Reduced cortical gray matter density in human MDMA (Ecstasy) users: a voxel-based morphometry study

Drug and Alcohol Dependence 72 (2003) 225–235

Reduced cortical gray matter density in human MDMA (Ecstasy) users: a voxel-based morphometry study夽 Ronald L. Cowan a,b,∗ , In Kyoon Lyoo a,c , Seung Mo Sung a,c , Kyung Heup Ahn a,c , Minue J. Kim a,c , Jaeuk Hwang c , Erika Haga a , Ram Lakhan Panday Vimal a , Scott E. Lukas b , Perry F. Renshaw a a

Brain Imaging Center, Department of Psychiatry, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA b Behavioral Psychopharmacology Research Laboratory, Department of Psychiatry, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA c Department of Psychiatry, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, South Korea Received 12 December 2002; received in revised form 21 May 2003; accepted 17 July 2003

Abstract The popular recreational drug, 3,4-methylenedioxymethamphetamine (MDMA) exerts its actions in part via blockade of serotonin and dopamine reuptake. Many animal and human studies have demonstrated long-lasting reductions in measures of central nervous system (CNS) serotonin function following MDMA administration. One emerging role of serotonin function in the CNS is a positive trophic effect via stimulation of intracellular signaling pathways and trophic factors. We hypothesized that human MDMA users might display neocortical gray matter reductions due to loss of serotonergically mediated trophic effects on cortical cells. However, unlike animal models, most human MDMA users worldwide are polydrug users, thereby complicating the assessment of MDMA toxicity in this group. Structural magnetic resonance imaging (MRI) scans of 31 MDMA polydrug users versus 29 non-MDMA users were compared using voxel-based morphometry (VBM) to assess regional brain gray and white matter concentration. VBM employs gray/white matter segmentation and statistical parametric mapping (SPM) analysis to calculate a voxel-wise comparison of regional gray or white matter concentration. Using this method, we consistently found several brain regions having decreased gray matter concentration in MDMA polydrug users. These regions were localized to neocortex in bilateral Brodmann area (BA) 18, left BA 21, and left BA 45, as well as bilateral cerebellum, and midline brainstem. Overall, these preliminary findings suggest that MDMA polydrug users have multiple regions of gray matter reduction, potentially accounting for previously reported neuropsychiatric impairments in MDMA users. Additional animal model and human studies of the CNS effects of MDMA and combined MDMA-polydrug toxicity are needed to further explain these findings. Potential explanations for our results including pre-existing brain differences predisposing to MDMA polydrug use, direct MDMA and polydrug toxicity, indirect changes due to MDMA and polydrug toxicity, or combinations of all these factors. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: MDMA; Neurotoxicity; Imaging; Polydrug abuse

1. Introduction 1.1. MDMA The substituted amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) has been used recreationally since 夽 Presented in part 12 June 2002 Meeting of The College on Problems of Drug Dependence, Quebec City, Que., Canada. ∗ Corresponding author. Present address: Division of Adult Psychiatry, Vanderbilt University Medical Center, 1500 21st Avenue South, Suite 2200, Nashville, TN 37212. E-mail address: [email protected] (R.L. Cowan).

the early 1970s, with surging popularity in the past few years. Acute ingestion of MDMA causes a transient marked increase in serotonin and dopamine at central synapses (reviewed in Bankson and Cunningham, 2001) producing stimulant and euphorigenic effects. Studies in rodents and non-human primates have clearly indicated that MDMA at some doses produces a global acute depletion of central nervous system (CNS) serotonergic markers, followed by a long-lasting distal axotomy of fibers arising from the dorsal raphe nucleus (DRN) (Ricaurte et al., 1998). There remain concerns that chronic use of MDMA may result in persistent neuropsychiatric problems (Montoya et al., 2002).

