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from Ecstasy: The Complete Guide ed. Julie Holland Spring 2001 This article, written in early 2001, is the best currently available overview of MDMA neurotoxicity. It is from the excellent book Ecstasy: The Complete Guide edited by Julie Holland which contains a number of very interesting articles on the topic of MDMA, its complications, and its potential as a psychiatric medication. Mattew Baggott's article on neurotoxicity was written as he was finishing his truly epic MDMA literature review published by MAPS as part of their work to explore MDMA as a potential therapeutic agent. Erowid had a very small part in helping Matt with his literature review and its dense 376 pages are a must-read for anyone truly dedicated to understanding the scientific complexities of this challenging psychoactive. This summary of the neurotoxicity issue for Dr Holland's book, however, is much more accessible and should be understandable by anyone with college-level reading skills and interest.
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Introduction MDMA Can Induce Long-term Serotonergic Changes Serotonergic Changes are Accompanied by Structural Changes to Axons Are these Serotonergic and Axonal Changes Evidence of Damage? The Role of Oxidative Stress in MDMA neurotoxicity Proposed Sources of Oxidative Stress Extent of Neurotoxicity Depends on Dose, Route of Administration, and Species Why are such High Doses Used and Can They be Justified? Extent of Neurotoxicity in Rats is Influenced by Environment, Especially Ambient Temperature 10. Time Course of Changes and Extent of Recovery 11. Behavioral and Functional Correlates of MDMA Exposure in Animals 12. Studies Comparing Ecstasy Users and Nonusers 13. Personality Differences between Ecstasy users and nonusers 14. Neurofunctional Differences between ecstasy users and nonusers 15. Cognitive Differences between ecstasy users and nonusers 16. Possible Significance of Cognitive Differences and MDMA Neurotoxicity 17. Findings in Prospective Clinical MDMA Studies 18. Potential Strategies for Reducing Risk of Neurotoxicity in Clinical Settings 19. Need for More Research 20. Summary
Introduction
The acute toxic effects of MDMA are well documented by hundreds of case reports of adverse
events in illicit users. Considering how many people use MDMA, serious acute adverse events seem rare. MDMA appears generally similar to psychostimulants such as methamphetamine with respect to the risks of acute toxicity. With trained personnel, properly screened volunteers, and established protocols for monitoring and treating adverse events, these acute risks appear modest and do not present a strong argument against carefully conducted clinical research with MDMA. On the other hand, the risks associated with possible long-term brain damage are more difficult to assess. Numerous studies in animals have shown that MDMA can produce long-lasting decreases in brain functions involving the neurotransmitter serotonin. It is unclear what these changes mean. Lasting behavioral changes in MDMA-exposed animals have been seldom detected and are fairly subtle when they are found. Though limited in scope, studies of ecstasy users present a strong probability that similar serotonergic changes occur in many humans. Studies comparing ecstasy users and nonusers support an association between modestly-lowered intelligence testing, or cognitive performance tests, and ecstasy use, but clinically significant performance decreases have not been detected. In other words, there is no increased incidence of clinical complaints or findings. The modest findings in behavioral studies of MDMA neurotoxicity have led some to dismiss concerns about MDMA neurotoxicity as politically-motivated alarmism. It is commonly pointed out that though fenfluramine and methamphetamine produce similar changes, their status as prescription medications was not affected by this finding. However, it is reasonable to note that the truly long-term effects of MDMA exposure are unknown. In 15 years of research on MDMA neurotoxicity, no published studies have investigated whether MDMA exposure can cause significant toxicity that only becomes apparent with aging. This fact must be taken into account when considering the risks and benefits of possible clinical studies. Perhaps the single most worrisome issue surrounding MDMA neurotoxicity is that there may be significant toxicity associated with serotonergic changes that is currently undetected. Although millions of people have taken millions of doses of ecstasy, controlled studies of users have not been large enough to detect any but the most common chronic adverse effects. Possible adverse effects such as an increased incidence of affective disorders, like depression, may have gone unnoticed. Because so little is known about possible long-term clinical implications of MDMA neurotoxicity, we believe it is important to minimize the risks of neurotoxicity in research volunteers. It is hoped that the information presented here may contribute to assessments of, and perhaps reductions in, the risks associated with MDMA use. This chapter will discuss (1) the nature and meaning of MDMA-induced serotonergic changes; (2) the possible mechanisms of these changes; (3) factors influencing the severity of these changes (such as dose, route of administration, species and animal strain, and environment); and (4) the time course of these changes and recovery. The latter part of this chapter will focus on the implications of long-term serotonergic changes by discussing (5) the behavioral and functional effects of MDMA-induced serotonergic changes in animals; (6) studies comparing ecstasy users to nonusers (including personality, cognitive, and functional comparisons); (7) available data from clinical studies in which MDMA was administered; and (8) potential strategies for reducing risk to human volunteers. Limitations of space unfortunately prevent a full discussion of every important paper and aspect
of this complex topic. For a broader sense of the range of views on MDMA neurotoxicity, the reader is therefore advised to consult other review articles (Boot, 2000; Burgess, 2000; Green, 1995; Hegadoren, 1999; McKenna , 1990; Morgan 2000; O'Callaghan, 2001; Seiden, 1996; Sprague, 1998; Steele, 1994), and the issue of Neuropsychobiology (Vol. 42, 2000) dedicated to MDMA neurotoxicity.
Thus, decreased 5-HT synthesis and subsequent SERT down-regulation initially appear to be a plausible explanation for MDMA-induced serotonergic changes. By examining the structure of serotonergic axons in MDMA-exposed animals, however, it is clear that MDMA can also cause axonal loss.
