, the peak occurring 60 to 90 min after ingestion, and the primary effects last for 3 to 5 h. MDMA usually produces a relaxed, euphoric state, including emotional openness, empathy, reduction of negative thoughts, and a decrease in inhibitions (Peroutka et al., 1988; Davison and Parrott, 1997; Parrott and Stuart, 1997; Hegadoren et al., 1999; Liechti and Vollenweider, 2000b; Morgan, 2000). Sounds and colors can also appear more intense (see Davison and Parrott, 1997). Accompanying physiological changes can result in severe adverse events (see below).
|
II. Epidemiological Studies on the Use of MDMA |
|---|
|
TopPreviousNext |
|---|
Williamson et al. (1997) studied 158 known current drug users (average age 30 years, 62% male, 93% white, 76% unemployed) in the United Kingdom. Over half the subjects had used one or more illicit drugs (MDMA, cocaine, or amphetamines) during the past year, 82% of subjects using MDMA within this time, taking an average of 2 tablets on each occasion.
Solowij et al. (1992) recruited 100 subjects in Sydney, Australia, to assess the extent of recreational use of MDMA. Subjects were aged 16 to 48 years (male: 61%); 68% of subjects had used MDMA more than three times and the longest duration of use was more than 5 years. Approximately one-third of subjects reported using MDMA between once a month and once every three months, while 18% used MDMA mainly on "special occasions."
Peroutka (1987) studied a randomly selected group of 369 U.S. university undergraduates and reported that 39% had used MDMA at least once (range: 1-38). Webb et al. (1996) performed a similar study of 3075 British 2nd-year undergraduate students (average age: 20 years) from 9 different faculties in 10 universities. Approximately equal numbers of male and female subjects from a cross-section of ethnic origins and religions took part; 5.2% of subjects had used MDMA more than once or twice, and 2.7% used MDMA at least once per week. In 1998 the National Institute of Alcohol and Drugs Research in Norway reported that 4.8% of people aged 15 to 20 years in Oslo had used MDMA at least once (Christophersen, 2000), while the estimated nationwide use of MDMA was 2.6% compared to 0.3% in 1994 (Mørland, 2000).
The U.S. National Institute on Drug Abuse publishes annual results of the "Monitoring the Future Study," conducted at the University of Michigan's Institute for Social Research. This study examines trends in drug abuse within different populations of individuals—school children, college students, and adults aged 19 to 40. In 2001, 44,346 school children completed the survey, being recruited from 424 schools across the United States, and including 8th-, 10th-, and 12th-grade students (aged 13-14, 15-16, and 17-18 years, respectively). The use of any illicit drug, at least once during a subject's lifetime, was 26.8, 45.6, and 53.9% by 8th-, 10th-, and 12th-grade students, respectively, and the use of MDMA at least once in an individual's lifetime was reported to be 5.2, 8, and 11.7%, respectively. While the overall use of illicit drugs had marginally declined since 1999, the use of MDMA had increased in each age group; in 1999, MDMA had been used at least once by 2.7, 6, and 8% of 8th-, 10th-, and 12th-grade students, respectively.
Similar trends were observed in college students (aged 19-22) and all young adults (aged 19-28). In both of these populations there has been little change in lifetime use of any illicit drug over the past ten years. For example, illicit drug use by college students has ranged from 45.5% in 1994 and 1995 to 53.7% in 2000, while use by all young adults has ranged from 56.4% in 1996 to 62.2% in 1991 (use was reported to be 58.1% in 2001). In contrast, use of MDMA at least once in an individual's lifetime has increased dramatically from 2 and 3.2% in 1991, by college students and all young adults, respectively, to 14.7 and 13% in 2001 (NIDA, 2002).
In a recent UK study aimed to "generate information on patterns and trends among regular recreational drug consumers," 1151 subjects were recruited via advertisements in a popular dance music magazine (60% male, average age 24 years). Ninety-six percent of subjects had used MDMA at least once, in addition to at least a single use of amphetamines (92%), cannabis (91%), cocaine (75%), and LSD (71%). The average duration of MDMA use was 4 to 5 years, 8% of users having taken the drug for over 10 years; 58% of users bought 4 or fewer tablets on each occasion. Since the subjects were self-nominating, the sample could be subject to bias and not a representative sample of drug users associated with the dance music scene in general. The majority of subjects were poly-drug users and over 70% also reported "harmful" levels of alcohol consumption (Winstock et al., 2001).
The "UK Drug Situation 2000" report to the European Monitoring Centre for Drugs and Drug Addiction was recently published by DrugScope, a government-designated body for drugs information in the United Kingdom (www.drugscope.org.uk). This reported that in England and Wales approximately one-third of adults aged 16 to 59 had used illicit drugs at least once in their lifetime. While cocaine use is on the increase, MDMA and amphetamine use has leveled off and there are indications that use is declining, particularly among individuals under age 20. MDMA use has been reported by approximately 10% of individuals in this age group. In the United States, in contrast, ecstasy use may be increasing. A very recent study on ecstasy use and related behavior in a group of over 14,000 college students found that use rose from 2.8% to 4.7% (an increase of 69%) between 1997 and 1999 (Strote et al., 2002).