0376-8716/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.drugalcdep.2003.07.001

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1.2. Neuroimaging in MDMA users Neuroendocrine, single photon emission tomography (SPECT) (Reneman et al., 2001b), positron emission tomography (PET) (e.g. McCann et al., 1998), and electrophysiological studies have provided indirect evidence supporting the conclusion that serotonergic function is altered in cortical and subcortical brain regions in human MDMA users. Magnetic resonance spectroscopy (MRS) studies have shown reductions (Reneman et al., 2002) or no change (Chang et al., 1999) in the putative neuronal marker N-acetyl-aspartate (NAA). 1.3. Neuropsychiatric findings in MDMA users As with other studies of MDMA users, reports analyzing neuropsychiatric measures in MDMA users have been confounded by other drug use, particularly cannabis and the difficulty of differentiating acute from long-term effects. Several case reports and group studies have found increases in low grade psychiatric pathology in MDMA polydrug users, but the level of psychopathology does not generally meet clinical diagnostic criteria (Dughiero et al., 2001; Daumann et al., 2001; Parrott et al., 2000; Soar et al., 2001; Parrott et al., 2001; Turner and Parrott, 2001). Cognitive deficits in MDMA users generally reflect impaired memory, executive processing, and increased impulsivity (reviewed in Parrott et al., 2000). 1.4. CNS serotonin CNS serotonin has at least three important roles: (1) classical neurotransmitter actions in cell–cell signaling (e.g. Hoyer et al., 2002), (2) vasoconstrictive effects via innervation of intracranial blood vessels (Marco et al., 1999), and (3) cytoskeletal effects, including positive coupling to the activity of growth factors (brain derived nerve growth factor (BDNF) and S100B) implicated in maintaining neuronal integrity (Duman et al., 2001). Thus, MDMA use could potentially result in absolute or relative reductions in CNS gray matter via transmitter-mediated toxicity, vascular ischemia or hemorrhage, or loss of neurotrophic effects. 1.5. Voxel-based morphometry (VBM) The emerging technique of voxel-based-morphometry (VBM) (Ashburner and Friston, 2000) has recently been employed to analyze regional gray and white matter concentrations in cocaine users (Franklin et al., 2002). We used VBM to compare gray and white matter concentration in the brains of human MDMA users compared to control subjects without MDMA use to test the hypothesis that MDMA users would have regional brain gray matter concentration reductions. Because MDMA is thought to affect primarily fine-diameter unmyelinated axons arising from the dorsal raphe nucleus (Ricaurte et al., 1988), we further

hypothesized that there would be no differences in white matter concentration between MDMA polydrug users and controls.

2. Methods 2.1. Subjects Subjects for this study enrolled as part of a broader study examining structural, functional, and spectroscopic neuroimaging measures in MDMA users. We recruited subjects via advertisements in local college newspapers advertising a brain imaging study for 18–35-year olds who had used drugs. Because studies of MDMA users worldwide have indicated that they are almost universal users also of marijuana, and because their rate of use of other drugs is also higher, we attempted to recruit age and sex-matched controls that also had used a variety of drugs. We used two different advertisements to recruit the MDMA and comparison groups. The ads were identical in their description of the study, reimbursement, etc. but differed in that one advertisement mentioned a study in individuals who had used “drugs” and the other mentioned having used “MDMA (Ecstasy)” or other drugs. We screened the subjects initially by telephone and those who met provisional inclusion criteria were screened in person by a psychiatrist using a semi-structured interview to obtain personal, medical, and psychiatric history. To permit the assessment of potential cumulative drug toxicity, subjects also completed drug use history questionnaires assessing the frequency of drug use as lifetime “episodes” of drug use. Responses on the questionnaires were reviewed with the subjects by a psychiatrist. Subjects were provided written informed consent approved by the McLean Hospital Human Subjects committee. Subjects were compensated for their participation. Exclusion criteria were contraindications to MRI scanning, general medical illness, pregnancy, history of substance dependence, and current Axis I psychiatric illness (except substance abuse). Not all subjects received urine or alcohol screens on the day of the structural MRI, but were required to have negative urine drug screens (using the Triage Drugs of Abuse panel) and negative alcohol breath test on the screening day or on days when functional MRI was performed. MDMA use within 3 weeks of enrollment for the overall study (but not specifically the structural study) was also an exclusion factor. 2.2. Drug use assessment In this initial study, we attempted to assess cumulative MDMA exposure using self-report of MDMA and other drug use obtained as lifetime episodes of use. Episodes were defined as within a 24 h period starting with the onset of MDMA ingestion. For entry into the study, MDMA users had to report at least five episodes of lifetime MDMA