hippocampus (Colado, 1999b; 1997b), two areas of the brain involved in movement, and memory, respectively. There is strong evidence that oxidative stress is involved in the mechanisms of MDMA neurotoxicity. Antioxidants are substances which inactivate free radicals; the antioxidants ascorbate and cysteine each reduce MDMA neurotoxicity in rats without altering levels of MDMA or MDMA-stimulated dopamine release (Gudelsky 1996; Schmidt, 1990). The free radical scavenger N-tert-butyl-alpha-phenylnitrone decreases both MDMA-induced hydroxyl formation and MDMA neurotoxicity in rats; this latter effect, however, may be partially due to an attenuation of MDMA-induced high body temperature, or hyperthermia (Che, 1995; Colado, 1998; 1995; Yeh 1999). Pretreatment with the antioxidant alpha-lipoic acid blocks both MDMAinduced serotonergic neurotoxicity and increased GFAP expression in the rat hippocampus without altering MDMA-induced hyperthermia (Aguirre, 1999). Mice that have been genetically altered to have large amounts of the human antioxidant enzyme, copper/zinc superoxide dismutase, are protected from MDMA-induced dopamine depletions, probably because of the increased trapping of superoxide radicals (Cadet, 1994; Cadet,1995; Jayanthi, 1999). At the same time, these genetically modified mice are protected from the acute inactivation of antioxidant enzymes and free radical changes seen in normal mice after a neurotoxic MDMA regimen (Jayanthi, 1999). Early evidence that MDMA caused significant oxidative stress came from Stone (1989a) who reactivated TPH which had been inactivated in rats at 3 hours after high dose MDMA by using sulfhydryl reducing conditions. This showed that the acute inactivation of TPH by MDMA was due to intracellular oxidative stress. Intracellular oxidative stress appears to be an effect of MDMA that requires sustained brain concentrations of MDMA (or a centrally formed metabolite). While a single injection of MDMA into the brain had no effect on TPH activity, slow infusion of 1 mg/kg MDMA into the brain over 1 hr produced enough oxidative stress to acutely reduce TPH activity (Schmidt, 1988). The acute decrease in TPH activity is an early effect of MDMA and can be seen 15 minutes after administration (Stone, 1989b). TPH inactivation can also be produced by non-neurotoxic MDMA doses (Schmidt, 1988; Stone, 1989a; 1989b). It therefore appears that MDMA rapidly induces oxidative stress but only produces neurotoxicity when the brain's free radical scavenging systems become overwhelmed. In summary, MDMA neurotoxicity involves an initial period of oxidative damage, where an increase in free radicals damages neural lipids. This damage seems to be part of the sequence of events producing serotonergic neurotoxicity since treatments that decrease MDMA-induced oxidative stress also decrease the long-term serotonergic changes (Aguirre, 1999). While MDMA can cause loss of axons, some serotonergic down regulation cannot be ruled out. Research on methamphetamine-induced dopaminergic neurotoxicity has led some to conclude that long-term dopaminergic changes can occur without significant axonal loss (Harvey, 2000; Wilson, 1996). Whether this is also the case with MDMA is unknown. For now, it seems reasonable to consider long-term serotonergic alterations after MDMA exposure as indicating that some degree of damage has occurred, while remembering that one is also measuring the response of the serotonergic system to acute drug effects and loss of axons.
which, in turn, may produce hydroxyl radicals. A quinone-like dopamine metabolite may also be formed with potential to generate further free radicals (Cadet and Brannock 1998; Graham, 1978)). Among many other potential toxic effects on cells, dopamine oxidation products have been shown to impair mitochondrial functioning (Berman and Hastings 1999). There is currently little direct evidence to support a role for dopamine metabolites in MDMA neurotoxicity. Some dopaminergic drugs alter MDMA neurotoxicity, but it is not clear that this is due to increasing or decreasing dopamine release. Many dopaminergic drugs are now thought to affect MDMA neurotoxicity through nonspecific mechanisms such as altering body temperature (Colado, 1999a; Malberg, 1996) or scavenging free radicals (Sprague and Nichols 1995a; b; Sprague, 1999). However, dopamine release does seem to play a poorly understood role in MDMA neurotoxicity (Nash and Brodkin 1991; Schmidt, 1990; Shankaran, 1999b; Stone, 1988).
Ricaurte, 1992a; but see also De Souza, 1990, for slightly different results). Many MDMA neurotoxicity studies have used squirrel monkeys as subjects. The threshold dose for producing long-term 5-HT depletions in squirrel monkeys is somewhere between 2.5 and 5 mg/kg oral MDMA. Two weeks after a single 5.0 mg/kg oral MDMA dose to this species, 5-HT levels were decreased to 83% of control levels in the hypothalamus and 79% of controls in the thalamus but were not changed in other examined brain regions (Ricaurte, 1988a). In contrast, no long-term serotonergic changes occurred after 2.5 mg/kg MDMA was given orally every two weeks for four months to squirrel monkeys (Ricaurte, unpublished, cited in Vollenweider, 1999). [Editor's note: a therapeutic dose of 125 mg in a 150 pound person would translate to 1.66 mg/kg] Another commonly studied nonhuman primate species is the rhesus monkey. Determining the threshold dose for 5-HT depletions in this species is difficult since all published studies using rhesus monkeys have employed multiple dose neurotoxic regimens. In one study, 1.25 mg/kg oral MDMA did not produce any long-term serotonergic changes when given twice daily for 4 consecutive days. Similarly repeated doses of 2.5 mg/kg MDMA lowered hippocampal 5-HT (to about 80% of controls) but did not affect levels in 6 other brain regions at post one month (Ali, 1993). In another experiment, Insel (1989) found that 2.5 mg/kg MDMA given intramuscularly twice daily for 4 days to rhesus monkeys produced extensive (possibly short term) 5-HT depletions but did not alter SERT density at 16 to 18 hours after the last drug exposure. Since SERT was unaffected, the researchers concluded that axonal loss had not occurred, despite the 5HT depletions. In a study that raises interesting questions about possible tolerance to MDMA neurotoxicity, Frederick (1995) investigated the long-term effects of escalating doses of MDMA. Intramuscular MDMA (0.1-20 mg/kg) was given twice daily for 14 consecutive days at each dose level and followed by three dose-response regimens using single MDMA doses up to 5.6 mg/kg. One month after the final dose-response determination and 21 months after the initial escalating dose regimen, animals were sacrificed. Few significant serotonergic effects were found. MDMA exposure did not produce significant 5-HT depletions in any brain region and decreased SERT to about 60% of control levels only in the hippocampus (and not two other brain regions). Thus, data on rhesus monkeys are complex and perhaps all that can be said with certainty is that the threshold dose for long-term 5-HT depletions appears to be above 1.25 mg/kg oral MDMA in this species.
Why are such High Doses Used and Can They be Justified?
Research on MDMA neurotoxicity has sometimes been criticized for the repeated high dose regimens that are commonly used. Some have questioned whether repeated injections of 20 mg/kg MDMA in rodents can provide useful information about the toxicity of single oral doses of 1.7 to 2.0 mg/kg MDMA in humans. It is true that many of the neurotoxic regimens are not designed to be clinically relevant but were intended to maximize the serotonergic neurotoxicity of MDMA in order to better understand its mechanisms and consequences. However, comparing dose on the basis on body weight can be misleading. In general, smaller species excrete drugs more quickly and form metabolites in greater amounts than larger species.
