PROJECT SUMMARY Psychedelics are compounds that produce an atypical state of consciousness characterized by altered perception, cognition, and mood. Among psychedelics, psilocybin has gained attention recently because early clinical trials indicated potential antidepressant effects, leading to a ‘breakthrough therapy’ designation from the FDA to test psilocybin for major depressive disorder. However, despite the promise, the biological mechanisms underpinning psilocybin’s potential therapeutic action are poorly understood. Our lab employs subcellular- resolution two-photon microscopy to visualize dendritic structure and function in head-fixed mice. The goal of this project is to characterize how a single dose of psilocybin may alter dendritic architecture in the medial frontal cortex of the mouse and the associated cellular mechanisms. The hypothesis is that psilocybin promotes spine formation by activating specific serotonin receptor subtypes and exerts differential effects on distinct subtypes of pyramidal neurons. To test the hypothesis, we propose a series of experiments that combine subcellular-resolution optical imaging, conditional knockouts, and causal perturbations in mice. The results will answer crucial questions regarding psilocybin’s ability to promote structural plasticity in vivo and delineate receptors and cellular factors that underlie the plasticity-promoting actions. We expect the mechanistic insights will be important as the field evaluates psychedelics as a potential treatment option for neuropsychiatric disorders and searches for novel antidepressants.