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2.4. Statistics

use and control subjects reported no lifetime MDMA use (subjects with one to four episodes of MDMA use were excluded). Subjects were instructed to estimate their lifetime prior use of recreational drugs and nicotine in categories of 1–10, 11–39, and 40 or more episodes of drug use. The drugs surveyed were non-MDMA hallucinogens (LSD, mescaline, peyote, or psilocybin), cocaine, cannabis, sedative hypnotics, non-heroin (other) opiate, alcohol, heroin, non-MDMA amphetamines, phencyclidine (PCP), and nicotine. Subjects were not aware of inclusion/exclusion criteria, or of the 3-week exclusion window.

Descriptive graphical statistics were computed for subject sex, demographics, handedness, and episode of drug use measures. T-tests were used to compare age distributions between the MDMA using and control group. Chi-square analysis was used to assess for significant differences in gender, handedness, and categorical proportions of drug use between the groups. Smoothed brain images were compared between MDMA users and controls using the statistical comparison modules of SPM99. T-maps from the statistical comparisons were transformed to z-values and the significance of the differences estimated using the theory of random Gaussian fields. We used a P-value of P < 0.05 corrected for global comparisons for the initial contrast between MDMA polydrug users and non-MDMA users and a P-value of P < 0.0001 uncorrected for global comparisons (Friston et al., 1996; Worsley et al., 1996) for the analysis adding covariates to the baseline comparison. Sample size limitations and heterogeneity of substance use prevented combined statistical modeling of dose–response effect for MDMA and for the effects of drugs of abuse. Therefore, we conducted sequential comparisons of gray and white matter density between the MDMA and non-MDMA groups sequentially covarying for the effects of other drugs of abuse (by categorical episode of use) and demographic variables. Covariates were controlled in the analysis of covariance (ANCOVA) model of the SPM using SPM99’s statistical modules.

2.3. Image acquisition Using a 1.5 T General Electric whole body MR scanner and a standard quadrature head coil, 124 T1-weighted volumetric 3D Spoiled Gradient Recall (SPGR) contiguous coronal anatomical images were obtained with parameters of: flip angle = 45◦ , TR = 35 ms, TE = 5 ms, one number of excitations (NEX) slice thickness = 1.5 mm, field of view (FOV) = 24 cm×24 cm, matrix size 256 pixels×192 pixels. Structural images were reviewed by a neuroradiologist prior to analysis and were excluded if they had visually observable structural abnormalities. VBM methods were implemented according to Ashburner and Friston (2000). Coronal image volumes were registered into a standardized proportional stereotaxic space using the Montreal Neurological Institute 152 brain-averaged space (Abell et al., 1999; SPM 99) that were approximately aligned into Tailarach stereotaxic space (Talairach and Tournoux, 1988). Spatial normalization was performed using a 12-parameter affine transformation (Ashburner and Friston, 1999) to reduce variability in non-linear shape differences, followed by gray and white matter segmentation using a modified mixture model cluster analysis technique (Ashburner and Friston, 1997). Segmented gray images were smoothed using an 8 mm full width at half maximum isotropic Gaussian kernel.