This is due to many factors including the proportionally larger livers and kidneys and faster blood circulation times in smaller mammals (Lin 1998; Mordenti,1989). As a result of such factors, the time it takes to lower the plasma levels of MDMA by half is about 1.5 hours in a rat (Cho, 1990) and about 8 hours in a human (Mas, 1999). This suggests that small species may require higher doses to achieve drug exposures comparable to those seen in larger species. These considerations at least partially justify the apparently high doses commonly used in rodent toxicity studies. Unfortunately, higher doses tend to alter the character of the drug exposure. While they lengthen the time smaller animals are exposed to the drug, they also tend the produce higher peak blood concentrations of drug and greater acute effects than occur in larger species at lower doses. A number of techniques have been developed for estimating equivalent drug doses in different species (Ings 1990; Lin 1998; Mahmood 1999; Mordenti, 1989). One of the most commonly used techniques, allometric interspecies scaling, involves administering a drug to different species and measuring resulting blood concentrations of drug. These measurements are then used to determine the relationships between species weight, drug exposure, and dose. Drug exposure in humans can then be estimated from these relationships. In these estimates, equivalent drug exposures are assumed to produce equivalent drug effects, including neurotoxicity. Recently, Ricaurte (2000) estimated that as little as 1.28 mg/kg MDMA may produce long-term 5-HT depletions in humans if interspecies dose conversions for MDMA follow a pattern that is common for drugs that are not extensively metabolized. Estimates of this sort are useful for emphasizing that the MDMA dose required to produce neurotoxicity in humans may be within the range of commonly administered doses, despite the seemingly higher doses used in rodent studies. However, such estimates require making assumptions about the mechanisms of neurotoxicity. For example, it is necessary to assume that the different species experience comparable drug effects when blood concentrations of drug are the same. This may not be true of neurotoxicity. Several other possible reasons for species differences in MDMA neurotoxicity have already been given. In addition, species may differ in the brain concentration of drug produced by a given blood concentration. It is not known if this is the case with MDMA, although it does seem to be true for fenfluramine (Campbell 1995). Furthermore, if MDMA neurotoxicity is caused by a toxic metabolite, as some have suggested, then the more extensive metabolism of MDMA expected in smaller animals will lead to increased neurotoxicity. Formation of specific drug metabolites in different species is difficult to predict and few data are available on MDMA. Research on species differences in fenfluramine metabolism have led some to conclude that no nonhuman species provides a good model of possible human fenfluramine neurotoxicity (Caccia, 1995; Marchant, 1992). Because current data suggest that both MDMA and metabolite exposure may mediate neurotoxicity, more data are needed from more species before interspecies dose conversions can be made with any confidence. Data from clinical MDMA studies show that there is a complex relationship between MDMA dose and blood levels of the drug and its metabolites (de la Torre, 2000; Mas, 1999). It appears that MDMA inactivates one of the enzymes in the liver that is important to its metabolism (an enzyme known as cytochrome p450 isozyme 2D6 or 'CYP 2D6') (Brady, 1986; Wu, 1997). As a result, small increases in dose can lead to large increases in drug exposure. When dose was
increased from 120 mg to 150 mg, drug exposure almost doubled in human volunteers, as measured by area under the curve of MDMA plasma concentration verses time (de la Torre, 2000). However, formation of some metabolites remained approximately constant. These complex dose-dependent pharmacokinetics in humans further increase the difficulty of estimating dose conversions between species. Nonetheless, these human studies with MDMA do suggest that doses above 120 mg may be associated with unexpectedly increased drug exposure and therefore risks of toxicity.
between 3 and 12 hours after drug administration in rats. Slow recovery of serotonergic functioning can be seen following a neurotoxic dose of MDMA. The extent of recovery is different in different species. In rats, there is extensive recovery of indicators of serotonergic functioning 1 year after drug exposure (Battaglia, 1988; Lew, 1996; Sabol, 1996; Scanzello, 1993), although there is significant variation in recovery between individual animals (Fischer, 1995). In primates, some recovery of serotonergic function occurs but is less extensive than in the rat. Altered serotonergic axon density was still detectable 7 years after MDMA exposure in one study of squirrel monkeys (Hatzidimitriou, 1999). Therefore, despite some recovery, MDMA-induced serotonergic changes are likely permanent in this primate species. This apparent species difference may be partially related to the more severe initial serotonergic damage usually seen in primates compared to rats, but also likely indicates a species difference in regrowth of serotonergic axons.
Table I: Studies of Long-term Changes after Neurotoxic MDMA Regimens in Nonhuman Animals Measures showing Species and Significant Differences in MDMA Regimen no significant Reference Strain MDMA-treated animals difference Right shift in MDMA and d-fenfluramine doseRhesus 10 mg/kg IM, twice response curve for time Baseline performance Frederick et Monkeys a day, for 4 days estimation, learning task, on all tasks. al., 1998 and motivation tasks at post 1 mo. Escalating doses of Right shift in MDMA 0.10, 0.3, 1.0, 1.75, dose-response curve for 3.0, 5.6, 7.5, 10.0, time estimation, short-term Rhesus Baseline performance Frederick et 15.0, and 20 mg/kg, memory, color and position Monkeys on all tasks. al., 1995 IM, twice daily for discrimination, and 14 consecutive days motivation tasks at post 21 at each dose. mo. None, although researchers note that 2 of 8 MDMAAcquisition of and Rats, exposed rats failed to Byrne, 20 mg/kg SC, twice behavior on a leverSpragueacquire lever pressing with Baker, & a day for 4 days press responding task Dawley 20 sec reinforcement Poling 2000 at post 14 days. delays during the 8 hr session. Significant pretreatment x treatment x crossing times Drug-free locomotion Rats, 10 mg/kg SC, twice interaction, suggesting at 21 days; RU24969- Callaway & Spraguea day for 4 days altered S-MDMA -induced induced behavioral Geyer 1992 Dawley behavioral activation at activation at 21 days. post 21 days. Increased core temperature when placed in either 22 10 mg/kg SC per Dafters & Rats, Wistar C or 28 C ambient None day for 4 days Lynch 1998 temperature at post 4 or 14 wks. Sexual behaviors at Dornan, Rats, Long- 40 mg/kg SC, twice post 10 days; Katz, & None Evans a day for 4 days Spontaneous motor Ricaurte activity. 1991 Electrical-stimulated Rats, Decreased electrical5-HT release in MRN Gartside, 20 mg/kg SC, twice Spraguestimulated 5-HT release in or hippocampus at McQuade, & a day for 4 days Dawley DRN at post 2 wks. post 2 wks; Number Sharp 1996 and firing pattern of
classical 5-HT neurons and burstfiring neurons in DRN. DOI-induced head twitch responses, locomotion, and rearing activity.
