In humans, brain regional 5‐HT synthesis can be estimated in vivo with PET imaging of α‐[ 11 C]methyl‐l‐tryptophan ( 11 C‐AMT) as a tracer, using the blood‐to‐brain clearance/trapping (K*) of the tracer (Diksic and Young 2001 ). The specificity and validity of the method has been demonstrated in both healthy and patient populations (Leyton et al . 2001 , 2005 ; Rosa‐Neto et al . 2005 , 2007 ; Lundquist et al . 2007 ; Berney et al . 2008 , 2011 ; Booij et al . 2010 ). In this study, we used the 11 C‐AMT method to compare brain 5‐HT synthesis capacity in regular MDMA polydrug users who were free of drugs at the time of scan, with non‐MDMA users. Our primary hypothesis was that polydrug users of MDMA would have ‘altered’ regional 11 C‐AMT trapping in cortical and/or subcortical areas, compared to age‐ and gender‐matched individuals who had never used MDMA. On the basis of previous reports emphasizing differences in brain 5‐HT synthesis between males and females (Nishizawa et al . 1997 ; Sakai et al . 2006 ), we also examined the regional K* data in MDMA polydrug users for a putative gender effect. Finally, we examined whether 11 C‐AMT trapping would be modulated by specific characteristics of MDMA use, such as an estimate of the cumulative lifetime MDMA dose, the duration of use and the time elapsed since the last MDMA dose.

Whether 5‐HT alterations in MDMA users are pre‐existing, because of MDMA or the other drugs used by MDMA users, or a combination of these, remain unknown. Nonetheless, given the important role of 5‐HT in brain development, mood, behavior, and cognitive function (Parrott 2013 ), these alterations could have significant consequences for human functioning. Using acute tryptophan depletion (ATD), we recently reported that women who are polydrug MDMA users may be more susceptible than men to the effects of lowering 5‐HT (Young et al . 2014 ). This study focused on exploring further the likelihood of putative differences in 5‐HT neurotransmission between MDMA polydrug users and controls, using an in vivo measure of regional 5‐HT synthesis capacity with PET, in real‐time .

Use of the drug 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) declined in the last decade in the United States (US), but consumption rates recently started rising again, possibly reflecting a reduced perception of risk (Johnston et al . 2012 ). The increase in prevalence may not be limited to the US, as there was also an increase in serious MDMA‐related incidents in the Netherlands in 2011 (Krul et al . 2012 ). In 2010, the lifetime prevalence for MDMA use in the US was 13.5% for individuals between ages 19–30 (Johnston et al . 2011 ).

A greater lifetime MDMA intake was associated with decreased 11 C AMT trapping in a very small frontal cortical area in the right hemisphere, although only at a marginally significant level (Peak T = 3.38, k max = 41). A greater lifetime MDMA intake was also associated with higher normalized 11 C‐AMT trapping in clusters of voxels projecting in the raphe area or in close approximation to it (anterior and superior right cerebellar hemisphere adjacent to the brainstem) (Peak T = 3.48, k max = 28). There was no significant correlation between the total dose of any of the other drugs of abuse and normalized 11 C AMT trapping (Fig. 2 ).

Overall, VOI analyses were largely consistent with results drawn for whole brain analyses. Male MDMA polydrug users had lower normalized K* in the pre‐central gyrus, relative to control males [ F (1,16) = 5.73, p = 0.029, d = 1.14]. Group by hemisphere interactions were observed in the pre‐central gyrus [ F (1,16) = 5.79, p = 0.029] and in the pre‐cuneus [ F (1,16) = 9.26, p = 0.008]. Univariate one‐way GLM analyses for the left and right hemispheres for each of these regions indicated that male MDMA polydrug users had lower normalized K* for both the pre‐central gyrus and pre‐cuneus in the right hemisphere ( d = 1.47 and d = 1.10, respectively), relative to male controls. Female MDMA polydrug users had lower normalized K* in the lateral orbitofrontal gyrus [ F (1,15) = 5.45, p = 0.03, d = 1.14], relative to female controls. In addition, increased normalized K* in the raphe observed in the MDMA polydrug users, was mainly because of an increased normalized K* in this region in female MDMA polydrug users [ F (1,15) = 8.55, p = 0.01, d = 1.43; Table 5 ].

>MDMA polydrug users had, relative to controls, higher regional normalized K* in the superior temporal gyrus [ F (1,33) = 9.54, p = 0.004, d = 1.06] and middle frontal gyrus [ F (1,33) = 5.08, p = 0.03, d = 0.77] in both hemispheres. Trends of increased normalized K* in MDMA polydrug users relative to controls, were also observed across the brainstem [ F (1,33) = 3.58, p = 0.07, d = 0.65] and for the raphe specifically [ F (1,33) = 2.94, p = 0.096, d = 0.71]. There were significant group by hemisphere interactions for the superior frontal gyrus [ F (1,33) = 4.53, p = 0.04]. Compared to controls, MDMA polydrug users had higher normalized K* in the left superior frontal gyrus (relative to the right superior frontal gyus) and lower normalized K* in the right pre‐cuneus (relative to the left pre‐cuneus), although not to a statically significant degree when analyzing hemispheres separately ( d = 0.60 and d = 0.61, respectively).