3. Results 3.1. Subject characteristics There were 14 females and 17 males in the MDMA group versus 11 females and 18 males in the non-MDMA group. The proportion of males and females did not differ between groups (Chi-square = 0.77, P = 0.38). The MDMA group was comprised of 25 right-handed and 6

Table 1 Drug use frequencies in non-MDMA users and MDMA users Drug

Hallucinogen Cocaine Cannabis Opiates PCP Sedatives Alcohol Amphetamine Heroin MDMA

Non-MDMA users (n = 29)

MDMA users (n = 31)

None

Low

Medium

High

None

Low

Medium

High

23 27 13 27 29 25 1 26 28 0

4 1 5 2 0 4 3 2 1 0

2 1 6 0 0 0 3 1 0 0

0 0 5 0 0 0 22 0 0 0

5 9 0 16 25 16 0 14 31 0

15 14 1 13 6 10 0 10 0 8

9 4 5 2 0 5 1 4 0 15

2 4 25 0 0 0 30 3 0 8

Chi-square

P

24.35 26.03 29.06 12.82 6.24 9.49 6.17 13.68 1.09 NA

<0.0005 <0.0005 <0.0005 0.002 0.013 0.009 0.104 0.003 0.30 NA

Categories are none = 0 episodes use of a drug, low = 1–10 episodes use of a drug, medium = 11–39 episodes use of a drug, high = 40 or more episodes use of a drug. PCP: phencyclidine, MDMA: 3,4,methylenedioxymethamphetamine.

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left-handed individuals whereas the control group contained 26 right-handed and 3 left-handed subjects. The proportion of subjects with a given handedness did not differ between groups (Chi-square = 0.954, P = 0.33). The subjects ranged in age from 18–35. The MDMA user group was significantly younger than the non-MDMA group (t = 3.03, P = 0.004) with a mean age of 24.3 years old (standard deviation ± 3.5 years) in the control group and

a mean of 21.7 years old (standard deviation ± 3.3) for the MDMA group. Drug use history data are summarized by episodes of use for MDMA and non-MDMA users in Table 1. All subjects had been abstinent from MDMA at least 3 weeks prior to enrollment for the study. MDMA users reported statistically greater episodes of drug use for all drug classes assayed with the exception of alcohol and heroin.

Fig. 1. (A) Glass brain projection from SPM 99 depicting overlay of regions demonstrating reduced gray matter concentration in MDMA users (contrast was MDMA vs. non-MDMA). SPM map threshold was P < 0.05 corrected for multiple whole brain comparisons. P: posterior, R: right. A: anterior. BA: Brodmann area. (B) Lateral view of regions showing reduced gray matter concentration in MDMA users overlayed on idealized brain, as described in B. (C) Ventral view of regions (in red for threshold significance of P < 0.05 corrected for multiple whole-brain comparisons) showing reduced gray matter concentration in MDMA users overlayed on idealized brain using SPM 99.

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Fig. 2. Transaxial slice series depicting regions showing reduced gray matter concentration in MDMA users overlayed on averaged subject brains. Upper left is ventral most slice with slices ascending from left to right, top to bottom. Z numbers indicate mm below the anterior commisure. L: left, A: anterior, BA: Brodmann area. Display threshold is set at T-value = 5.36 or above, P < 0.05 corrected.

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3.2. VBM outcomes 3.2.1. Primary comparison We initially compared the regional gray and white matter concentrations in MDMA polydrug users versus non-MDMA polydrug users with a statistical threshold of P < 0.05 corrected for multiple whole brain comparisons. We view this significance threshold as relatively conservative because it also corrects for the many non-brain voxels included in the volume matrix. This analysis revealed multiple areas of reduced gray matter concentration in the MDMA polydrug users (Fig. 1). At this significance threshold, the neocortical regions showing reduced gray matter concentration in MDMA users included bilateral Brodmann area (BA) 18 (a portion of secondary visual cortex, V2) in the occipital lobe; left BA 21 in the temporal lobe; and left BA 45 in the frontal lobe. A midline region in the brain stem and bilateral areas of the cerebellum also had reduced gray matter concentration in the MDMA group. There were no detectable differences in white matter density between groups. The regions of gray matter concentration reduction are depicted on the glass and rendered brain projections in Fig. 1 and in a series of axial images of the actual brain data with overlayed SPM maps in Fig. 2. 3.2.2. Secondary comparisons Next, we systematically analyzed other variables that might contribute to altered gray matter concentration between the two groups. These included age, sex, handedness, and other drug use (Fig. 3). Because adding a covariate reduced the available degrees of freedom by 1, we used non-binarized, uncorrected P-values for the entire brain volume comparison (P < 0.0001). This approach, namely using a less conservative P-value to threshold the SPMs while simultaneously lowering the degrees of freedom, appeared to offer a reasonable compromise between potential Types I and II error. The SPMs displaying the effect of covarying for age, sex, and handedness (upper row, Fig. 3) are useful as baselines for comparing the contributions of other drugs of abuse. At the less conservative statistical threshold, the area of the affected regions was somewhat larger, and some new areas of reduced gray matter concentration appeared. Of these new areas, the anterior cingulate gyrus (AC) consistently showed reduced gray matter concentration in all analyses. Each drug-use covariate had some impact on the SPMs as demonstrated in Fig. 3. In general, while the spatial extent of regional brain gray mat-