All the foregoing indicates that MDMA use by young people is widespread; indeed, it has been estimated that in the United Kingdom alone 500,000 young people ingest the drug every weekend. Fatalities following ingestion of the drug are estimated to be approximately 12 persons per year.
|
III. Acute Effects of MDMA in Experimental Animals |
|---|
|
TopPreviousNext |
|---|
1. Release and Depletion of Serotonin in the Brain.
MDMA administration to rats induces an acute and rapid release of 5-HT. This has been demonstrated using in vivo microdialysis (Gough et al., 1991; Yamamoto et al., 1995; Gudelsky and Nash, 1996; Sabol and Seiden, 1998; Shankaran and Gudelsky, 1999; Nixdorf et al., 2001; Mechan et al., 2002a) and is also reflected by the fact that the 5-HT concentration in brain tissue decreases markedly during the first few hours following drug administration (Schmidt et al., 1986; Stone et al., 1987a; Logan et al., 1988; McKenna and Peroutka, 1990; Gough et al., 1991; Colado and Green, 1994; Aguirre et al., 1995; Connor et al., 1998). For example, Gudelsky and Nash (1996) demonstrated a dose-related increase in extracellular 5-HT concentrations in the striatum and medial prefrontal cortex following peripheral administration of MDMA. 5-HT release in both the striatum (Gudelsky and Nash 1996) and hippocampus (Mechan et al., 2002a) is markedly attenuated by pretreatment with the serotonin uptake inhibitor, fluoxetine, indicating that MDMA-induced 5-HT release involves a carrier-mediated mechanism. Depletion of vesicular stores with reserpine also produces a significant attenuation of 5-HT release (Sabol and Seiden, 1998).
Acute 5-HT release has also been demonstrated in vitro following addition of MDMA to brain slices (Johnson et al., 1986; Schmidt et al., 1987; Schmidt, 1987b; Berger et al., 1992; Crespi et al., 1997; Koch and Galloway, 1997) or synaptosomal preparations (Berger et al., 1992; O'Loinsigh et al., 2001). Johnson et al. (1986) first demonstrated an acute release of [3H]5-HT from rat hippocampal slices by MDMA and reported that there was no significant difference in the releasing effects of the two MDMA enantiomers. Schmidt (1987b) demonstrated similar dose-dependent release of [3H]5-HT from rat striatal slices following superfusion with MDMA, MDA, or MDEA. At the highest concentration (10 µM), MDA was the most potent compound, followed by MDMA and MDEA. Berger et al. (1992) also examined the potencies of several compounds on [3H]5-HT release from synaptosomes. Dose-dependent release of [3H]5-HT was observed, with p-chloroamphetamine (PCA) and fenfluramine being the most potent (EC50 = 3 µM), MDMA slightly less so (EC50 = 8 µM), and methamphetamine being the least potent (EC50 = 23 µM). Fluoxetine significantly attenuated the [3H]5-HT-releasing actions of all four compounds (Berger et al., 1992). O'Loinsigh et al. (2001) recently reported that MDMA, MDA, and MDEA were equipotent at inducing a dose-dependent release of [3H]5-HT from frontal cortex/hippocampal synaptosomes, while 3,4-methylenedioxybutylamphetamine (MDBA) the N-butyl analog of MDMA, only induced significant release at a concentration of 100 µM.
2. Effect on Tryptophan Hydroxylase and Monoamine Oxidase.
It has been known for some time that the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme required for 5-HT synthesis, is inhibited by MDMA administration (Stone et al., 1987a,c; 1988; Schmidt and Taylor, 1988; Johnson et al., 1992; Che et al., 1995). Stone et al. (1987c) demonstrated that TPH activity started to decline in the neostriatum, frontal cortex, hippocampus, and hypothalamus within 15 min after MDMA administration. Inhibition of the enzyme has been reported to still be detectable more than 2 weeks following a single dose of MDMA (Schmidt and Taylor, 1987).
Depletion of central dopamine content by administration of 
-methyl-p-tyrosine (AMPT) or reserpine, or by selectively destroying nigrostriatal dopamine projections with 6-hydroxydopamine, provides partial blockade of the MDMA-induced reduction of TPH activity (Stone et al., 1988). Although a single, direct, central injection of MDMA did not alter cortical or striatal TPH activity, a continuous i.c.v. infusion of MDMA for 1 h resulted in a significant reduction in TPH activity (Schmidt and Taylor, 1988). These data may indicate that the peripheral generation of an active metabolite is responsible for the acute neurochemical effects of MDMA, a proposal that is supported by the observation that MDMA has no inhibitory effect on the enzyme in vitro (Schmidt and Taylor, 1987). The possible involvement of calcium influx in MDMA-induced decreases in TPH activity has been demonstrated by pretreatment with flunarizine (thereby blocking calcium influx through non-NMDA calcium channels), which significantly attenuated the inhibitory effect of MDMA (Johnson et al., 1992). The fact that MDMA can be metabolized to a quinone led Rattray (1991) to suggest that the quinone could combine with sulfhydryl groups within the enzyme molecules leading to deactivation. This proposal is supported by the observation that enzyme activity can be restored by reduction with sulfhydryl reagents under anaerobic conditions (Stone et al., 1989).
The MDMA-induced decrease in TPH activity is influenced by body temperature. Che et al. (1995) demonstrated that MDMA administration at an ambient temperature (Ta) of 25°C produced a hyperthermic response, while administration at a Ta of 6°C produced a hypothermic response. A significant reduction in TPH activity was observed in the hippocampus, striatum, and frontal cortex of hyperthermic animals, whereas TPH activity was unaltered in hypothermic animals. This observation indicates the possible involvement of free radicals in the inactivation of the enzyme, since MDMA-induced free radical formation is enhanced by hyperthermia (Colado et al., 1999b).
