Increased anxiety and impaired memory in rats 3 months after administration of 3,4-methylenedioxymethamphetamine (“Ecstasy”)

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Abstract

Male Wistar rats were administered either (a) a high dose regime of 3,4-methylenedioxymethamphetamine (MDMA) (4×5 mg/kg, i.p. over 4 h on each of 2 consecutive days), (b) a moderate dose regime of MDMA (1×5 mg/kg on each of 2 consecutive days), (c) d-amphetamine (4×1 mg/kg over 4 h on each of 2 days), or (d) vehicle injections. The high MDMA dose regime and the amphetamine treatment both produced acute hyperactivity and hyperthermia. Twelve weeks later, all rats were tested in the drug-free state on a battery of anxiety tests (elevated plus maze, emergence and social interaction tests). A further 2 weeks later they were tested on a novel object recognition memory task. Rats previously given the neurotoxic dose of MDMA showed greater anxiety-like behaviour on all three anxiety tests relative to both controls and d-amphetamine-treated rats. Rats given the moderate MDMA dose regime also showed increased anxiety-like behaviour on all three tests, although to a lesser extent than rats in the high dose group. In the object recognition task, rats given the high MDMA dose regime showed impaired memory relative to all other groups when tested at a 15-min delay but not at a 60-min delay. Rats previously exposed to amphetamine did not differ from saline controls in the anxiety or memory tests. These data suggest that moderate to heavy MDMA exposure over 48 h may lead to increased anxiety and memory impairment 3 months later, possibly through a neurotoxic effect on brain serotonin systems.

Introduction

3,4-Methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) is an increasingly popular recreational drug in many countries of the world. Concern continues to mount about the long-term neurotoxic effects of the drug and its possible deleterious effects in humans (Boot et al., 2000). In animals, MDMA markedly decreases regional brain serotonin (5-HT) content and produces 5-HT nerve terminal degeneration in forebrain areas of rats Battaglia et al., 1987, Molliver et al., 1990, Ricaurte et al., 1992 and primates (Fischer et al., 1995). Human MDMA users also show several possible markers of 5-HT depletion such as blunted endocrine responses to d-fenfluramine challenges, decreased cerebrospinal 5-hydroxy-indolacetic acid (5-HIAA) concentrations and a reduced density of brain 5-HT transporter sites Gerra et al., 2000, McCann et al., 1999, Semple et al., 1999.

Recent studies suggest that heavy MDMA users may suffer from both long-term cognitive deficits Reneman et al., 2000, Verkes et al., 2001, Wareing et al., 2000 and a number of psychiatric sequale such as anxiety and depression Parrott et al., 2000, Verkes et al., 2001, Wareing et al., 2000. Although these studies provide increasing evidence of clinical problems associated with long-term ecstasy use, they suffer from certain difficulties of interpretation (Boot et al., 2000). It is not possible for such studies to control for the amount of MDMA used, the purity of the MDMA consumed and the polydrug use of human subjects. In addition, pre-existing abnormalities in 5-HT systems and in cognitive and emotional function cannot be ruled out in human MDMA users. It is therefore likely that preclinical studies will be useful in allowing a definitive account of the long-term behavioural and emotional effects of MDMA.

Surprisingly, there has been a relative paucity of such preclinical studies particularly in relation to anxiety and memory. Slikker et al. (1989) found a trend towards increased anxiety-like behaviours in rats given neurotoxic doses of MDMA 2–4 weeks previously. With respect to memory, Marston et al. (1999) documented impairment in a delayed matching to position working memory task in rats at up to 19 days following MDMA treatment. However, Ricaurte et al. (1993) found that the choice accuracy of rats in a T-maze delayed alternation task was generally unaffected following exposure to neurotoxic doses of MDMA.

The present study aimed to further investigate the long-term effects of MDMA on anxiety and memory in rats using a range of behavioural paradigms. A battery of three different anxiety tests were employed for the first paradigm (see Morley and McGregor, 2000), which are thought to reflect different anxiety states in humans (File, 1995). These consisted of: the emergence test (Crawley and Goodwin, 1980), the elevated plus-maze test (Pellow et al., 1985) and the social interaction test (File, 1980). To assess memory, the novel object recognition test was used. This model is thought to measure non-spatial working memory in rats Blanchard et al., 1970, Aggleton, 1985, Aggleton, 1993.

In addition to a vehicle control group, the present study also utilized a control group given d-amphetamine. Although d-amphetamine has a similar chemical structure to MDMA and similar stimulant and hyperthermic properties, it is not generally thought to be neurotoxic except at very high doses (e.g. Linder et al., 1995, Ryan et al., 1990). Thus, use of this treatment group therefore allowed additional control for any long-term effects of stimulant administration on behaviour (cf. Robinson and Becker, 1986) that are unrelated to neurotoxicity.

Section snippets

Subjects

A total of 64 inbred male albino Wistar rats (Concord Hospital, Sydney, Australia) were used in the experiments, aged between 75 and 95 days. The rats weighed an average of 273 g at the start of treatment and 360 g at the start of behavioural testing 12 weeks later. The rats were housed in large plastic tubs in groups of 8 per cage in a temperature-controlled environment (average temperature 22 °C). A 12-h reversed light cycle was in operation (lights off at 8:30 a.m.) and all testing took

Temperature

Body temperatures on the 2 days of drug administration are depicted in Fig. 1. For day 1, ANOVA showed no significant group effect for h 0 (F<1), or h 1 (F(3, 60)=2.69, P=0.054), but revealed a significant group difference for h 2 (F(3, 60)=6.45, P<0.001), h 3 (F(3, 60)=20.00, P<0.0001) and h 4 (F(3, 60)=38.64, P<0.0001). For day 2, ANOVA showed no significant effect for h 0 (F<1), but revealed significant differences for h 1 (F(3, 60)=3.26, P<0.05), h 2 (F(3, 60)=8.80, P<0.0001), h 3 (F(3,

Discussion

The results of the present study indicate that exposure to MDMA can produce pronounced long-term effects on anxiety-like behaviours, social interaction and memory in rats. No long lasting effects were seen after exposure with another chemically similar stimulant d-amphetamine, suggesting that these behavioural effects are relatively specific to MDMA.

Acute hyperactivity was observed in both MDMA and d-amphetamine groups, as previously demonstrated Gold et al., 1989, Callaway et al., 1990,

Acknowledgements

Supported by a National Health and Medical Research Council grant to ISM and GEH. Kirsten Morley is the recipient of an Australian Postgraduate Award.

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