Purpose: Brain imaging, inter-species comparison; To examine post-synaptic 5HT2A receptor density in current and abstinent regular ecstasy users, and also in rats given a neurotoxic regimen of MDMA, and to investigate changes in receptor density after time since last use or last dose. Specific hypotheses tested - That 5HT2A receptor density will decline shortly after MDMA or ecstasy, but that 5HT2A receptor density will increase after a longer period of time has elapsed since MDMA or ecstasy administration, with expected effects occurring in rats and humans.

Design: Humans: (Addressed in this summary) Non-experimental (retrospective) between-groups design, with drug use (current regular ecstasy user, abstinent ecstasy user or non-ecstasy user) serving as a between-group variable, and with all participants undergoing SPECT scans with [123-I]R91150 to measure 5HT2A receptor density. Rats (not addressed in this summary; see summary under "Research in Non-Human Animals")

Subjects: 17 current ecstasy users, 7 abstinent ecstasy users and 11 non-user controls residing in the Amsterdam (Netherlands) area, and recruited via advertisements placed in local newspapers. Matching - All 3 groups were matched on gender and age (within same age range rather than exact match). Examining group demographics suggests that at least some participants appeared in a previously presented study also using SPECT scans measuring SERT site density (Reneman et al. 2001), and a sub-sample of this sample may have appeared first in an earlier comparison of 5HT2A receptor density (Reneman et al. 2000a).

Criteria for Inclusion - Ecstasy Users - Self-reported lifetime use of at least 50 ecstasy tablets. Abstinent Ecstasy Users - Self-reported lifetime use of at least 50 ecstasy tablets, but abstinence from ecstasy for at least 2 months. Non-User Controls - No self-reported lifetime use of ecstasy, though use of other drugs was permitted. All Groups - Healthy, as apparently indicated through medical examination and no past or current psychiatric illness, between the ages of 18 and 45 years old, not pregnant, and abstinent from all psychoactive drugs for 1 wk before study day, with abstinence verified through urinary drug screen.

Drug Use Parameters - Current ecstasy users reported taking 224 +/- 311 tablets over a lifetime, with reported average dose per use of 2.2 +/- 1.7 tablets. No information is provided concerning frequency of use. Current ecstasy users reported a duration of use, in months, of 70.8 +/- 34.8 months. Self-reported time since last use for current ecstasy users, in days, was 23.1 +/- 16.8 days. Abstinent ecstasy users reported taking 274 +/- 238 tablets over a lifetime, with reported average dose per use of 1.1 +/- 2.1 tablets per use. No information is provided about frequency of use. Duration of use for abstinent ecstasy users, in months, was reported at 55.2 +/- 28.8 months. Abstinent ecstasy users reported time since last use, in days, was 137.2 +/- 81.2 days. Other Drug Use - Extent of all other drugs used reported in amount of specific units (i.e. cigarettes, joints, etc.) used in the last 3 months. Alcohol - Current ecstasy users reported drinking 30.1 +/- 31.4 units in the last 3 months, abstinent ecstasy users reported drinking 30.3 +/- 33 units in 3 months and non-ecstasy users reported drinking 43.1 +/- 36.2 units in last 3 months. Tobacco - Current ecstasy users reported smoking 52 +/- 59.5 cigarettes in 3 months, abstinent ecstasy users reported smoking 78.6 +/- 34.8 cigarettes in 3 months and non-users reported smoking 16.5 +/- 20.2 cigarettes in the last 3 months. Cannabis - Current ecstasy users reported smoking 20.5 +/- 38.3 joints in the last 3 months, abstinent ecstasy users reported smoking 43.1 +/- 36.6 joints in the last 3 months and non-users reported smoking 3 +/-5.6 joints in the last 3 months. Cocaine - Current ecstasy users reported using cocaine 0.8 +/- 0.4 times in 3 months, abstinent ecstasy users reported using cocaine 1.1 +/- 0.9 times in 3 months and non-users reported using cocaine 1.7 +/- 0.2 times in 3 months. Amphetamine - Current ecstasy users reported using amphetamine 1.5 +/- 2.2 times in 3 months, abstinent ecstasy users reported using amphetamine 0.3 +/- 0.5 times in the last 3 months and non-users did not report using amphetamine in the last 3 months.

Group Demographics and Matched Variables - The authors reported matching groups for gender and age. In this case, age matching refers to the age range for all groups. There were significant between group differences in participant age. Gender, as M/F ratio - Current ecstasy users = 12/5, abstinent ecstasy users = 5/2, non-users = 5/6. Age - Average age of current ecstasy users was 27.9 +/- 3.8 years. Average age of abstinent users was 22.7 +/- 4.6 years. Average age of non-users was 22.5 +/- 2.7. Current ecstasy users were significantly older than abstinent ecstasy users. Education - No information is provided concerning average educational attainment for any of the 3 groups. Other Variables - DART-IQ (used to estimate verbal IQ) - Current users had an average DART score of 92.8 +/- 8.2, abstinent users had an average DART score of 86.2 +/- 11.5 and non-users had an average DART score of 96.4 +/- 5.6.