In common with other amphetamine analogs, MDMA inhibits the catabolic enzyme monoamine oxidase (MAO). Potency was approximately 10 times greater at MAO-A (IC50 = 44 µM) than MAO-B in a rat brain homogenate preparation (Leonardi and Azmitia, 1994). Such inhibition reduces the metabolism of 5-HT and dopamine within the nerve terminal and therefore contributes to the increased release of active neurotransmitter by MDMA.
3. Release and Depletion of Dopamine in the Brain.
MDMA also rapidly increases dopamine release from cerebral tissue, as has been shown by both in vivo microdialysis (Yamamoto and Spanos, 1988; Gough et al., 1991; Nash and Brodkin, 1991; Nash and Yamamoto, 1992; Gudelsky et al., 1994; Yamamoto et al., 1995; Koch and Galloway, 1997; Sabol and Seiden, 1998; Colado et al., 1999a; Nixdorf et al., 2001) and by in vitro studies using tissue slices (Johnson et al., 1986; Schmidt, 1987b; Crespi et al., 1997). In vivo studies have generally found the striatal tissue concentration of dopamine to be raised and the metabolite concentration lowered in the first few hours after MDMA administration (Logan et al., 1988; Yamamoto and Spanos, 1988; Gough et al., 1991; Schmidt et al., 1991; Colado and Green, 1994).
Yamamoto and Spanos (1988) placed voltammetry electrodes in the caudate and nucleus (n.) accumbens to enable measurement of dopamine release in awake-behaving rats. Following peripheral administration of MDMA there was a dose-dependent release of dopamine in both brain areas, release being significantly greater in the caudate compared to the n. accumbens at the highest dose of MDMA, but of similar magnitude at the two lower doses. The peak release occurred within 120 min after drug administration and returned toward baseline values within 180 min. Colado et al. (1999a) administered MDMA to male Dark Agouti rats and, using in vivo microdialysis, demonstrated a rapid, significant increase in extracellular dopamine concentrations in the striatum, and a sustained depletion of DOPAC and HVA.
Although there is consistent evidence that 5-HT release induced by MDMA results from an interaction of MDMA with the 5-HT uptake carrier, since fluoxetine blocks MDMA-induced 5-HT release (Gudelsky and Nash 1996; Mechan et al., 2002a), the involvement of the dopamine uptake site in MDMA-induced dopamine release is controversial. When Nash and Brodkin (1991) infused MDMA directly into the brain they observed that the dopamine uptake inhibitor GBR 12909 antagonized the enhanced dopamine release. In addition, Koch and Galloway (1997) showed that GBR 12909 prevented MDMA-induced dopamine release using an in vitro brain slice preparation. In contrast, Mechan et al. (2002a), using an in vivo microdialysis technique and peripheral MDMA administration found that GBR 12909, far from inhibiting dopamine release, in fact produced a further increase in extracellular dopamine. This suggests that MDMA enters the dopamine terminal by diffusion, not the uptake carrier, a conclusion supported both by the fact that the dopamine uptake inhibitor mazindol fails to block the dopamine releasing actions of the MDMA-related compound methamphetamine (Marek et al., 1990) and evidence that MDMA can enter nerve-ending tissue by diffusion (Zaczek et al., 1990; O'Shea et al., 2001).
Hansen et al. (2002) demonstrated that multiple doses of MDMA resulted in a 35 to 55% reduction in [3H]dopamine uptake in synaptosomes prepared from treated animals 1 h post-administration, this effect being reversed by 24 h. These data are in contrast to the effects of methamphetamine, where a 70 to 80% reduction in plasmalemmal [3H]dopamine uptake has been reported 1 h post-administration and where a 60% reduction is still apparent at 24 h (Kokoshka et al., 1998). Binding of [3H](-)-2-
-carbomethoxy-3--(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate ([3H]WIN 35,428) to the dopamine transporter was only reduced by 10% following MDMA administration and persisted for at least 24 h. In vitro, incubation of striatal synaptosomes with MDMA also resulted in a 35-55% reduction in [3H]dopamine uptake, an effect which was prevented by pretreatment with two PKC inhibitors, S-2,6-diamino-N-[[(1-oxotridecyl)-2-piperidinyl]methyl]hexanamide dihydrochloride (NPC 15437) and 2-[1-3(aminopropyl)indol-3-yl]-3(1-methylindol-3-yl)maleimide acetate (Ro 31-7549), indicating the possible involvement of PKC activation in this response (Hansen et al., 2002). These data highlight some of the differences between the effects of MDMA and methamphetamine on dopaminergic systems.
The significant attenuation of MDMA-induced striatal dopamine release by pretreatment with fluoxetine suggests an involvement of 5-HT in the response, at least in this brain region (Koch and Galloway, 1997). Gudelsky et al. (1994) demonstrated that MDMA-induced release of striatal dopamine was significantly potentiated by pretreatment with either the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), or the nonselective 5-HT agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). These data indicate that stimulation of 5-HT2 receptors enhances MDMA-induced dopamine release. 5-HT release was unaltered by pretreatment with either the noradrenaline uptake inhibitor, desipramine, or N-(2-chloroethyl)-N-ethyl-2-bromo benzylamine (DSP4), a compound that selectively depletes brain noradrenaline (Shankaran and Gudelsky, 1998). In contrast, dopamine release from the hippocampus was inhibited by both compounds, indicating that the MDMA-induced increase in hippocampal extracellular dopamine may result from dopamine release from noradrenergic nerve terminals. MDMA may therefore be taken up by the noradrenaline transporter into noradrenergic nerve terminals and increase efflux of cytosolic dopamine (Shankaran and Gudelsky, 1998).