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ter reductions was influenced by some covariates, areas of cerebellum, brainstem, and neocortex (left BA 45, 21, and bilateral BA18) consistently demonstrated reduced gray matter in the MDMA polydrug group.

4. Discussion 4.1. Overview Using VBM to assay gray and white matter concentration in MDMA polydrug users, we found significantly lower gray matter concentrations in multiple brain regions of MDMA polydrug users when compared to non-MDMA using controls. 4.2. Relation to clinical findings 4.2.1. Neocortex MDMA polydrug users have been demonstrated to have multiple cognitive impairments, including increased impulsivity (Morgan, 1999), and impaired verbal and auditory memory (Bolla et al., 1998; Morgan, 1999; Reneman et al., 2000; Verkes et al., 2001). In this context, it is interesting to note that neuroimaging studies suggest that, among other functions, both left BA 45 and left BA 21 (along with other areas) play an important role in semantic memory retrieval (e.g. Lee et al., 2002; Booth et al., 2002; Friederici et al., 2000). BA 18 (among other roles) is involved in visual learning involving face-name association (Herholz et al., 2001). In the primary analysis contrasting MDMA users versus non-users with a threshold of P < 0.05 we found regional cortical gray matter concentration decreases restricted to the left hemisphere for BA 45 and BA 21 but bilaterally for BA 18. 4.2.2. Brainstem We found a single midline region of reduced gray matter concentration in a region that approximately overlaps and extends beyond the location of the midline raphe nuclei. The resolution of the present technique, coupled to the known variety of nuclear groups in the brainstem region, precludes a conclusion regarding direct gray matter loss in the raphe nuclei per se. MDMA is not thought to be strongly toxic to serotonergic neuron cell bodies in animals studied thus far; however, Ricaurte et al. (1988) reported cytotoxic changes (but not overt neuron death) in dorsal raphe neuron soma

Fig. 3. Glass brain projections depicting the effects of individually controlling for covariates. To account for the loss of one degree of freedom by adding a covariate to the initial model contrasting MDMA versus non-MDMA users, P-value is set at an uncorrected P < 0.0001. Label for each three-image grouping indicates covariate controlled for. Each group of SPM maps depicts regions having reduced gray matter in MDMA users after controlling for covariates. These figures are best interpreted by using the age, sex, or handedness SPMs as baseline comparisons and referencing the effects of covariate control to these projections. Overall, the less conservative P-value revealed an additional region of reduced gray matter concentration in the anterior cingulate gyrus of MDMA users. Controlling for hallucinogen, cocaine, and cannabis use reduced the spatial extent of reduced gray matter, but several regions showed consistent gray matter concentration reductions in the MDMA group. BA: Brodmann area, AC: anterior cingulate gyrus, PCP: phencyclidine. Opiates indicates non-heroin opiate. Amphetamine indicates non-MDMA amphetamine.

