The serotonin 2 receptor (5HT2R) agonist psilocybin has demonstrated rapid and long-lasting efficacy across neuropsychiatric disorders characterized by cognitive inflexibility. Psilocybin may accomplish this by inducing rapid and stable dendritic plasticity. However, the impact of psilocybin on patterns of neural activity underlying sustained changes in cognitive and behavioral flexibility has not been characterized. To test the hypothesis that psilocybin enhances behavioral flexibility by rapidly and persistently altering activity in cortical neural ensembles, we performed longitudinal single-cell calcium imaging in the retrosplenial cortex across a five-day trace fear learning and extinction assay. Leveraging tensor component analysis to identify neurons that modulate activity on multiple temporal scales, we found that a single-dose of psilocybin induced cortical ensemble turnover between fear learning and extinction days while oppositely modulating activity in fear- and extinction-active neurons. The extent of suppression of fear-active neurons and recruitment of extinction-active neurons were both predictive of psilocybin-enhanced fear extinction. These results both align with hypotheses that psilocybin enhances behavioral flexibility by recruiting new populations of neurons and introduce a new mechanism involving the suppression of fear-active populations in the retrosplenial cortex.

Psilocybin-enhanced fear extinction linked to bidirectional modulation of cortical ensembles