Horan, 20 mg/kg SC, twice Increased conditioned place preference response Gardner, & a day for 4 days to cocaine in MDMA group at 2 post wks. Ashby 2000 Increased motor stimulant effects of 5.0 mg/kg SC MDMA in both MDMA5 mg/kg sc once per treated groups at post 11 day or 20 mg/kg Rats, days; Increased motor Basal DA in nucleus Kalivas, SC, twice a day for Spraguestimulant effects of 15.0 accumbens at post 2 Duffy, 4 days, followed by Dawley mg/kg IP cocaine in both wks. White 1998 5 mg/kg MDMA 2 MDMA-treated groups at days later post 11 days; Increased MDMA-stimulated DA release in the nucleus Basal activity of nigrostriatal DA Loss of rate-dependence of Rats, neurons; Quipazine- Kelland, response of nigrostriatal Sprague15 mg/kg IP induced inhibition of Freeman, & cells to either quipazine or Dawley nigrostriatal DA cell Chiodo 1989 apomorphine at post 1 wk. firing for all cells at post 1 wk. Rats, Left shift in MDMA dose6 mg/kg SC, twice Li et al., Spragueresponse curve on DRL None a daily for 4 days 1989 Dawley task in MDMA group. ascending regimen Spontaneous of 10, 15, and 20 Decreased performance in behavior, body Rats, Lister Marston et mg/kg IP, each operant delayed match to temperature, and Hooded al., 1999 dose given twice nonsample task. skilled paw reach daily for one day ("staircase task"). Increased cocaine-induced Rats, dopamine release in 20 mg/kg SC, twice Morgan et Spraguenucleus accumbens in None a day for 4 days al., 1997 Dawley MDMA group at 2 wks after neurotoxic regimen. Rats, Increased morphineNencini, 20 mg/kg SC, twice Baseline behavior in Spragueinduced antinociception Woolverton, a day for 4 days tail flick test. Dawley (assessed by tail flick test) & Seiden
Inhibitory effects of GABA on glutamateRats, evoked firing in Spraguenucleus accumbens Dawley cells at post 9-15 days. Basal ACTH and Increased 8-OH-DPATprolactin Rats, induced prolactin release at concentrations and Sprague20 mg/kg SC post 2 weeks. Decreased ACTH and prolactin Dawley 8-OH-DPAT-stimulated response to saline ACTH release at 2 weeks. injection. Increased d,ld,l-FenfluramineFenfluramine-stimulated stimulated ACTH at Rats, prolactin release at post 2 12 months; d,lSprague20 mg/kg SC and 4 months. Decreased FenfluramineDawley d,l-Fenfluraminestimulated prolactin stimulated ACTH release at 8 and 12 months. at 2-8 months. Saline-stimulated Increased d,lACTH and prolactin Fenfluramine-stimulated release at post 2 Rats, prolactin release at post 4 20 mg/kg SC, twice weeks; d,lSpragueand 8 months. Decreased a day for 4 days FenfluramineDawley d,l-Fenfluraminestimulated prolactin stimulated ACTH release release at post 12 at post 4, 8, and 12 months. months. Performance in a spatial memory task 20 mg/kg SC, twice using a T-maze and Rats, Lister a day for 4 days None scopolamine-induced Hooded with entire regimen changes in repeated 2 wks later performance on this task. Spatial navigation task after 1st trial, Increased time to find skilled reaching task, Rats, 10 mg/kg IP, twice hidden platfrom in first place navigation Spraguea day for 4 days trial of spatial navigation learning-set task, Dawley task at post 2 days. foraging task, with or without atropine pretreatment. Rats, 20 mg/kg SC twice Decreased discrimination Discrimination of 0.5 Spraguea day for 4 days of 1.0 mg/kg MDMA from or 1.5 mg/kg;
at post 2 wks. Decreased inhibitory effects of DA and 20 mg/kg SC, twice SKF38393 on glutamatea day for 4 days evoked firing in nucleus accumbens cells at post 915 days.
Poland 1990
Schechter 1991
Dawley
Rats, SpragueDawley
Rats, SpragueDawley
Decreased d-fenfluramine20 or 40 mg/kg SC Series, stimulated 5-HT release in twice a day for 4 None Cowen, & frontal cortex at post 2 days Sharp 1994 wks. Decreased behavioral, hyperthermic, and 5-HTShankaran & 10 mg/kg IP, every releasing effects of MDMA None Gudelsky 2 h for 4 injections at 1 wk after neurotoxic 1999 regimen. Increased cerebral glucose Cerebral glucose utilization in molecular utilization in 20 or 40 mg/kg SC layer of dentate gyrus and Sharkey, neocortex, raphe twice a day for 4 in CA2 and CA3 fields of McBean, & nuclei, and some days Ammon's horn in Kelly 1991 hippocampal areas at hippocampus at post 14 post 14 days. days. Auditory startle, emergence from darkened chamber, complex maze 5 or 10 mg/kg PO Slikker et None navigation, response daily for 4 days al., 1989 to hot plate, FI 90 operant behavioral task at post 2 to 4 weeks.. Behavioral responses to the 5-HT1a agonist Decreased rate of 10 mg/kg SC every 8-OH-DPAT, the 5ultrasonic vocalization Winslow & 12 hrs for 4 or 7 HT1b agonist measured up to post 11 Insel 1990 injections TFMPP, and the 5days. HT2 agonist DOI at post 8 days.