Sample characteristics are shown in Table 1 . Self‐reported MDMA consumption varied significantly from 28 to 1015 tablets over the subject's lifetime (mean ± SD: 236 ± 282; median: 100), over periods of time ranging from 1 to 9 years (4 ± 2.3 years; median: 3.5). Compared to other samples reported in the literature, this corresponds to a ‘moderate’ to a ‘significant’ pattern of use. The controls all reported never having used MDMA. Consistent with what is normally observed in the general population, all MDMA users reported using other drugs as well (Table 2 ). There were no gender differences in reported lifetime MDMA intake, duration of MDMA use or time elapsed since the last MDMA dose, nor in the lifetime consumption of alcohol or of any other drugs. All participants were free of drugs when participating in the study, as verified by the urine drug tests obtained during screening and on the morning soon prior to the PET measurement.

All of these analyses were carried out in statistical parametric mapping (SPM) version 8 (SPM8; Wellcome Functional Imaging Laboratory, London, UK). To confirm and quantify the results obtained from whole brain SPM analyses, Volumes of interest (VOI) analyses were performed in those specific brain regions that showed significant group differences in the whole brain SPM analyses and were large enough to be reliably identified on each participant's MRI using an automatic segmentation method (Collins et al . 1999 ). Regional K* was expressed both as normalized and non‐normalized values; the need to normalize the regional K* data in this study was supported by the observed trend in gender differences in the control group in plasma free tryptophan values (see below). Group differences in normalized K* extracted from the VOI analyses were analyzed using general linear models (GLM) for repeated measures, with hemisphere as a within subject factor, and group (MDMA polydrug user vs. control) as between subjects factor. Significant group by hemisphere interactions were further investigated using one‐way GLM models, using the left and right hemisphere for the specific VOI investigated, as a dependent variable, and group, as a between subject factor. One‐way GLM models, without a hemisphere term, were run for the analyses of the raphe and brainstem given that these are very small regions. As was the case for whole brain SPM analyses, all GLM analyses were rerun for males and females separately. The magnitude of group differences in normalized regional K* identified by VOI analyses was further quantified by calculating Cohen's d effect sizes using the G‐power program (Faul et al . 2007 ).

Both the co‐registration and normalization were conducted according to standardized procedures used at the MNI (e.g. Leyton et al . 2001 ; Rosa‐Neto et al . 2004 ; Booij et al . 2010 , 2012 ; Berney et al . 2011 ). Briefly, coregistration of the individual PET and MRI images was performed using an automatic procedure (Woods et al . 1993 ), which uses averaged tissue activity images obtained during the time period of 5–60 min of dynamic PET data acquisition (Okazawa and Diksic 1998 ). Parametric K* images of 11 C‐AMT trapping were generated (Okazawa and Diksic 1998 ) and re‐sampled into MNI305 2‐mm isotropic stereotaxic space using a standard automatic algorithm (Collins et al . 1994 ). The images were subsequently smoothed to a 14‐mm resolution FWHM, using an isotropic Gaussian filter. To cancel out the effects of individual global effects on regional 11 C‐AMT trapping (K*) values, statistical analyses with proportional scaling was used and regional K* values were normalized by the mean global K* of the gray matter to 100, as in our previous studies (Leyton et al . 2001 ; Rosa‐Neto et al . 2004 ; Booij et al . 2010 , 2012 ; Berney et al . 2011 ). The rationale for normalizing to the mean global K* of the gray matter is to remove changes because of brain size, higher blood flow, etc. within and between groups. The number 100 was chosen to express normalized K* in percentages.