Yamamoto et al. (1995) demonstrated a complete blockade and significant attenuation of MDMA-induced dopamine release in the substantia nigra and striatum, respectively, following central infusion of the 5-HT2A/2C receptor antagonist, ritanserin, indicating modulation of MDMA-induced dopamine release by 5-HT2A/2C receptors. In addition, MDMA administration decreased the extracellular GABA concentration in the substantia nigra, a change that was prevented by ritanserin. The authors suggested that MDMA-induced striatal dopamine release could be modulated through an interaction between 5-HT and GABA. Administration of tetrodotoxin attenuated MDMA-induced dopamine release, indicating that release is an impulse-mediated response (Yamamoto et al., 1995).
Nixdorf et al. (2001) demonstrated a significant potentiation of MDMA-induced striatal dopamine release following co-administration of malonate and suggested that augmentation of MDMA-induced transporter-mediated dopamine release might have resulted from either malonate-induced increases in intracellular calcium or intracellular sodium accumulation due to inhibition of sodium/potassium adenosine triphosphatase (Na/K ATPase). In addition, malonate-induced inhibition of energy production might have rendered dopaminergic nerve terminals vulnerable to MDMA.
Crespi et al. (1997) demonstrated acute [3H]dopamine release in striatal synaptosomes following incubation with amphetamine, PCA, MDMA, and fenfluramine (in descending order of potency), and showed the response to be calcium-dependent. In a similar type of study, O'Loinsigh et al. (2001) found the order of potency to be MDA > MDMA > MDEA > MDBA.
4. Release and Depletion of Norepinephrine in the Brain.
In vitro MDMA has been shown to induce the release of norepinephrine (NE) from brain tissue. Induction of both basal and stimulated [3H]NE release from preloaded rat brain slices was blocked by desipramine (Fitzgerald and Reid, 1990). In a synaptosomal preparation, MDMA induced NE release with similar potency to 5-HT and greater than that for DA (Rothman et al., 2001). However the effectiveness of MDMA on NE release in vivo is unclear in the absence of microdialysis studies. MDMA depresses the firing of noradrenergic neurons in the locus ceruleus (Piercey et al., 1990), but it is unclear whether this results from the local release of NE, direct activation of 2-autoreceptors, or indirect mediation via serotonergic mechanisms. In isolated rat atrial and rabbit perfused ear preparations MDMA induced NE release, causing a positive chronotropic effect and vasoconstriction, respectively, both effects being blocked by desipramine (Fitzgerald and Reid, 1994). Although cardiovascular effects of MDMA are also seen in humans (see Section V) these are mostly inhibited by prior administration of citalopram, suggesting that they are mediated predominantly via indirect serotonergic mechanisms (Liechti and Vollenweider, 2000a).
Following administration of a neurotoxic regimen of MDMA there is generally reported to be no long-term depletion of tissue NE levels in either rat or monkey (Battaglia et al., 1987; Slikker et al., 1988; Insel et al., 1989) and no change in density of catecholamine uptake sites labeled by [3H]mazindol (Battaglia et al., 1987, 1991). Using a more intensive MDMA regimen (20 mg/kg for 10 consecutive days), Mayerhofer et al. (2001) observed a significant depletion of both 5-HT and NE, but not DA, in the n. accumbens 4 weeks after the treatment.
5. Effects on Neurotransmitter Receptors and Transporters.
MDMA binds to all three presynaptic monoamine transporters, exhibiting highest affinity (submicromolar) for the 5-HT transporter. Affinities for the noradrenaline and dopamine transporters are at least 10-fold less (Steele et al., 1987; Battaglia et al., 1988). Binding at both the 5-HT and DA transporters is stereo-selective, the S-(+) isomer being the more potent, whereas no stereoselectivity is evident at the NE transporter (Steele et al., 1987).
Binding affinities for the classical neurotransmitter receptors can be divided into three groups on the basis of KDi values: 1 to 10 µM range for 5-HT2, 2-adrenergic, M1 muscarinic, and histamine H1 receptors; 10 to 100 µM range for M2 muscarinic, 1-adrenergic, -adrenergic and 5-HT1 receptors; and above 100 µM for dopamine D1 and D2, opioid receptors, and benzodiazepine sites (Battaglia et al., 1988). The affinities of MDA are broadly comparable (within a factor of 2) to those of MDMA at these sites. Acute administration of MDMA to rats at doses of 10 to 20 mg/kg results in brain concentrations in the micromolar range (Battaglia et al., 1990; Esteban et al., 2001), so effects at the higher-affinity group of receptors may be pertinent to the psychotropic and neurotoxic actions of MDMA.
Affinity of MDMA at 5-HT2 receptors labeled by the agonist ligand [3H]1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB) is more than 10 times greater than that indicated by antagonist radioligands (Lyon et al., 1987), suggesting an agonist role. This has been confirmed by the demonstration that MDMA induces phosphatidylinositol turnover in cells expressing 5-HT2A or 5-HT2C receptors. These responses are highly stereospecific, the R-(-) isomer exhibiting greater potency and efficacy at the 5-HT2A receptor than the S-(+) isomer, which has negligible efficacy, whereas the opposite isomerism applies at the 5-HT2C receptor (Nash et al., 1994). Agonism at the 5-HT2A receptor is associated with the hallucinogenic effects of substituted amphetamines and ergolines (Egan et al., 1998) and, although efficacy at the 5-HT2A receptor is low (21%), this is also true for LSD (Newton et al., 1996). However, the affinity of MDMA at the human 5-HT2A receptor is slightly less than that for the rat receptor (Sadzot et al., 1989), corresponding with the low incidence of hallucinations induced by MDMA in humans.