of frequent illicit drug users and those specifically associated with ecstasy use. [Editor's note: It is also important to realize that ecstasy users may not be ingesting MDMA alone or sometimes at all, as there is no guarantee that purchased ecstasy contains MDMA. It is possible that polydrug use could contribute to any detected problems.] Other frequent methodological limitations of these studies include: poorly described recruitment and matching of volunteer groups; reliance on self-reports of drug use; failure to separate residual effects of recent drug use from long-term effects; use of the same volunteers in multiple publications; and the difficulty relating serotonergic differences to toxicity. Despite these limitations, some conclusions can be drawn from studies comparing ecstasy users and nonusers. Findings can be grouped into personality, neurofunctional, and cognitive performance differences. These areas are discussed below. Consistent reports link repeated ecstasy use to depressed mood (Cohen 1995; Curran, 1997; Davison, 1997; Gamma, 2000; Gerra, 2000;1998; Morgan, 1999; Parrott 2000; 1998; Solowij, 1992). Because dysphoric mood is a known residual effect of other psychostimulant drugs (Coffey, 2000), it is likely that ecstasy use plays a causal role in this phenomenon. In a survey of 158 polydrug users, Williamson (1997) found that similar numbers of users reported depression, anxiety, and related adverse effects after cocaine as compared to MDMA. Thus, in some ways, MDMA is very similar to other psychostimulants. In addition, there are a number of case reports of psychiatric disorders, such as psychosis, depression, and panic attacks in ecstasy users (reviewed in McGuire, 2000). Given that other psychostimulants are associated with psychiatric disorders in illicit users, it would not be surprising if this were also true of MDMA. For example, it is well established that stimulantinduced psychosis can occur in cocaine or methamphetamine users (Angrist, 1994). Reports of MDMA-related psychosis have also been published (Creighton, 1991; McCann, 1991a; McGuire, 2000; 1991). These psychiatric disorders need not be related to the selective neurotoxicity discussed in this chapter. For example, methamphetamine can produce chronic behavioral disturbances resembling psychosis in primates using regimens that are not neurotoxic to dopaminergic or serotonergic systems (Castner, 1999).
one could compare ecstasy users with different total ecstasy exposures or compare polydrug users with and withoutecstasy experience. Findings from these comparisons are ambiguous and, at best, such comparisons can provide only limited support for possible MDMA-induced alterations in personality. Studies in which the same individuals are examined at different time points are necessary to properly examine this issue. There is mixed evidence that MDMA use is associated with increases in self-reported impulsivity. Morgan (1998) reported that a post hoc comparison of more (30+ tablets ingested) and less experienced (20 - 30 tablets ingested) ecstasy users revealed heightened impulsivity (measured with Eysenck's self-report IVE questionnaire) in the more experienced group. Parrott (2000) reported a non-significant trend towards greater IVE impulsivity in polydrug-usingecstasy users with an average of 371 (30-1000) exposures compared to a group of users with an average of 6.8 (1-20) exposures. Tuchtenhagen (2000) found that ecstasy users with an average of 93.4 119.9 (20-500) exposures has significantly higher scores for the nonplanning impulsivity (measured with the self-report Barratt Impulsiveness Scale) compared to controls matched for other drug use. The researchers also noted a trend towards increased experience seeking (measured with the self-report Sensation Seeking Scale) which reached statistical significance only when ecstasy users were compared to nonusers. These findings differ from those of McCann (1994) who compared ecstasy users, with an average of 94.4 +/- 90.6 (25-300) reported ecstasy exposures, to nonusers (without controlling for other drug use). McCann, reported decreased impulsivity (measured as increases in the Control subscale of the Multidimensional Personality Questionnaire) but failed to find significant differences in self-reported impulsivity with a second questionnaire (the self-report Eysenck Personality Questionnaire). There are less data examining behavioral impulsivity, which is thought to be different from selfreported impulsivity (Evenden, 1999). Gouzoulis-Mayfrank (2000), using the same volunteers as in the Tuchtenhagen (2000) report, did not find evidence of behavioral impulsivity in ecstasy users undergoing a cognitive test battery. In contrast, Morgan (1998) reported that ecstasy users made increased errors in a Matching Familiar Figures task, a difference he interpreted as evidence of increased impulsivity. Morgan suggested his behavioral findings indicated a decreased capacity to cope with high levels of cognitive demands.
from those of non-user volunteers. Does this mean that serotonergic neurotoxicity has taken place? This seems the most likely possibility. Several studies have shown differences in measures of serotonergic functioning between users and nonusers. Two groups have reported decreased cortical SERT binding in ecstasy users (McCann, 1998; Semple, 1999), although there is some question about the specificity of the measurement technique (Heinz, 2000; Kuikka, 1999). Three of four studies have found CSF levels of 5HIAA to be lower in users than nonusers (decreased in McCann, 1999b; 1994; Ricaurte, 1990; unchanged in Peroutka, 1987). These differences are consistent with animal studies in which neurotoxic MDMA exposure similarly altered these indicators (Insel, 1989; Ricaurte, 1988b; Scheffel, 1998). Such parallel findings in humans and nonhumans provide some evidence that selective serotonergic neurotoxicity has occurred. However, all published studies in humans have been retrospective. Without knowing what ecstasy users were like before using drugs, we can only guess whether unusual serotonergic functioning is the result of damage. Unfortunately, these serotonergic measures are sufficiently new that we do not know the full range of "normal" values for them. It is therefore difficult to decide whether the values seen in ecstasy users are truly 'abnormal' and indicative of damage. Alternatively, they may be simply 'unusual' for non-drug users but 'usual' for the kind of person who is likely to use ecstasy repeatedly. [Editor's note: There are many complicating factors in measuring 5HT levels in living humans including such as people with depression have been shown to have decreased numbers of SERT in other areas of the brain (Mann, 2000) and decreased levels of 5HT and 5HIAA (Meltzer, 1990) prior to being treated with antidepressants.] In addition, typical indicators of serotonergic function may be affected by influences other than neurotoxicity. Some theories suggest that individuals who abuse psychostimulants are more likely to have unusual serotonergenic functioning (Laviola, 1999; Zuckerman 1996). These interpretive difficulties can be illustrated using studies that investigate the amount of hormone released after serotonergic drug administration in different populations. Measuring the amount of hormone released in response to a serotonergic drug is one way to test for changes in the serotonergic system. This tactic has uncovered statistically significant usernonuser differences in 4 of 6 studies (differences detected in (Gerra, 2000; 1998; McCann, 1999a; Verkes, 2000; no significant differences in McCann, 1994; Price, 1989). However, other studies have established that both personality and use of other drugs, such as cocaine, may modulate this serotonergic measure. High sensation-seeking humans have been shown to have blunted hormone response to the partial 5-HT1a agonist, ipsapirone (Netter, 1996). Similarly, the prolactin response to the 5-HT releaser, fenfluramine, in a group of cocaine-dependent individuals was significantly increased between the first and third weeks after discontinuing cocaine use (Buydens-Branchey, 1999), suggesting recovery from cocaine-induced alterations. Therefore, one could argue that factors other than MDMA neurotoxicity still might explain some apparently serotonergic differences between users and nonusers. This issue can only by solved using prospective studies that assess the same individuals at different time points. One strong argument that MDMA neurotoxicity occurs in many human users is simply that estimated doses ingested by some users exceed those known to produce 5-HT depletions in squirrel monkeys (Ricaurte, 1988a). Given that approximately similar doses are associated with similar changes in serotonergic indices in nonhumans and humans, it seems likely that the same
phenomenon is occurring in both species. Furthermore, if one is considering administering MDMA to humans, it may be more important to be conservative in risk assessment than to wait for conclusive scientific proof of neurotoxicity. This is especially important because some individuals may be more susceptible to neurotoxicity than others. Studies comparing ecstasy users to non-users suggest that neurotoxicity may occur with MDMA exposures that are selfadministered by humans. MDMA neurotoxicity and its largely unknown possible long-term consequences must therefore be considered when evaluating the risks of clinical MDMA research.
