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5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a potent serotonergic psychedelic with rapid therapeutic potential for depression and anxiety disorders. Despite growing clinical interest, the neurobiological mechanisms underlying 5-MeO-DMT's acute brain effects remain poorly understood, and potential sex differences in response have not been investigated. We conducted the first functional MRI study of 5-MeO-DMT in awake, drug-na ve rats using BOLD imaging. Male (n = 24) and female (n = 24) rats received I.P. injections of vehicle or 5-MeO-DMT (0.01, 0.1, or 1.0 mg/kg) during scanning sessions. BOLD signal changes and resting-state functional connectivity were assessed across 169 brain regions. Negative and positive BOLD volume activations were quantified during acute (1-10 min) and sustained (11-20 min) time windows post-injection, with sex-stratified analyses performed. Females demonstrated markedly enhanced sensitivity to low-dose 5-MeO-DMT (0.1 mg/kg), exhibiting extensive negative BOLD responses that were largely absent in males at the same dose. The 1.0 mg/kg dose induced widespread negative BOLD responses across multiple brain regions in both sexes during the acute window (1-10 min post-injection), with effects substantially diminishing thereafter. Global functional connectivity was significantly reduced across all doses (p < 0.0001), with regional specificity observed in hypothalamic and cerebellar networks. Temporal analysis revealed peak neurobiological effects within the first 10 min, consistent with 5-MeO-DMT's known rapid pharmacokinetics. To our knowledge, this is the first fMRI characterization of 5-MeO-DMT in any species, and it reveals a previously unreported sex difference in psychedelic response. The rapid onset and brief duration of peak effects align with 5-MeO-DMT's unique pharmacological profile and clinical reports.
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