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following MDMA administration in monkeys. MDMA has been demonstrated to cause death of human serotonin neurons in cell culture. Simantov and Tauber (1997), and Allen et al. (1993) reported persistent decreased stage 2 sleep accounting for decreased total sleep time in MDMA users, potentially secondary to brainstem serotonergic toxicity. 4.2.3. Cerebellum We found bilateral gray matter concentration reductions in the cerebellar hemispheres. The cerebellum has a well-established role in the fine control of motor activity and emerging data increasingly supports a broader cognitive role for this structure. For example, the cerebellum plays a role in motor-associative learning (Frings et al., 2002), visuo-spatial judgment (Fink et al., 2000), and influences auditory, visual, language, and spatial abilities during development (Riva and Giorgi, 2000; Scott et al., 2001). 4.2.4. Anterior cingulate gyrus The anterior cingulate gyrus did not have significantly reduced gray matter concentration in the more statistically conservative VBM analysis using a corrected P-value of 0.05 but showed reduced gray matter concentration (predominantly right-sided) under all conditions using an uncorrected P-value of 0.0001. The anterior cingulate gyrus appears to have a major role in conflict monitoring during information processing (Van Veen and Carter, 2002). While our study did not permit the precise localization of the particular subregion of the anterior cingulate having reduced gray matter, the anatomical location of the gray matter deficits here approximately overlaps with the vocalization “VOA” subdivision (reviewed in Gruber et al., 2002). The right VOA appears to play a specific role in inhibiting incorrect (unwanted) responses to stimuli in the Stroop task (Gruber et al., 2002). The anterior cingulate (along with orbitofrontal cortex) is implicated in various phases of drug use, including intoxication, craving, bingeing, and withdrawal (reviewed in Goldstein and Volkow, 2002). 4.3. Relation to other neuroimaging studies 4.3.1. MDMA The regions of reduced gray matter concentration reported here do not consistently overlap with brain regions shown to have altered serotonergic function or altered neurochemistry or metabolism in other neuroimaging studies. Evidence for reduced serotonin (5-HT) transporter concentration in a variety of brain regions, including frontal and occipital cortex, pons, and cerebellum has been demonstrated by McCann et al. (1998) using positron emission tomography (PET) and the ligand McN-5652. McCann et al. (1998) did not report reduced 5-HT-transporter in temporal cortex. Other regions having reduced 5-HT-transporter in this study included hypothalamus, midbrain, caudate/putamen, cingulate and parietal cortex. These regions did not show reduced gray matter concentrations in the present report except for the finding of

anterior cingulate concentration loss using the more sensitive threshold to produce SPMs. Chang et al. (2000) reported no change in global or regional cerebral blood flow (rCBF) in abstinent MDMA users, but found reduced rCBF following high-dose MDMA administration in caudate, superior parietal, dorsolateral frontal, and middle and superior temporal gyrus compared to controls. It is unclear whether these findings were due to primary vascular changes, or secondary to neuronal changes. Reneman et al. (2001a) reported increased apparent diffusion coefficient and increased relative cerebral volume in the globus pallidus of MDMA users, but other studied brain regions included frontal and occipital cortex, which appeared normal. Proton magnetic resonance spectroscopy studies have indicated normal N-acetylaspartate (NAA), Creatine (CR), and Choline (Cho) in the hippocampi of MDMA users (Obergriesser et al., 2001). Chang et al. (1999) did not specifically study the same regions reported here (their occipital voxel was midline and did not overlap with the BA 18 occipital region reported here), but did report increased parietal and occipital myoinositol with normal NAA. These areas were not affected in the present study. Conversely, Reneman et al. (2002) examined midfrontal and midocciptal gray, and midparietal white matter using 1 H MRS, finding only reduced frontal NAA/CR and NAA/Cho ratios. In a positron emission tomography (PET) study of regional cerebral glucose metabolic rate in MDMA users (Obrocki et al., 2002), metabolism was significantly reduced in left amygdala, and bilateral caudate/putamen, with global, non-significant reductions in all areas studied except BA 10, where there was a non-statistical trend for increased metabolism in MDMA users. 4.3.2. Age, sex, and handedness We did not detect appreciable effects of age on our outcome measures. Age-related alterations in brain gray and white matter have been demonstrated in a large (465 subjects) sample size VBM study with subjects ranging in age from 18–79 (Good et al., 2001b). The absence of age effects on regional brain volume in our study is likely due to the narrow age range of subjects enrolled (18–35), greater variance introduced by polydrug effects, and the smaller sample size. Our groups did not differ statistically in handedness, and we did not detect appreciable effects of handedness on our outcome measures. In a separate VBM study using the same sample of 465 subjects (Good et al., 2001a), handedness had no effect on gray or white matter. In this same study, sex predicted a variety of differences in gray and white matter concentration, but these regions of difference do not specifically overlap with the brain regions having reduced gray matter concentration in our study. Although covarying for sex effects influenced the degree of regional brain gray matter decrease in MDMA users compared to age or handedness maps, the location of the affected regions did not change. Therefore, our male and female results were pooled to enhance the sample size and statistical power. The failure of our present study to detect robust sex differences is likely