studies on these other possible factors (Cho, 2000; Dinges,1991; Kretsch, 1997) would not lead us to expect an effect comparable to what we see in studies of ecstasy users. These other possible factors seem likely to be significant only if the ecstasy-using volunteers in these international studies engage in a particularly 'hard-partying' lifestyle. In the first published study that properly controlled for lifestyle, Verkes (2000) found that 'moderate' ecstasy users (with 73 +/- 68 reported exposures to ecstasy) had lower performance scores than nonusers attending a similar number of 'raves' in the previous 12 months. Pre-existing differences and effects of lifestyle seem unlikely to fully explain the reported cognitive performance differences. Average performance in immediate declarative verbal memory tasks was decreased by about 0.8 standard deviation units in several studies (GouzoulisMayfrank, 2000; Morgan 1999; Parrott, 1998a; 1998b). This means that the average ecstasyusing volunteer in these studies scored in the bottom 21% of what was expected based on the comparison volunteers. While possible, it seems improbable that primarily the quarter of the population with the worst memory goes on to use ecstasy several times a month (and participates in these studies). Use of drugs other than MDMA has not always been properly taken into account in studies of ecstasy users. In particular, cannabis use has often been greater in ecstasy-using volunteers than in ecstasy-nave volunteers. This is significant because chronic cannabis use can cause longlasting residual decreases in cognitive performance (Pope,1996). Three studies have compared users of both ecstasy and cannabis to users of cannabis alone (Croft, 2000; Gouzoulis-Mayfrank, 2000; Rodgers 2000). Two of these studies have suggested that MDMA is associated with lowered cognitive performance beyond that expected for cannabis (Gouzoulis-Mayfrank, 2000; Rodgers 2000). In contrast, Croft (2000) was unable to detect performance differences between cannabis users and users of both cannabis and ecstasy using a battery of cognitive tests. Furthermore, covariate analysis suggested that performance decreases were more closely related to cannabis than ecstasy use. In another study that attempted to control for the influence of other drugs, Morgan (1999) detected lower memory performance in ecstasy-experienced polydrug users compared to ecstasy-nave polydrug users. However, matching of drug use between comparison groups was imperfect in this study. It is clear that future studies should control for use of cannabis and that the apparent magnitude of the MDMA-associated cognitive performance decrease is likely exaggerated by cannabis use. The lower ocognitive performance of ecstasy users may be due to serotonergic neurotoxicity or some other neurochemical alteration. It has been demonstrated that acute serotonergic depletion (by dietary manipulation) can impair declarative verbal memory in healthy volunteers (Riedel, 1999). Two studies of ecstasy users have reported correlations between alterations in serotonergic measures and decreased cognitive performance (Bolla, 1998; Reneman, 2000a; Verkes, 2000). This suggests a relationship between lower cognitive performance and MDMAinduced serotonin depletions or neurotoxicity. On the other hand, if MDMA-induced loss of serotonin or damage to serotonergic axons were sufficient to impair memory to the degree suggested by human studies, one would expect this effect to have been readily detected in prospective animal studies. It appears possible that the reported lower cognitive performance is related to the volunteers'
chronic, repeated patterns of ecstasy use. Because MDMA exposures are limited (usually 4 consecutive days or less) in most animal experiments, this could explain the apparent discrepancy between these studies and ecstasy user studies. Furthermore, it is well established that chronic psychostimulant use lowers cognitive performance (McKetin,1999; Ornstein, 2000). For example, repeated cocaine use is associated with impaired cognitive functioning (Beatty, 1995; Bolla, 1999; O'Malley, 1992), although cocaine use per se does not necessarily produce deficits (Bolla, 1999). Cocaine is not a selective neurotoxin but, like MDMA, can cause both serotonergic (Jacobsen, 2000; Little, 1998) and cerebrovascular (Bartzokis, 1999; Herning, 1999) alterations. Since repeated exposure to other psychostimulants can impair cognitive functioning, it is credible that repeated MDMA use might be associated with cognitive deficits. Suggesting this leaves open the question of whether this effect is due to repeated neurotoxic damage or residual drug effects. Specific evidence linking the lower cognitive performance of repeated ecstasy users to serotonergic neurotoxicity could come from studies of the time course of these differences. Residual drug effects might be expected to improve more quickly than changes due to serotonergic neurotoxicity. Unfortunately, too few studies have looked for evidence of recovery to draw any conclusions. Morgan (1999) reported that a subset of three ecstasy users who had not taken ecstasy in over 6 months had significantly better immediate and delayed recall (of ideas from stories taken from the Rivermead Behavioral Memory Test) than users with more recent use. In contrast, Wareing (2000) were unable to find evidence of a significant abstinencerelated improvement in working memory and executive functioning tasks when 10 current ecstasy users were compared to 10 volunteers who reportedly had not used ecstasy in 6 months. It is therefore not clear if there is recovery from this lower cognitive performance. In conclusion, repeated ecstasy exposure is associated with lowered cognitive performance. The apparent magnitude of the effect may be exaggerated by limitations in published studies, particularly the confounding effects of cannabis [and perhaps other substances] on performance. There are insufficient data to decide whether there is recovery of performance with abstinence. The question also remains open as to whether this is due to a residual drug effect or a frank neurotoxic change. Table II: Reported Neurofunctional Differences Between Ecstasy Users and Nonusers Correlated Selective for Relevant Evidence with Measure Serotonergic Animal for References MDMA Differences? Literature? Recovery? Exposure? Putative Serotonergic Measures Decreased up to 2 weeks after MDMA in No squirrel monkeys (Ricaurte et al., 1988) and 14 Decreased in McCann et al.,1999, 1994; Ricaurte et al.,1990. Unchanged in Peroutka et al., 1989
No
weeks after MDMA in rhesus monkeys (Insel et al., 1989). Decreased then Increased 5-HT2a Yes receptor density in 1 of 1 studies Decreased neuroendocrine response to Yes serotonergic drugs, in 3 of 5 studies Disputed ligand Decreased SERT density, kinetics may estimated with be altered by PET, in 2 of 2 other changes studies (Kuikka & Ahonen 1998). Increased stimulus dependence for ERP EEG N1/P2 amplitudes in 1 of 1 studies Decreased at 24 hr, normal at 21 d after MDMA Yes in rats (Scheffel et al., 1992). Increased at 2 months, normal Yes in at 12 months in Gerra et rats (Poland et al., 2000 al., 1991) PET measures apparently decreased in one baboon up to 14 weeks after MDMA (Scheffel et al., 1998). Increased in Reneman et al., 2000a; Decreased Not reported then increased in Reneman et al., 2000b Decreased in Gerra et al., 2000, 1998; McCann et al., No 1999a. Unchanged in Price et al., 1989; McCann et al., 1994.
