INTEGRATED BEHAVIORAL AND MOLECULAR ANALYSIS OF PSILOCYBIN EFFECTS IN C57BL/6 WT MICE

Psilocybin, a serotonergic psychedelic, induces profound behavioral and molecular changes, yet the direct relationship between altered behavior and underlying transcriptional programs remains poorly understood. Here, we bridge this gap by integrating high-resolution behavioral phenotyping with spatial transcriptomics in the same animals to map psilocybin-induced behavioral states onto their corresponding molecular signatures across key brain regions. Male and female C57BL/6 mice received 0.125, 1, or 3 mg/kg psilocybin i.p. and were subjected to 60-minute Open Field Tests (OFT), followed by brain collection for Visium 10x Spatial Transcriptomics. Leveraging pose estimation (DeepLabCut), classical OFT analysis, and unsupervised behavioral classification (B-SOiD), we uncovered dose-dependent and temporally dynamic effects: 1 mg/kg induced early hyperlocomotion (0-10 min), while 3 mg/kg produced delayed hypolocomotion (20-40 min) accompanied by anxiogenic-like preference towards the periphery of the open field box. B-SOiD identified over 10 distinct behavioral motifs. Motifs including sniffing and rearing were selectively modulated by psilocybin in a dose-dependent manner. Spatial transcriptomics resolved molecular clusters corresponding to cortical layers, hippocampus, amygdala, thalamus, hypothalamus, lateral habenula, and striatum, representing regions critically involved in affective, cognitive, and innate behaviors. Critically, we identified cluster-specific psilocybin-induced differentially expressed genes (DEGs). The number of DEGs within anatomical structures was positively correlated with the expression of psilocybin’s main molecular target – Htr2a. Canonical correlation analysis uncovered the underlying transcriptomic signature that significantly influenced behavioral patterns, revealing potential molecular substrates underlying psychedelic-induced behavioral states. This integrative approach provides a framework for mechanistically linking the behavioral and transcriptional dimensions of psychoactive compounds.