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due to polydrug use in both our subject groups and also due to sample size limitations. 4.3.3. Polydrug effects In general, covarying for polydrug use affected the extent of the affected zone of gray matter but did not substantially alter the location of the regions having reduced gray matter in the direct MDMA versus non-MDMA comparison. Among the categories of substance use we surveyed, cocaine, hallucinogens, and cannabis most strongly affected the SPM maps. 4.3.3.1. Cocaine. While many MDMA users in our study reported at least one episode of cocaine use, it is notable that the group of MDMA users reported in the present study did not generally have gray matter concentration changes overlapping with those of cocaine-dependent subjects (Franklin et al., 2002). In the Franklin et al. study, affected regions were restricted to insula, orbitofrontal cortex, anterior cingulate gyrus, and superior temporal cortex. The one area of overlap between the MDMA cohort reported here and the cocaine-using group reported by Franklin et al. occurred in the anterior cingulate gyrus (in the analysis using the uncorrected P < 0.0001), and remained present, although reduced in extent, when the data were controlled for prior cocaine use. 4.3.3.2. Hallucinogens. In this study, statistical control for the effects of hallucinogen use produced results similar to those of controlling for cocaine and cannabis. LSD and MDMA both alter serotonergic function. MDMA raises serotonin at axon terminals arising from both the dorsal and median raphe nuclear groups, and thus likely produces a global effect on serotonin receptors of all subtypes. LSD, conversely has a relative specificity for partial agonist activity at serotonin (5-HT) 5-HT2A and 5-HT2C receptors (reviewed in Aghajanian and Marek, 2000; Backstrom et al., 1999). While hallucinogens such as LSD are known to induce long lasting functional brain changes in the form of hallucinogen-induced persisting perceptual disorder, we are unaware of reports citing structural brain changes in hallucinogen (LSD and mushroom) users. Neuropsychological studies are equivocal regarding the long-term sequelae of hallucinogen use (reviewed in Halpern and Pope, 1999). LSD has been shown to have neuroprotective properties (potentially mediated via 5-HT2A actions) in animal models examining its ability to inhibit NMDA antagonist-induced toxicity (Farber et al., 1998). 4.3.3.3. Cannabis. All of the MDMA users in this study reported cannabis use, and 25 of the 31 subjects reported high levels of use. Controlling for cannabis produced results similar to those of controlling for hallucinogens and cocaine. A single study using contemporary MRI analysis of regional brain volume in cannabis users did not detect evidence of volume loss in cannabis users when