While the presence of the 3,4-methylenedioxy substituent increases the affinity of MDMA for serotonergic sites compared to the parent amphetamine, affinity for the 2-adrenergic receptor is correspondingly decreased. Blockade of central presynaptic 2-adrenergic receptors may account for the increase in both systolic and diastolic blood pressure caused by MDMA in humans (McCann et al., 1996) and since such receptors are located on some serotonergic terminals this may also contribute to the induction of 5-HT release. In the vas deferens, however, MDMA exhibits agonist effects similar to xylazine, reducing stimulus-evoked contractions (Rajamani et al., 2001).
In addition to these classical receptors, MDMA has recently been reported to possess high affinity (EC50 = 1.7 µM) and efficacy for a novel receptor that is positively coupled to adenylyl cyclase, for which the endogenous agonist may be a trace amine such as tyramine (Bunzow et al., 2001). Unlike the normal monoamine receptors, this receptor is located within the cell cytosol, possibly on vesicular membranes (Borowsky et al., 2001). Since MDMA is rapidly transported into and concentrated within the serotonergic terminal, it may be expected to express significant intrinsic activity at this receptor. However, the level of expression of this receptor in rat brain is low, so its relevance to the psychotropic actions of MDMA is unclear.
6. Induction of Immediate Early Genes.
The regional expression of immediate early genes (IEGs) such as Fos, in response to neurochemical stimulation, provides a means of mapping neuronal activation (Hughes and Dragunow, 1995). MDMA induces localized but widespread induction of c-fos mRNA and Fos protein in rat brain, particularly throughout the cerebral cortex, striatum, lateral septum, n. accumbens, amygdala, and paraventricular nucleus of the thalamus (Hashimoto et al., 1997; Erdtmann-Vourliotis et al., 1999; Stephenson et al., 1999). Colocalization studies indicated that in the striatum no neurons were double-labeled with Fos and parvalbumin or neuropeptide Y following MDMA (Dragunow et al., 1991) and in the raphe nuclei very few of the Fos-positive cells were serotonergic neurons as labeled with a 5-HT antibody (Stephenson et al., 1999). Similar patterns of Fos expression were seen following administration of fenfluramine or PCA but a differentially stronger effect of MDMA was noted in the n. accumbens, supraoptic hypothalamic nucleus, and dorsal raphe (Moorman and Leslie, 1996; Rouillard et al., 1996). Fos expression in the striatum and n. accumbens was inhibited by pretreatment with the NMDA antagonist MK-801 and the dopamine D1 antagonist SCH 23390, but not by fluoxetine (Dragunow et al., 1991; Hashimoto et al., 1997).
Induction of egr-1 mRNA, which is constitutively expressed at higher levels than c-fos in several brain regions, resulted in a similar pattern of expression by MDMA in prefrontal cortex and striatum but additionally in the dentate gyrus of the hippocampus. This response was inhibited by pretreatment with MK-801, SCH 23390 or paroxetine but not by the 5-HT2A receptor antagonist SR46349B (Shirayama et al., 2000). Arc mRNA, which is implicated in the development of synaptic plasticity (Steward and Worley, 2001), again generated a broadly similar pattern of expression by MDMA, but notably included the hippocampal CA1 region but not the dentate gyrus (Aston et al., 2002). Pretreatment with paroxetine inhibited arc mRNA expression in the frontal but not parietal cortex (Aston and Elliott, 2002).
The localized expression of IEGs induced by MDMA may be particularly useful in mapping brain areas associated with specific functional or behavioral effects, such as Fos induction in the pontine reticular nucleus oralis, an area concerned with the control of masticatory muscles, corresponding with the frequent observation of bruxism reported in subjects taking ecstasy (Stephenson et al., 1999). The differences in expression revealed by individual IEGs may suggest important differences associated with the functional role of the corresponding proteins. Pharmacological studies of the neurotransmitter control of IEG expression by MDMA implicate glutamate acting at NMDA receptors and dopamine at 5-HT receptors in the striatum and n. accumbens and serotonin at some, but not all, cortical sites. Further analysis of this type using more specific receptor antagonists should lead to a clearer understanding of the biochemical mechanisms and neuronal circuitry underlying both the acute and the neurotoxic effects of MDMA.
7. Effects on Free Radical Production in the Brain.
The first indication that neurotoxic damage produced by amphetamines results from free radical formation was the paper of Steranka and Rhind (1987), which reported that the free radical scavenger cysteine attenuated brain damage produced by administration of PCA and amphetamine. Sprague and Nichols (1995) subsequently showed that MDMA administration increased lipid peroxidation, a marker of free radical-induced damage. This finding was confirmed by Colado et al. (1997a), although they found that increased lipid peroxidation occurred much earlier following MDMA than Sprague and Nichols showed (1995). In 1995 it was also reported that administration of the nitrone radical trap -phenyl-N-tert-butyl nitrone (PBN) attenuated MDMA-induced damage to cerebral 5-HT nerve endings (Colado and Green, 1995). PBN was further shown to lessen the damage produced by PCA, but not fenfluramine (Murray et al., 1996).
