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3,4-Methylenedioxymethamphetamine (MDMA or ‘ecstasy’) administration to a variety of animal species results in a longterm neurotoxic degeneration of 5-hydroxytryptamine (5-HT) nerve terminals in several regions of the brain (Steele et al., 1994; Green et al., 1995). The degeneration has been demonstrated histologically and is reflected in the marked loss in the concentration of 5-HT and its metabolite 5-hydroxy-indoleacetic acid (5-HIAA) and by the loss of [3H]-paroxetine binding to the presynaptic transporter (see review of Green et al., 1995). There have been a variety of compounds which, when given concurrently with the MDMA, have been shown to protect against the neurodegenerative loss of 5-HT (see Green et al., 1995). It is also well established that hypothermia will attenuate or prevent neurodegeneration in a variety of animal models of acute ischaemic stroke (Busto et al., 1987; Buchan & Pulsinelli, 1990; Corbett et al., 1990; Nurse & Corbett, 1996). However much of this evidence became available either before or during the time that many of the neuroprotective studies were being conducted on MDMA. Consequently, until recently few studies on MDMA-induced neurodegeneration examined the body temperature of the animals under investigation.
In the last few years Seiden and colleagues have presented compelling data to support their proposal that several compounds previously shown to be neuroprotective against MDMA-induced damage (including certain NMDA antagonists) only had this property because, when combined with MDMA, they produced a significant hypothermia (Malberg et al., 1996; Farfel & Seiden, 1995a;b). However, there is a further complication in that MDMA administration alone produces a significant hyperthermia (Nash et al., 1988; Gordon et al., 1991; Colado et al., 1993; Dafters, 1994) and Broening et al., (1995) have shown that hyperthermia plays a significant role in the expression of 5-hydroxytryptaminergic neurotoxicity following MDMA.
In our earlier studies on clomethiazole (INN: clomethiazole; BAN: chlormethiazole) and dizocilpine we noted that at the doses used neither compound when given with MDMA appeared to produce frank hypothermia (Colado et al., 1993; Hewitt & Green, 1994). Nevertheless both compounds were neuroprotective. However, both compounds did abolish the MDMA-induced hyperthermia (Hewitt & Green, 1994). We have now therefore re-examined the effect of clomethiazole to determine whether its neuroprotective effect against MDMA-induced damage is due to its effect on body temperature or whether this compound had an additional neuroprotective action in this neurodegenerative model as it has in animal models of acute ischaemic stroke (Green & Cross, 1994; Green, 1998). In addition we examined the effect of the novel low affinity NMDA receptor channel blocker AR-R15896AR (Greene et al., 1996; Cregan et al., 1997; Palmer et al., 1997) to see whether the proposal of Farfel & Seiden (1995a) that NMDA antagonists may not be neuroprotective against MDMA induced damage in the absence of hypothermia could be confirmed and extended. Some of these results were given in preliminary form to a meeting of the British Pharmacological Society (Colado et al., 1997a).
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There have been several studies, including those conducted in our laboratory, which have demonstrated a protective effect of various NMDA antagonists against MDMA-induced neurotoxicity (Finnegan et al., 1990; Farfel et al., 1992; Colado et al., 1993; Hewitt & Green, 1994). However, recently Farfel & Seiden (1995a) observed that the combination of dizocilpine or CGS 19755 and MDMA produced frank hypothermia and suggested that it was the hypothermia that produced the neuroprotection. They strengthened this proposal by showing that dizocilpine was no longer neuroprotective when rats were kept at high ambient temperature, thereby preventing a decrease in body temperature in the dizocilpine/MDMA treated animals. We have now found that the novel low affinity NMDA channel blocker AR-R15896AR did not attenuate the MDMA-induced hyperthermia and also had no neuroprotective effect at a dose known to be neuroprotective in animal models of ischaemic stroke (Palmer et al., 1997; Cregan et al., 1997). All these data therefore suggest that the neurodegeneration which follows MDMA may not involve an excitotoxic cascade involving glutamate, as has been suggested to occur in ischaemia-induced neurodegeneration (see Small & Buchan, 1997).
These data raised the possibility that other drugs previously shown to be neuroprotective might also have induced protection by lowering body temperature, rather than through a specific neuroprotective mechanism. We therefore examined clomethiazole, another compound we had previously investigated, and which has been found to be neuroprotective in various animal models of acute ischaemic stroke (see Green & Cross, 1994; Green, 1998).
In our previous studies clomethiazole did not produce hypothermia in the MDMA-treated Lister Hooded rats, but nevertheless clearly abolished the hyperthermia induced by MDMA and was an effective neuroprotective drug (Colado et al., 1993; Hewitt & Green, 1994). In this study, using Dark Agouti rats this finding was confirmed.
When the temperature of the rats given clomethiazole plus MDMA was kept elevated to approximately that of those given only MDMA, there was a reduction in the degree of neuroprotection. Nevertheless, there was still considerable protection, in contrast to the studies with dizocilpine (Farfel & Seiden, 1995a) where it was abolished. This observation also indirectly supports our evidence that the neuroprotective action of clomethiazole does not involve an action of the drug at NMDA receptors (Cross et al., 1993; Green et al., 1997).
Damage following MDMA occurs in normothermic animals (Broening et al., 1995; Farfel & Seiden, 1995a) but hyperthermia worsens the damage (Broening et al., 1995). Our data are consistent with this indicating that the protection afforded by clomethiazole at normal ambient temperatures comprises two additive mechanisms. One is a reduction of the rat temperature to normothermia and the second is an unrelated neuroprotective mechanism.
There is now a substantial body of evidence that increased free radical formation is responsible for MDMA-induced neurotoxicity (Cadet et al., 1994; Colado & Green, 1995; Sprague & Nichols, 1995; Murray et al., 1996; Colado et al., 1997b,c). This, in turn, would explain why hyperthermia exacerbates the damage, since it has been shown to enhance free radical production in the brain (Globus et al., 1995; Kil et al., 1996). The MDMA-induced increase in free radical formation only becomes apparent 1 h after administration and continues for several hours thereafter (Colado et al., 1997c). It seems unlikely therefore that the lower body temperature of the clomethiazole/MDMA treated rats compared to the MDMA treated rats in the first hour explains the substantial neuroprotection seen.
What remains uncertain is the mechanism(s) by which clomethiazole affords protection against MDMA-induced damage. Although there is good evidence that the damage results from increased free radical formation in the brain (see above), recent studies have indicated that clomethiazole is not a free radical scavenger (Colado et al., unpublished observations). Clomethiazole is known to potentiate the action of γ-aminobutyric acid (GABA) in the brain and does so in a way that differs from benzodiazepines and barbiturates (see Cross et al., 1989; Green et al., 1996), compounds that are not neuroprotective against ischaemic stroke (Green, 1998). There are reasonable grounds for believing that it is this GABA potentiating action that is responsible for the drug being neuroprotective in animal models of acute ischaemic stroke (see Green, 1998 for review). However, whether this property is involved in the neuroprotective action of the drug against MDMA-induced damage requires further investigation.
Finally, the current data and that of others (Farfel & Seiden 1995a,b; Malberg et al., 1996) suggest that re-evaluation is required of some of the earlier reports on the neuroprotective properties of various compounds against MDMA-induced neurodegeneration, if the studies were conducted in the absence of good measures of body temperature.