Yes, though Mixed (Yes McCann in Semple; Semple et al., 1999; included No in McCann et al., 1998. controls. McCann)
Disputed 5HT depletion did not change Unknown measure in one study (Dierks et al., 1999).
No
Not reported
Nonspecific Neurofunctional Measures Increased brain myoinositol measured as by 1H MRS in 1 of 1 studies Decreased total sleep time (non-REM and stage 2 sleep) in 1 of 1 study Altered
Unknown
Yes
Unknown
No
Yes
Altered in Reneman
et al., 2000b and in Chang et al., 2000 (prospective study). Unchanged in Chang et al., 2000 (user-nonuser study) and Gamma et al., in press.
Increased in some hipppocampal areas 2 wks (Sharkey et al., No 1991) after MDMA and 6-9 wks after MDA (McBean et al., 1990) in rats Unknown Yes
No
Not reported
serotonergic-related behaviors, although the maximal serotonergic response to drugs or other stimuli is likely to be reduced (reduced electrically-stimulated 5-HT release in MDMA-exposed rats was documented by Gartside, 1996). Second, the concept of cognitive reserve has been developed to explain why greater education, intelligence, or brain size is associated with less severe impairment in conditions such as Alzheimer's disease, AIDS, and normal aging (Alexander, 1997; Coffey, 1999; Graves, 1996; Stern, 1996). This cognitive reserve may be seen as a surplus of processing capacity that protects the individual against loss of functioning when that capacity is decreased. Cognitive reserve could be the result of more extensive functional brain tissue, density of neural connections, or cognitive strategies for problem solving. Individuals with less cognitive reserve could be expected to undergo larger cognitive decreases from MDMA exposure than users with greater cognitive reserve. Support for this possibility comes from Bolla (1998) who reported a significant interaction between dose and vocabulary (measured with the WAIS-R). ecstasy users with lower vocabulary scores showed greater decreases in delayed visual memory performance, while users with higher vocabulary had largely preserved performance. Although the absolute magnitude of performance decrease was small, this study suggests that cognitive reserve could play a role in expression of MDMA neurotoxicity. Whether symptoms of MDMA neurotoxicity are likely to increase as users age is difficult to predict. Some have speculated that aging ecstasy users might have increased risk of depression and other affective disorders. From a neurochemical perspective, age-related decrease in SERT density appears modest (estimated at 4.3% per decade in one recent study (van Dyck, 2000)), while 5-HT receptors undergo more complex age-related changes (reviewed in (Meltzer, 1998). One would hope that these changes will not cause ecstasy users to exceed a hypothetical threshold for developing symptoms of neurotoxicity. However, we simply do not understand 5HT or affective disorders sufficiently to make predictions with any confidence. Late onset affective disorders are probably influenced by many nonserotonergic factors, such as social isolation and cerebrovascular disease. These are serious and legitimate concerns and there is insufficient research to adequately address them. On the other hand, there is no direct evidence to support these concerns. Neurotoxic phenethylamines have been self-administered by humans for over 60 years. In this time, no evidence has been published suggesting that methamphetamine or amphetamine increase risk of Parkinson's disease, despite damaging dopaminergic axons. In contrast, the link between Parkinson's disease and MPTP, a meperidine analogue and dopaminergic neurotoxin that destroys cell bodies, was rapidly discovered (Davis, 1979; Langston, 1983). This suggests that there may be fundamental differences between neurotoxic phenethylamines, which selectively damage a subset of monoaminergic axons but not cell bodies, and other neurotoxins. Similarly, concerns about the selective serotonergic neurotoxicity induced by MDMA and other drugs are not fueled by a toxic syndrome identified in users. Instead, they are motivated by the intuition that the dramatic decreases in indices of serotonergic functioning must have some adverse behavioral consequences. Some have suggested that MDMA neurotoxicity may be related to its putative therapeutic effects. Although this is technically possible, there are a number of reasons to doubt this
hypothesis. The acute intoxication induced by MDMA is unusual. In contrast, similar serotonergic neurotoxicity can be produced by many other drugs. The events associated with MDMA neurotoxicity occur in rats between approximately 3 and 12 hours after drug administration, when subjective effects are decreasing or absent in humans. Thus, the acute intoxication produced by MDMA appears to be fully separable from long-term serotonergic effects. If MDMA proves useful as an adjunct to psychotherapy, it seems more likely that this utility will be associated with the unusual acute intoxication produced by MDMA than with the chronic serotonergic changes produced by many drugs.