Direct evidence for MDMA administration increasing free radical formation in the brain was provided by Colado et al. (1997a). This group perfused salicylic acid through a microdialysis probe implanted in the hippocampus and demonstrated that peripheral MDMA injection increased the conversion of salicylate to 2,3-di-hydroxybenzoic acid (2,3-DHBA). Since this reaction only occurs in the presence of free radicals (Halliwell et al., 1991; Halliwell and Kaur, 1997), these data provided the first direct evidence for MDMA increasing free radical formation in the brain. This study also showed a similar increase in free radical formation following injection of PCA, but not fenfluramine. Administration of PBN inhibited free radical formation and attenuated neurotoxic damage and was shown to do so without altering MDMA-induced hyperthermia. The protective effect of PBN against MDMA-induced damage was confirmed by Yeh (1999).
The failure of fenfluramine administration to increase free radical formation is supported by the fact that PBN injection fails to provide protection against fenfluramine-induced damage to 5-HT nerve endings (Murray et al., 1996). It was suggested that fenfluramine, in contrast to MDMA and PCA was not metabolized to catechol or quinone compounds, which are capable of forming free radicals on further degradation. Although the mechanism of fenfluramine-induced neurotoxicity still appears uncertain, these data do indicate that one cannot extrapolate from the apparent clinical safety profile of fenfluramine to a projected safely profile for MDMA, as has sometimes been done (Saunders, 1996). In any event, the weakness of this argument is emphasized by the fact that fenfluramine has never been ingested in high recreational doses.
Other free radical scavenging drugs have also been found to protect against MDMA-induced damage. Gudelsky (1996) reported that administration of large doses of sodium ascorbate or L-cysteine prevented the long-term depletion of 5-HT induced by MDMA injection, and subsequently Shankaran et al. (2001) found that ascorbic acid administration suppressed the MDMA-induced formation of hydroxyl radicals, as indicated by the inhibition of 2,3-DHBA formation from salicylic acid in the striatum. MDMA also produced a significant reduction in vitamin E and ascorbate in the striatum and hippocampus. Aguirre et al. (1999) administered a high dose of the metabolic antioxidant -lipoic acid before MDMA injection and found that it fully protected against damage to 5-HT nerve endings, again supporting the suggestion that free radical formation is responsible for MDMA-induced neurotoxicity. Shankaran et al. (1999b) using in vivo microdialysis observed that mazindol suppressed the MDMA-induced increase of both 2,3-DHBA and dopamine in the striatum and stated that this result supported the suggested role of extracellular dopamine in producing free radicals and neurotoxic damage. However, other recent data fail to support this contention (see below). Finally, Yeh (1997) reported that salicylate administration did not produce neuroprotection and suggested that MDMA-induced neurotoxicity might occur more through production of superoxides than hydroxyl radicals. However, these data are somewhat at variance with the other data presented above.
In a study demonstrating that clomethiazole did not act as a neuroprotective agent by a free radical scavenging action, Colado et al. (1999b) also observed that free radical formation was markedly inhibited when the MDMA-induced hyperthermic response was prevented. This result provides a plausible explanation as to why hypothermia or normothermia is neuroprotective against MDMA-induced damage and is perhaps analogous to the observation that hypothermia is neuroprotective against ischemia-induced damage and also attenuates free radical production (Globus et al., 1995; Kil et al., 1996).
Finally, the fact that a prior 5-HT lesion (produced by pretreatment with fenfluramine) prevented the MDMA-induced rise in free radical formation, as measured by the conversion of salicylate to 2,3-DHBA in a hippocampal probe, suggests that the 5-HT nerve endings are the site of the enhanced free radical formation (Colado et al., 1997a). This proposal was supported by the subsequent study by Shankaran et al. (1999a), who found that MDMA-induced free radical production was attenuated by fluoxetine, which indicates that free radical production occurs following activation of the 5-HT transporter.
8. Neuroendocrine and Immune Responses.
Administration of MDMA produces a significant elevation of rat serum corticosterone and prolactin concentrations 30 min post-injection (Nash et al., 1988). Serum corticosterone concentration remains elevated for over 4 h, whereas the peak prolactin response occurs at 60 min and concentrations return to control values by 4 h. Although the increase in serum corticosterone concentration was dose-dependent, such a relationship was not apparent with the prolactin response. Ketanserin, mianserin, or fluoxetine administration all attenuated the MDMA-induced increase in corticosterone but not prolactin, which indicates that MDMA-induced corticosterone secretion, at least, is mediated by serotonergic systems.
Aldosterone and renin secretion have also been shown to increase following MDMA administration to rats, and in vitro studies using adrenal capsules suggested that this effect was the result of MDMA increasing aldosterone secretion by potentiating the action of 5-HT on secretion (Burns et al., 1996). In vitro studies using isolated hypothalamic tissue have demonstrated that MDMA and some of its metabolites can stimulate release of both oxytocin and vasopressin, the response being dose-dependent (Forsling et al., 2001; 2002).
