Receptor-Enriched Analysis of functional connectivity by targets (REACT): A novel, multimodal analytical approach informed by PET to study the pharmacodynamic response of the brain under MDMA

MDMA (3,4-methylenedioxymethamphetamine) increases extracellular serotonin, dopamine and noradrenaline and produces prosocial and euphoric effects as well as mild visual hallucinations linked to 5-HT2A. Previous neuroimaging studies using resting-state fMRI (rs-fMRI) reported MDMA-related changes in circuits implicated in social and affective processing, but standard fMRI lacks molecular specificity: the haemodynamic signal does not directly index activity at particular receptor sites. This limits mechanistic inferences about which receptor targets mediate observed functional connectivity (FC) changes. Dipasquale and colleagues introduce Receptor-Enriched Analysis of functional Connectivity by Targets (REACT), a multimodal method that uses PET-derived maps of receptor/transporter density to inform rs-fMRI analysis. Using a high-resolution atlas of four serotonin receptors (5-HT1A, 5-HT1B, 5-HT2A, 5-HT4) and the serotonin transporter (5-HTT), the study tests whether MDMA-induced FC changes co-vary with the spatial distribution of these targets. The investigators hypothesised that FC informed by 5-HTT, 5-HT1A and 5-HT2A maps would be sensitive to MDMA, and also explored interactions between target distributions and relationships with subjective effects, MDMA plasma levels and oxytocin changes.

Twenty-one male volunteers with prior MDMA experience were recruited; 20 completed the study (mean age 24.8 years). Exclusion criteria included psychiatric illness, first-degree family history of psychosis, significant medical disorders, current medication, excessive caffeine or alcohol use, and positive drug screens. Participants had not used MDMA in the preceding three months. The protocol received ethical approval and participants provided written consent. A within-subject, double-blind, randomised, placebo-controlled crossover design was used. Each participant attended two experimental days at least one week apart. Subjective effects were measured with the 11-dimension Altered States of Consciousness Questionnaire (ASC). Plasma MDMA was sampled at 45 and 165 minutes post-dose; oxytocin was measured pre-dose and at the same two post-dose timepoints. MRI was acquired on a 3 T GE scanner with a multi-echo EPI resting-state sequence; the resting scan began approximately 90–100 minutes post-dose. Preprocessing combined the echoes into an optimally combined (OC) dataset and applied Multi-Echo ICA (ME-ICA) to denoise non-BOLD artefacts. Data were then spatially smoothed (8 mm FWHM), nuisance signals from eroded white matter and CSF masks were regressed out, high-pass filtered (cut-off 0.005 Hz), and normalised to a study-specific template and MNI space. Rs-fMRI was sampled at 1x1x1 mm3 for the first analysis stage and at 2x2x2 mm3 for the second. REACT implemented a two-step multivariate regression (analogous to dual regression) using a publicly available PET-derived atlas of 5-HT1A, 5-HT1B, 5-HT2A, 5-HT4 receptors and 5-HTT density maps as spatial regressors in the first step to extract subject-specific time series. These time series were then used in a second-stage temporal GLM to produce subject-specific spatial maps reflecting FC associated with each molecular target under placebo and MDMA. Analyses excluded cerebellar voxels because those PET maps used cerebellar grey matter as reference. An exploratory interaction analysis tested products of maps (5-HT1A*5-HT2A, 5-HT1A*5-HTT, 5-HT2A*5-HTT and the three-way product) entered alongside the single-target maps. A complementary resting-state network (RSN) analysis used 13 RSNs derived from an independent dataset (group ICA, model order 20) as templates in dual regression. Statistical comparisons of target-enriched spatial maps and RSNs between MDMA and placebo used permutation testing with Randomise (5,000 permutations) and threshold-free cluster enhancement (TFCE). Linear relationships between FC change (MDMA minus placebo) and behavioural measures, MDMA plasma at 45 and 165 minutes, and changes in peripheral oxytocin were tested voxelwise. Mean FC values from significant clusters were correlated with predictors using Pearson correlations with bootstrapping (1,000 samples). Multiple-comparison control across maps/contrasts was addressed with Bonferroni correction where reported.