All participants underwent a 60‐min dynamic PET scan, conducted with an ECAT HR+ (CTI Molecular Imaging, Inc/Siemens, Knoxville, TN, USA) in the late morning or early afternoon. The participants received a low‐protein diet the previous day and fasted overnight (limited water intake was allowed). Women underwent the PET scan in the follicular phase of their menstrual cycle. Two intravenous lines were secured, one in each arm, with one used for tracer injection and one for blood withdrawal. Before beginning the dynamic PET scan, transmission scans were performed using a 68 Ga source for attenuation correction. Participants received approximately 10 mCi of 11 C‐AMT over 2 min, at the beginning of the dynamic PET scan protocol. Twenty‐six frames were acquired using the following protocol: six frames of 30 s, seven frames of 60 s, five frames of 120 s, and eight frames of 300 s. All scanning was acquired with a Neuro‐Shield ® (Montreal, QC, Canada) placed around the participant's neck to reduce scattering. Blood samples were drawn throughout the PET scan from the ante‐cubital vein at progressively increasing time intervals to obtain the 11 C‐AMT plasma time activity curves. The input function was estimated using a validated non‐invasive procedure combining venous sinus activity (first 20 min) and venous plasma time‐activity data (Nishizawa et al . 1997 , 1998 ). Specifically, radioactivity‐time courses from the venous samples were drawn, and corrected by the venous sinus from dynamic PET images, as reported in Nishizawa et al . ( 1998 ). The slopes K* were then calculated using normalized exposure time points (Nishizawa et al . 1998 ).

Participants were asked to refrain from drug use for any drug, 3 weeks prior to the study sessions. They were required to test negative on two consecutive urine drug tests for the detection of illicit drugs, one at screening and one on the morning of the PET session (Triage, San Diego, CA, USA). All participants who were suitable and willing to give informed consent were scheduled. Once a full description of the study had been completed, written informed consent from all participants was obtained. The study was in conformance with the code of ethics of the World Medical Association. The Research Ethics Boards of the McGill University Health Centre and the Montreal Neurological Institute (MNI) approved the study.

Assessment of the controls was similar to that of the MDMA polydrug users. Potential control participants reporting some illegal drug use other than MDMA were not in principle excluded from the study; selecting controls reporting no drug use could have resulted in a control cohort that would be too different from that of our MDMA users. Participants with less than five total uses, and no use in the previous year, of illicit drugs other than cannabis, were deemed eligible. For cannabis, we only included controls, if their use in the previous year averaged less than once a month.

The main inclusion criterion for MDMA polydrug users was the use of MDMA on at least 25 occasions, a level of use which has been previously associated with evidence of brain alterations in the brain serotonergic system such as altered CSF 5‐hydroxyindoleacetic acid (5‐HIAA) levels, altered brain SERT binding and altered mood and neuroendocrine responses to the 5‐HT agonist probe meta‐chlorophenylpiperazine (mCPP) (McCann et al . 1999 ). These criteria were similar to those used in a previous study on MDMA polydrug users conducted in our laboratory, using ATD (Young et al . 2014 ). Exclusion criteria included the following: (i) a current or past major medical illness (determined by physical examination and laboratory tests); (ii) evidence for current axis I DSM‐IV disorders; (iii) current use of any prescription psychotropic drug; (iv) a beck depression inventory score of 12 or above; (v) reports suggesting evidence of a positive family history in first‐degree relatives for major depressive disorder; and (vi) being a sexually active woman and not using a reliable form of contraception.

Participants using MDMA, as well as MDMA‐naïve controls, were recruited through newspaper advertisements, posters distributed at local universities and flyers distributed at local clubs and raves. Following a telephone interview to assess initial eligibility, participants underwent a full screening procedure in person involving the following: (i) A psychiatric assessment, including a semi‐structured psychiatric interview (structured clinical interview for DSM‐IV: patient edition, SCID‐NP) (First et al . 2002 ), the beck depression inventory (Beck et al . 1961 ), and a determination of the presence or absence of psychiatric illness in the family using the family history method (Andreasen et al . 1977 ). (ii) A complete physical examination, including laboratory testing (glucose, electrolytes, alanine transaminase, aspartate aminotransferase, creatinine, thyroxine, thyroid‐ stimulating hormone, and complete blood count) and an electrocardiogram. (iii) Estimations of past and current drug use, using a timeline follow‐back procedure as in our previous PET studies on drug use (e.g. Boileau et al . 2006 ).

Discussion

This study points to regional differences between MDMA polydrug users and matched controls for 5‐HT synthesis capacity, with both increases and decreases in 11C‐AMT trapping in MDMA polydrug users relative to controls. While decreases were primarily observed in pre‐frontal–orbital and parietal regions, with somewhat more widespread decreases in males relative to females, increases in 11C‐AMT trapping was also noticeable, in particular in the brainstem.

Our results are consistent with studies in rodents and non‐human primates indicating that repeated administration of MDMA often results in long‐term region‐specific reduction in numerous 5‐HT markers, including brain tissue concentrations of 5‐HT, 5‐HIAA levels, TPH enzyme activity and SERT density (Biezonski and Meyer 2011; Urban et al. 2012). These reductions in 5‐HT markers have previously been interpreted as a result of distal axotomy of brain 5‐HT neurons (Molliver et al. 1990). However, it is noteworthy that in our study, the spatial extent of focal declines in cingulate cortex and mesencephalon is less than might have been expected on the basis of SERT imaging studies in similar populations or in pre‐clinical studies where a loss of small serotonin fibers in the forebrain (see also below) was exhibited as a result of MDMA. In addition to decreases in 11C‐AMT trapping in the frontal regions, increased normalized 11C‐AMT trapping was also observed, mainly in the brainstem as well as in the superior medial and temporal gyri.