An MDMA-induced alteration in immune function has been reported by Connor et al. (1998), who measured brain monoamine concentrations, serum corticosterone levels, total leukocyte counts, and concanavalin A-induced lymphocyte proliferation, 30 min and 6 h following MDMA administration. Serum corticosterone levels were significantly increased 30 min post-injection and had returned to control levels within 6 h. Total leukocyte counts were reduced by approximately 50% at 30 min and 6 h post-treatment, as was concanavalin A-induced lymphocyte proliferation. Thus, acute MDMA administration produced a rapid, sustained suppression of mitogen-stimulated lymphocyte proliferation and total leukocyte count and, therefore, a suppression of immune function. These changes suggest that recreational users of MDMA may be subject to a reduced immunocompetence. A subsequent study (Connor et al., 1999) demonstrated that mitogen-induced lymphocyte proliferation was suppressed at doses of MDMA that did not alter serotonergic function. However, MDMA-induced reductions in circulating lymphocyte numbers were only apparent at doses that caused an increase in serotonergic activity and plasma corticosterone levels. MDMA-induced alterations in lymphocyte functional activity might therefore be occurring via a glucocorticoid-independent mechanism, while reductions in circulating lymphocytes could be a glucocorticoid-mediated event.
9. Cardiovascular and Sympathetic Effects.
While clinical reports have linked MDMA use with cardiovascular toxicity, cardiovascular and sympathetic nerve responses in rats are still being characterized. MDMA was shown to produce a range of effects on cardiovascular function in the rat some time ago when Gordon et al. (1991) reported that the compound had cardiac stimulant effects, resulting in tachycardia and arrhythmia. The compound also facilitates vasoconstriction (Fitzgerald and Reid, 1994).
O'Cain et al. (2000) recently reported that MDMA (0.01-3 mg/kg i.v.) produced a dose-dependent increase in mean arterial pressure, significant bradycardia following administration of the highest dose of drug, and a significant decrease in renal sympathetic nerve activity. The increases in mean arterial pressure are consistent with reported increases in arterial pressure in humans following MDMA ingestion (e.g., Vollenweider et al., 1998), while bradycardia may have been due to pressor-mediated baroreceptor reflex activation, and the observed inhibition of sympathetic nerve activity could have been due to an action on medullary 2-adrenergic receptors (O'Cain et al., 2000). Repeated frequent administration of MDMA to rats followed by a period of abstinence (binge administration) appears to be particularly effective in altering cardiovascular function and inducing cardiac toxicity (Badon et al., 2002).
MDMA can displace noradrenaline from adrenergic nerve terminals (Fitzgerald and Reid, 1993; Lavelle et al., 1999) and appears to have direct 2-adrenoceptor-mediated actions both in the periphery (Lavelle et al., 1999) and at central 2-adrenoceptors mediating depressor responses (McDaid and Docherty, 2001). Rajamani et al. (2001) provided further evidence for the drug having potency at 2-, 2AD-, and 2C-adrenoceptor subtypes. Data suggest that MDMA may competitively block the noradrenaline transporter (Al-Sahli et al., 2001). In contrast, MDMA does not appear to significantly alter 5-HT-induced aortic contraction (Cannon et al., 2001; Murphy et al., 2002). However both 4-methylthioamphetamine and 4-methylthiomethamphetamine are potent inhibitors of 5-HT-mediated vascular contraction (Murphy et al., 2002).
The first study on the effect of MDMA on glucose utilization was that of Wilkerson and London (1989) who observed significant effects in several brain regions within 5 min of drug administration. Marked stimulation was seen in areas of the extrapyramidal motor system, while parts of the limbic system showed decrements. Some of these effects on glucose utilization in the brain resembled changes seen after cocaine, amphetamine, and phencyclidine administration. Recently, Quate et al. (2003) examined the effect of MDMA on intracerebral blood flow and intracerebral glucose utilization in Dark Agouti rats and obtained similar results. MDMA resulted in an increase in glucose utilization in many brain regions, particularly areas concerned with the motor system, together with decreases in blood flow in regions such as the limbic and primary sensory nuclei, thereby indicating an uncoupling of blood flow from metabolic demand. Darvesh et al. (2002) found that the glucose concentration increased following MDMA administration and demonstrated that this increase was linked to an increase in glycogenolysis, which in turn appeared to be linked to the MDMA-induced hyperthermia. The authors speculated that the altered cellular bioenergetics might be associated with the oxidative stress and subsequent neurotoxicity.
10. Body Temperature.
a. Effect on Body Temperature.
Under "normal" Ta conditions (20-22°C), MDMA administration to rats has generally been reported to produce a marked hyperthermic response of approximately +1-2°C, with a peak at about 40 to 60 min post-injection (Nash et al., 1988; Schmidt et al., 1990a; Colado et al., 1993; Dafters, 1994; Broening et al., 1995; Che et al., 1995; Malberg et al., 1996; O'Shea et al., 1998). However, an acute decrease in temperature has also been reported in a few studies. Marston et al. (1999) reported a hypothermic response in Hooded Lister rats, and Malberg and Seiden (1998) demonstrated a hypothermic response in Holtzman rats at a Ta of 20-22°C, no change from control animals at a Ta of 24-26°C, and a hyperthermic response at a Ta of 28-30°C.
The influence of ambient temperature on the effect of MDMA on body temperature seen by Malberg and Seiden (1998) has been observed by others. For example, Broening et al. (1995) administered MDMA to female Sprague-Dawley (SD) rats under Ta conditions of 10, 25, and 33°C on postnatal days (PND) 10, 40, and 70. There was no clear temperature response to MDMA administration in the PND-10 group under any of the temperature conditions. However, both PND -40 and -70 animals demonstrated a hypothermic response at a Ta of 10°C, and an acute hyperthermia following MDMA administered at a Ta of 25°C or 33°C. Dafters (1994) administered MDMA to male Wistar rats housed under Ta conditions of either 11 or 24°C. At a Ta of 11°C there was a dose-dependent hypothermic response, while at a Ta of 24°C a dose-dependent hyperthermic response was seen. When rats were administered MDMA under Ta conditions of 24°C, and subsequently transferred to a "cool" room (Ta = 11°C), their hyperthermic response was significantly attenuated. In a subsequent study, Dafters and Lynch (1998) found that MDMA produced hyperthermia when given to rats in a 22°C environment and a hypothermic response when they were in a 17°C environment, indicating a high sensitivity to small changes in Ta.