How long do these decreases last? This is not clear. Two volunteers who underwent repeated SPECT scans showed evidence of possibly increased cerebral blood flow at later time points (43 and 80 days after MDMA, respectively). This suggests that the decreased cerebral blood flow is either a subacute drug effect of limited duration or part of a lasting biphasic effect (with decreases followed by increases). Chang states that decreased regional cerebral blood flow was generally less in volunteers with greater time from last MDMA exposure, providing evidence of recovery. In addition, the authors did not find differences in cerebral blood flow when 21 ecstasy-experienced volunteers were compared to 21 nonusers (in press). Similarly, Gamma (2001) saw no significant differences between 16 ecstasy users (most of whom had used ecstasy at least 100 times) and 17 nonusers when regional cerebral blood flow was measured during a vigilance task using [H2 15O]-PET. Finally, it should also be pointed out that Vollenweider and colleagues reportedly did not detect changes in regional cerebral blood flow using [H2 15O]PET in a retrospective analysis of a study in which volunteers received 1.7 mg/kg MDMA (Dr. Alex Gamma, personal communication). One possible mechanism for subacute alterations in regional cerebral blood flow is suggested by two preliminary reports of a study by Dr. Liesbeth Reneman and colleagues (Reneman, 2000a; 2000b). These researchers used [123I] R91150 SPECT to measure cortical 5-HT2a receptors and found evidence of decreased 5-HT2a receptors in ten ecstasy users with 7 5 weeks from last ecstasy exposure. In contrast, a group of five ecstasy users with at least two months from reported last exposure (18 +/- 15 weeks) showed a trend toward increased cortical 5-HT2a binding which did not reach statistical significance. Reneman, suggest that MDMA-induced 5HT release may have led to a downregulation of 5-HT2a receptors. Indeed, Scheffel (1992) reported a transient downregulation of these receptors in rats after a neurotoxic regimen of MDMA. Changes in 5-HT2a receptors are thought to play a role in regulation of cerebral blood vessel constriction (Nobler, 1999). Consistent with this idea, Reneman (2000b) reported correlations between apparent 5-HT2a density and regional cerebral blood volume in the occipital cortex and globus pallidus of a subset of five ecstasy users in whom cerebral blood volume was measured using MRI. Thus, the decreased cerebral blood flow/volume seen in Grob's volunteers and Reneman's ecstasy users may be the result of transient 5-HT2a downregulation due to MDMA-induced 5-HT release. This hypothesis does not, however, explain the trends toward increased cerebral blood flow or volume seen by both groups at later time points. Given that this trend occurred in very few volunteers, it must be interpreted with caution until confirmed in a more detailed study. Nonetheless, the duration of this possible increase provides cause for concern. A rodent study by McBean (1990) found that a neurotoxic regimen of MDA increased regional cerebral blood flow in rats at six to nine weeks after drug exposure. Thus, although one could hypothesize nonneurotoxic mechanisms, long-term increases in cerebral blood flow have been associated with serotonergic neurotoxicity. Therefore, it appears possible that one or more of the doses received by these two volunteers is sufficient to produce neurotoxicity. The two volunteers each received 2.0 mg/kg MDMA in one session and either 1.75 or 2.25 mg/kg in another session. Increases in cerebral blood flow after MDMA may not be permanent since the MDMA-experienced volunteers in Grob's study did not have increased cerebral blood flow in comparison to non-users (Chang, 2000). Nonetheless, researchers may wish to consider carefully the risks and benefits of exposing volunteers to a drug that may have detectable effects 80 days later.
Overall, preliminary findings from clinical studies suggest that cognitive functioning is not likely to be significantly altered by one or two exposures to MDMA in a clinical setting. However, changes in cerebral blood flow lasting several weeks or longer may occur. Although the mechanism of these changes has not been directly investigated, MDMA neurotoxicity cannot be ruled out as a possible explanation for any changes lasting several months. Of course, it must be again noted that the numbers of volunteers in the studies described in this section are small and any conclusions must be tentative. Further research is necessary.
adverse interaction between these compounds (Lauerma, 1998). Thus, giving an SSRI at 3 or 4 hours after MDMA administration could be considered if MDMA pharmacokinetics are not being measured and SSRIs are not otherwise contraindicated in the relevant patient or volunteer population. Antioxidant supplements may also prove useful. Aguirre (1999) reported that twice daily administration of high dose alpha-lipoic acid for two days completely blocked the neurotoxicity of a subsequent single dose of MDMA in rats. Because the acute inactivation of TPH can occur after non-neurotoxic MDMA doses and is due to oxidative stress, it is plausible that antioxidants may also enhance recovery from even low MDMA doses. One consideration with antioxidants is that high doses of some acids, such as ascorbic acid (vitamin C), may alter urinary pH and thus affect excretion of MDMA. Aside from such possible pharmacokinetic interactions, doses of antioxidants that are known to be well tolerated appear unlikely to increase risk of adverse events and may decrease risks of chronic toxicity. Although these potentially neuroprotective strategies are worth considering, appropriate doses and timing are largely unknown. It would furthermore be technically and ethically difficult to establish whether a given intervention has been successful in reducing MDMA neurotoxicity. These interventions should therefore be considered experimental and not be used to reassure potential volunteers that the risks of MDMA neurotoxicity are reduced.
Summary
High or repeated-dose MDMA regimens can produce long-term changes in indices of serotonergic and axonal functioning in animals. Increasing evidence supports the view that these changes are at least partially the result of damage. The magnitude of these serotonergic changes varies with dose, species, and route of administration. Rodent studies have shown that changes in the core temperature of animals can increase or decrease MDMA neurotoxicity. While some recovery does occur, a study in squirrel monkeys suggests that there may be permanent changes in axonal distribution in some areas of the brain. Oxidative stress appears to play an important role in MDMA neurotoxicity, although the exact mechanisms are poorly understood. The sustained acute pharmacological effects of MDMA may exhaust neuronal energy sources and antioxidant defenses, leading to damage. Metabolites of MDMA are another possible source of oxidative stress. Very few behavioral correlates of MDMA exposure have been found in drugfree laboratory animals, despite dramatic serotonergic changes, alterations in neurofunctioning, and changes in response to drugs. A growing number of studies describe differences between ecstasy users and nonusers. These studies have serious limitations, but suggest that some ecstasy users experience serotonergic changes and cognitive alterations. In contrast to studies of illicit users, the few controlled clinical trials with MDMA in healthy volunteers have reportedly not found evidence of cognitive changes, despite cerebral blood flow alterations in one study. The possible risks of neurotoxicity must be considered when assessing the potential administration of MDMA to humans.