Measures: SPECT-Radioligand Binding - Scans performed with the 5HT2A receptor radioligand, [123-I]R91150, co-registered with MRI scans. Scans were recorded 2 h after injection of 140 MBq [123-I]R91150, with participant in supine position. Frontal, parietal and occipital areas were selected as regions of interest (ROIs) and the cerebellum served as non-serotonergic control. An investigator blind to subjects' drug use histories performed ROI analysis, with the cerebellum used as a reference due to its presumed lack of 5HT2A receptors. "Specific" binding for each ROI calculated as "mean" ROI binding / cerebellar binding = 5HT2A binding ratio.

Analyses: Demographic comparisons - Examined via 1-way between subjects (across groups) ANOVA, with comparisons made between current ecstasy users, abstinent ecstasy users and non-ecstasy user controls, with p. value set at 0.05. Differences between the two ecstasy user groups (current, abstinent) were compared via student's t test.

SPECT-Radioligand Binding - Differences in R91150 binding ratios within the 3 cortical areas were compared across groups (ecstasy user, abstinent ecstasy user or non-ecstasy user) via multiple analysis of variance (MANOVA), with correlations between brain regions and multiple comparisons taken into account. If group differences were found, R91150 binding ratios were further examined via 1-way ANOVA with drug use group serving as between-group factor. Post-hoc comparisons were made via Dunnett's test.

Drug Use Parameters and R91150 Binding Ratios - Possible associations between overall cortical ligand binding ratio (mean binding ratio) and 2 parameters of ecstasy use were examined via Spearman's rank coefficient. Parameters assessed were time since last use and overall lifetime ecstasy use (number of tablets taken).

Results - Significant Differences: SPECT-Radioligand Binding - [123-I]R91150 binding ratios were lower in recent ecstasy users, compared to non-ecstasy users, and [123-I]R91150 binding ratios were higher in abstinent ecstasy users when compared with non-users or with current ecstasy users (abstinent ecstasy users > controls > current ecstasy users). Further comparison of binding ratios found that R91150 binding ratios were lower for all cortical regions examined in current ecstasy users (current ecstasy users > controls). [123-I]R91150 binding ratios were higher in the occipital region of abstinent ecstasy users when compared with controls.

Drug Use Parameters and R91150 Binding Ratios - There was a significant correlation between mean global [123-I]R91150 binding ratios and time since last use, with amount of binding increasing with increasing length of abstinence from ecstasy use.

Results - No Significant Differences Found: SPECT-Radioligand Binding - Further comparisons between regional R91150 binding ratios found that abstinent ecstasy users did not have higher [123-I]R91150 binding ratios in frontal or parietal regions than did controls. Age and gender covariance effects were not significant (there was no significant difference in [123-I]R91150 binding ratio due to age or gender.

Drug Use Parameters and R91150 Binding Ratios - There was no significant relationship between number of tablets taken in a lifetime (extent of use) and mean binding ratios.

Overall Effects: A group of men and women who had recently used ecstasy had apparently lower 5HT2A receptor density than did non-ecstasy user controls, with receptor density measured via SPECT scan using the radioligand [123-I]R91150. 5HT2A receptor density was greater in people who had not taken ecstasy for at least 2 months before SPECT scan, with amount of ligand binding in occipital areas significantly higher in abstinent ecstasy users than in controls. No association was found between number of ecstasy tablets taken in a lifetime and 5HT2A receptor, but higher numbers of ligand binding sites were related to longer time since last use. Though no age-related effects on ligand binding were reported, current ecstasy users were significantly older than controls. Findings are somewhat similar, but not identical, to findings in rats. In rats, 5HT2A receptor density (measured in [123-I]R91150 binding directly observed in brain) is lowest 6 h after MDMA administration and highest 30 days after MDMA administration, with comparisons made with control rats. These effects were correlated with lower 5HT measured in frontal and occipital areas, with 5-HT and 5-HIAA lowest in cortical areas in rats 30 d after MDMA administration. However, ligand binding was low in occipital sites 6 h after MDMA administration. The authors' hypothesis was confirmed, with ligand binding ratios lowest in people with the most recent exposure to ecstasy or MDMA, and with ligand binding increasing as time since last exposure increased. This suggests that 5HT2A receptor density decreases acutely after MDMA, and that receptor density returns to normal or increases in density as time since last use increases.

Comments: This paper is the third of several reports from the same author and her colleagues concerning differences in 5HT2A receptor density measured across ecstasy users and non-user controls. The findings here seem to replicate findings reported in an earlier paper, with putative 5HT2A receptor site density decreasing after recent ecstasy use and increasing after less recent ecstasy use. However, consistency of findings may be partially or wholly due to the use of similar samples to those examined in previous reports. This study suffers from the usual limitations faced by retrospective studies with small samples, and there is a lack of information concerning comparative educational attainment across each group. Non-users also have reported using less cannabis and amphetamine than either group of ecstasy users. However, abstinence from ecstasy before the study day was confirmed via urinary analysis.