Using the same tracer and radio‐autography 14 days after rats were administered a total of eight doses of MDMA, there were significant decreases in 5‐HT in a variety of brain areas including parts of the cortex, striatum and hippocampus, and a significant increase only in the median raphe nucleus (Molliver et al. 1990). This suggests that, in addition to MDMA‐induced neurotoxicity in humans, compensatory feedback mechanisms in the cell bodies of the 5‐HT neurons might also occur. This observation was consistent with findings from a longitudinal study in MDMA polydrug users with repeated measurements of brain SERT binding, which reported increases in SERT binding after cessation of MDMA use (Buchert et al. 2006). Indeed, a large animal literature reports on the extent to which the effects of MDMA may be enduring/persistent, as opposed to reversible. For instance, although studies in rats have shown that there is almost complete recovery of 5‐HT markers 1 year after drug exposure, as a result of sprouting of axons (Molliver et al. 1990), studies in monkeys reported that the recovery is incomplete, and innervation patterns remain altered (Scheffel et al. 1998; Hatzidimitriou et al. 1999). In squirrel monkeys that had been treated with MDMA and monitored for up to 7 years thereafter, some of the 5‐HT ‘deficits’ ‘recorded’ at the end of the study were actually less severe than those observed at 18 months following exposure. Factors influencing recovery included the distance from the raphe nuclei to the purported affected site, the degree of initial axonal injury, and possibly the proximity of myelinated fibers (Hatzidimitriou et al. 1999). PET studies of SERT distribution in baboons also suggested that there is some recovery in regions closer to the raphe nuclei. Specifically, at 13 months after MDMA exposure, SERT levels had actually risen above control levels in the hypothalamus, but were still down respectively by 62%, 78% and 73% in the frontal, parietal and occipital cortex (Scheffel et al. 1998). A more recent autoradiography study in rats investigating the impact of MDMA on 5‐HT biosynthesis found that 14 days after chronic MDMA administration, TPH 2 enzyme immuno‐reactivity was decreased, while TPH 2 mRNA was increased in the cell bodies of 5‐HT neurons in the Dorsal Raphe Nucleus, suggesting some compensatory mechanisms (Bonkale and Austin 2008).

The impact of polydrug use on brain 5‐HT synthesis appeared to be more widespread in males than in females. Interestingly, there were no differences in reported lifetime MDMA intake, duration of MDMA use or time elapsed since the last MDMA dose, nor in the lifetime estimate of alcohol or of other drugs examined consumed. At first glance, these results seemed to be at odds with findings of previous studies, showing that women appear to be more susceptible to the neurochemical effects of MDMA on CSF 5‐HIAA (McCann et al. 1994) and SERT binding (Reneman et al. 2001), than men. They are also at first glance at odds with our recent ATD study, indicating a greater mood‐lowering response to ATD in female MDMA polydrug users than in males (Young et al. 2014). Yet, previous studies in animals did not find compelling evidence of gender effects of MDMA on the 5‐HT system (5‐HIAA or 5‐HT concentrations) (Walker et al. 2007). Methodological differences across studies, genetic differences, polydrug use, hormonal factors, and MDMA dose might account for some of those different observations. On the other hand, in spite of the observation that the effects of MDMA polydrug use in males may be more widespread than in females, interestingly, female MDMA polydrug users in the present study had, relative to female controls, a relatively specific lowering in regional K* in the orbitofrontal regions, a brain region highly involved in emotion regulation. Alterations in regional K* in the orbitofrontal regions were absent in male polydrug users. Previous PET studies assessing cerebral metabolism following ATD have shown that the mood‐lowering response to ATD in depression vulnerable populations correlates with alterations in metabolism in the orbitofrontal regions after ATD (Bremner et al. 1997; Smith et al. 1999; Neumeister et al. 2004). Taking the findings of our ATD study and from the current PET study together, it might be that the mood‐lowering effect of ATD in female polydrug users as observed in (Young et al. 2014) are due to sex‐specific, drug induced, 5‐HT effects in the orbitofrontal regions. Only four female MDMA polydrug users participated in both the ATD study and the current PET study and thus correlations between the mood response to ATD and 5‐HT synthesis capacity could not be reliably tested; yet, it is tempting to speculate that sex‐specific effects of drug use on 5‐HT neurotransmission in the orbitofrontal regions might make female users more susceptible to aversive behavioral manifestations, including clinical depression. Longitudinal studies in (former) drug users having consumed MDMA in significant quantities over long periods of time are needed to test such hypothesis.