Gordon et al. (1991) investigated the effects of MDMA on the thermoregulatory mechanisms of rats by monitoring metabolic rate (MR), evaporative water loss (EWL), and rectal temperature under three Ta conditions (10, 20, and 30°C). MR was significantly increased, compared to control animals, under Ta conditions of 20 and 30°C and was unchanged at 10°C. MDMA-treated rats demonstrated an increasing EWL with increasing Ta; EWL values in MDMA-treated rats were approximately 275% above control values at a Ta of 30°C. Rectal temperature increased with increasing Ta: hypothermia (-2°C) occurred at 10°C, while at 20°C there was no difference between MDMA- and saline-treated animals, and at 30°C hyperthermia was seen (+2°C). It therefore appears that MDMA administration has profound effects on the thermoregulatory system of the rat, involving increases in MR, EWL, and rectal temperature, and that such effects are apparently dependent on Ta. A recent study has further shown that the tail temperature of rats was unchanged following a hyperthermic dose of MDMA (Mechan et al., 2002a). Since vasodilation of the tail blood vessels is a major heat loss mechanism in rats (Grant, 1963), these data suggest that MDMA interferes with normal heat loss mechanisms, a proposal also advanced to explain the hyperthermic action of methamphetamine (Mohaghegh et al., 1997). Presumably, when the animal is kept in a low-temperature environment the loss of this mechanism is of little consequence and hyperthermia no longer occurs. Finally, Dafters (1995) also showed that 14-day administration of MDMA at a presumed non-neurotoxic dose resulted in an increase in peak temperature responses across the test days, indicating a sensitization effect.
b. Pharmacology of the Hyperthermic Response.
It is well established that hyperthermia can be produced by increasing 5-HT function by administering L-tryptophan plus an MAO inhibitor (Grahame-Smith, 1971a) or various 5-HT agonists such as 5-MeODMT (Grahame-Smith, 1971b), 6-chloro-2-(1-piperazinyl)pyrazine (MK212) (Yamawaki et al., 1983), or the 5-HT-releasing drug PCA (Colado et al., 1993). There has been an assumption, therefore, that the hyperthermia that follows MDMA administration is also 5-HT receptor-mediated (Shankaran and Gudelsky, 1999). However, methamphetamine-induced hyperthermia has been shown to involve dopamine release (Bronstein and Hong, 1995), which implies that dopamine could also be involved in MDMA-induced hyperthermia, given the fact that MDMA and methamphetamine release both 5-HT and dopamine.
A recent study has strongly supported the contention that MDMA-induced hyperthermia is a consequence of dopamine release. Methysergide, ritanserin, and selective 5-HT2A and 5-HT2C antagonists all failed to block MDMA-induced hyperthermia (Mechan et al., 2002a), and while MDL 11,939 did antagonize the hyperthermic effect, confirming an earlier report (Schmidt, 1987b), the authors suggested that this might be due to lack of receptor selectivity of this compound or its active metabolites. Crucially, it was shown that administration of the selective 5-HT uptake inhibitor fluoxetine almost totally inhibited the increase in extracellular 5-HT, as measured by in vivo microdialysis, but had no effect on the hyperthermic response in the same animals. This finding confirmed earlier studies that measured these two parameters in separate groups of animals (Schmidt et al., 1990a; Berger et al., 1992; Malberg et al., 1996). The separation of 5-HT release and hyperthermia strongly indicated that neurotransmitters other than 5-HT might be involved in the hyperthermic response. Furthermore, the observation that the dopamine D1 receptor antagonist SCH 23390 dose-dependently inhibited MDMA-induced hyperthermia leads to the conclusion that MDMA might be producing hyperthermia by enhancing the release of dopamine, which then acts on D1 receptors (Mechan et al., 2002a). Support for this proposal was supplied by another study published almost simultaneously which found that PCA-induced hyperthermia was also unaltered by fluoxetine or the 5-HT-depleting drug p-chlorophenylalanine (PCPA), but was antagonized by SCH 23390 (Sugimoto et al., 2001).
c. Aggregation Toxicity.
Over 60 years ago Gunn and Gurd (1940) reported that when mice were grouped or "aggregated" (as opposed to being housed singly), both the behavioral and toxic effects of amphetamine were enhanced. This observation was confirmed and extended by Chance, who also noted that toxicity was enhanced if mice were grouped even if each mouse was given the area allocated to a singly housed animal. He also noted that toxicity was increased by elevated ambient temperature, poor hydration, and loud noise (Chance, 1946, 1947; Morton et al., 2001).
Although the mechanism of toxicity has generally been assumed to be directly related to raised body temperature (Askew, 1961; Craig and Kupferberg, 1972), acute toxicity can occur without marked hyperthermia (Wolf and Bunce, 1973). However, the mechanism for the increased toxicity on exposure to loud noise is unknown.
While specific studies on aggregation toxicity have not been performed with MDMA, there is clear evidence that the phenomenon occurs when using this amphetamine derivative and indications are that the toxicity primarily relates to hyperthermia. Rats kept at elevated temperatures display a greater hyperthermic and neurotoxic response to MDMA (Dafters