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compared to controls (Block et al., 2000). This group did report decreased ventricular cerebrospinal fluid (CSF) volume in the cannabis group (increased ventricular CSF volume is often used as a marker of compensatory ventricular enlargement following neuronal loss. The implications of decreased CSF volume are unclear in this context). 4.4. Relation to serotonin As a premise for this study, we hypothesized that serotonergic axon loss in MDMA users would lead to loss of serotonin-mediated trophic effects and thus reduced gray matter concentration. This study does not permit us to conclude that this particular cohort of MDMA users suffered serotonin axon loss nor does it permit us to directly link serotonin to the observed effects. Serotonin has important roles in brain development but also in maintaining neural and glial function in the mature brain. As reviewed by Azmitia (2001), serotonin and other monoamine neurotransmitters serve as “Maintenance Growth Factors” in the brain. Antidepressant medications that alter serotonin also influence hippocampal neurogenesis (reviewed in Duman et al., 2001). Serotonin’s effects are mediated in part by 5-HT1A receptors and are linked to the trophic factors brain derived nerve growth factor (BDNF) and S100B (Nishi et al., 2000). Serotonin actions at these receptors induce intracellular signaling changes in postsynaptic neurons as well as stimulating glia to produce S-100B (Azmitia, 2001). Notably, S100B is a calcium binding protein having pro-growth effects at low concentrations but pro-apoptotic effects at higher concentrations (Rothermundt et al., 2003). 4.5. VBM VBM is an emerging method for comparing regional brain proportions of gray matter, white matter, and CSF. VBM relies on data from the deformation fields used to map segmented brain data onto a common template to assay brain gray and white matter concentration (Ashburner and Friston, 2000). Reduced gray matter concentration in this context implies a relative loss of gray matter neuropil as opposed to white matter. This could be secondary to loss or size reduction in the neuronal or glial components of the gray matter. We are not aware of studies validating VBM against controlled brain lesion experiments in animal models. VBM has been compared to other methods of assaying regional brain concentration and volume using hand-drawn region of interest (ROI) methods in a group of dementia patients. In this side-by-side comparison, VBM demonstrated a similar pattern of gray matter changes but differed from the ROI technique in regional sensitivity. Overall, the VBM technique was conservative in estimating gray matter changes when compared to the ROI method (Good et al., 2002).

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4.6. Limitations

References

Pre-exiting differences between MDMA polydrug users and controls may have accounted for these findings. As with other emerging techniques, the VBM method will require further validation and clarification to determine its utility in exploring the brain. We collected categorical drug use data as number of episodes of drug, thereby limiting our ability to make comparisons about the degree of exposure effects on our outcome measures. Sample size and methodological limitations prevented a comprehensive assessment of drug exposure versus outcome. While a problem common to other studies in MDMA users, this study is limited by a reliance on self-report data and inexact matching for polydrug use between the MDMA and control groups.

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4.7. Summary Brain differences in MDMA users compared to controls may arise from a variety of sources, including: (1) pre-existing differences not related to drug use, (2) neurotoxicity from drugs of abuse other than MDMA, (3) altered neuronal signaling directly resulting from loss of serotonin axons, (4) altered neuronal or glial structure due to loss of growth factor effects, (5) vasospastic ischemia, or (6) direct neurotoxic damage. Although we hypothesized that loss of serotonin-mediated trophic effects would result in the observed gray matter concentration reductions, the present technique does not permit a conclusion regarding the cause of the observed findings. Because MDMA users are genetically diverse; have varied dosage and use patterns of MDMA; and are generally polydrug users, it is not unexpected that different research methods conducted in different populations will have non-overlapping findings. In addition, because MDMA can affect the brain in a variety of ways, and because compensation, such as receptor changes, axonal sprouting, and other forms of neuronal plasticity will vary by region, it is difficult to anticipate the outcome of a particular research study. Further studies examining dose–response effects of MDMA and other drugs in animal models and human users, as well as studies correlating structural findings with other measures of brain function are necessary to address the issue of etiology and regional specificity.

Acknowledgements This work was supported by NIDA (Grants DA03994, DA00343, DA09448, DA15116, DA14178, and DA00366), the American Psychiatric Institute for Research and Education, and the Shervert Frazier Foundation. The authors would like to acknowledge the expert technical assistance of Eileen Connolly, RT(R)(MR), Robert P. Marquis, BSRT(R)(MR), and Anne M. Smith, RT(R)(MR).

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