Behaviourally, MDMA increased ASC scores in 10 of 11 dimensions (including spiritual experience and blissful state) relative to placebo (all Bonferroni-corrected p < 0.05). Oxytocin peripheral levels were higher after MDMA at both post-dose timepoints (Bonferroni-corrected p = 0.054 at 45 min; p < 0.001 at 165 min). Mean plasma MDMA concentrations on the active day were 91.7 μg/L (SD 60.2) at 45 min and 188.2 μg/L (SD 32.8) at 165 min. The REACT two-step analysis produced one subject-specific spatial map per target and condition. Comparing MDMA versus placebo, significant decreases in FC were observed in maps enriched by 5-HT1A and by 5-HTT (cluster-level TFCE, family-wise error corrected pFWE < 0.05, Bonferroni-corrected across maps/contrasts). The 5-HT1A-enriched FC decreases involved multiple cortical areas including bilateral precentral and postcentral gyri, left frontal pole, left insular and inferior frontal regions, left middle and supramarginal gyri, left Heschl's and planum temporale, right superior frontal and parietal regions and supplementary motor cortex. The 5-HTT-enriched decreases were localised to anterior and posterior cingulate cortex, precuneus, cuneal and calcarine cortices, lingual gyrus and lateral occipital cortex. No significant MDMA-induced FC changes were found in the primary analyses of 5-HT1B, 5-HT2A or 5-HT4-enriched maps. The RSN analysis (uninformed by PET maps) identified MDMA-related FC changes in primary visual, sensorimotor, medial temporal, salience and ventral stream networks, but none survived Bonferroni correction across RSNs and contrasts. The interaction analysis including products ofPET maps returned both decreases and increases: significant FC decreases were found in maps derived from 5-HT1A, 5-HTT and the 5-HT1A–5-HT2A interaction, and an increase was detected in portions of the 5-HT1A-enriched maps (all pFWE < 0.05 TFCE, Bonferroni-corrected). In correlational analyses, MDMA-induced FC change in 5-HT2A-enriched maps correlated negatively with the spiritual experience subscale (r = -0.782, p < 0.0001, 95% CI -0.909 to -0.572), localised to left lingual gyrus and occipital/temporo‑occipital fusiform areas: greater spiritual experience was associated with larger reductions in 5-HT2A-related connectivity. No linear relationships were found between main REACT FC maps and oxytocin levels. MDMA-induced FC increases in regions of the 5-HT1A-enriched maps correlated strongly with MDMA plasma at 45 minutes (r = 0.90, p < 0.0001, 95% CI 0.71 to 0.965), localised to right supramarginal gyrus, lateral occipital and cuneal cortices. Exploratory interaction analyses supported the 5-HT2A–spiritual experience relationship and additionally revealed correlations between MDMA-induced FC decreases in some regions of 5-HT1A, 5-HT1A–5-HTT and 5-HT1A–5-HT2A–5-HTT maps and increased oxytocin at 165 minutes; these interaction results are reported in supplementary materials. The RSN maps showed no significant correlations with subjective, MDMA or oxytocin measures.

Dipasquale and colleagues interpret their findings as evidence that augmenting rs-fMRI with PET-derived molecular maps can link haemodynamic responses to receptor distributions. REACT defined target-enriched networks using voxel-wise serotonin receptor and transporter density and detected MDMA-related FC changes that aligned with the compound's principal serotonergic targets, notably 5-HT1A and 5-HTT. The authors note that these target-enriched maps partly replicate prior MDMA studies implicating visual cortex, supplementary motor areas and posterior cingulate, while also extending effects to other regions. The investigators highlight two key receptor-related relationships. First, FC changes in 5-HT1A-enriched maps correlated with MDMA plasma levels at 45 minutes, suggesting that these posterior cortical interactions capture functional impact related to acute drug exposure. Second, reductions in 5-HT2A-enriched connectivity correlated with the spiritual experience subscale, consistent with the role of 5-HT2A in hallucinogenic/psychedelic subjective effects; affected regions included lingual and fusiform occipital areas associated with mental imagery. The interaction analysis showed partly convergent results but also additional effects when accounting for co-localisation of targets. The authors caution that interpreting interactions at the whole-brain level is complex: PET maps reflect regional co-localisation rather than cellular co-expression, no thresholds were applied to maps (so low-density voxels were included), and MDMA exerts non-serotonergic actions (dopamine, noradrenaline) that could contribute to effects. Limitations explicitly acknowledged include the lack of subject-specific PET data (the analysis used a high-resolution template rather than individual receptor maps), the all-male sample with prior MDMA exposure, and the complexity of receptor interactions that remain to be detailed. The authors propose further validation with other compounds and the development of high-resolution templates for other neurotransmitter systems (e.g. dopamine). They suggest REACT may provide a drug-specific ‘‘fingerprint’’ of functional connectivity changes by linking haemodynamic data to receptor distributions, with potential utility for mechanistic studies and characterising novel compounds.

Grounded in receptor occupancy theory, the authors conclude that REACT demonstrates a link between MDMA-induced haemodynamic responses and underlying serotonin receptor/transporter distributions. By enriching connectivity analyses with receptor maps, the method can define drug-specific topographies of functional connectivity and may offer a novel fingerprinting approach for characterising the functional impact of psychoactive compounds and improving understanding of heterogeneous treatment responses.