Background: Psychedelic compounds have long been known for their ability to profoundly alter human consciousness, even at microgram doses. Psilocybin, in particular, has recently garnered significant attention due to its potential therapeutic benefits in treating many mental health disorders. Despite promising clinical results, the neural mechanisms underlying psilocybin's effects remain poorly understood. Psilocybin acts on the brain via multiple neurotransmitters, including serotonin and dopamine. On a whole-brain level, psilocybin decreases activity in the default mode network (DMN) and medial prefrontal cortex (mPFC), while increasing connectivity between the DMN, frontoparietal network, and salience network. These changes are thought to underlie the "ego dissolution" and synesthesia-like effects often reported during psilocybin-induced mystical experiences. However, a major knowledge gap remains in linking these global network effects to the initial receptor-level actions of the drug. Methods: To better understand how psilocybin affects the brain, we are combining molecular biology, in vivo imaging, and novel behavioral assays. First, we treated mice with either psilocybin or saline and isolated nuclei from the medial prefrontal cortex (mPFC) 24 hours later for single-nucleus RNA sequencing. This allowed us to identify distinct neuronal cell types (e.g., excitatory vs. inhibitory, different cortical layers) based on their unique gene expression profiles. Next, we performed live 1- and 2-photon microscopy in freely-moving and head-fixed mice with implanted lenses over the mPFC to visualize excitatory neural activity during and after psilocybin administration. Finally, we developed a novel behavioral assay using a high-fidelity camera to record mouse movement on a 2-dimensional linear track, enabling a detailed, AI-driven analysis of kinematic parameters. Results: Our molecular profiling experiment revealed that psilocybin treatment upregulated genes involved in excitatory synapse assembly. Excitatory neurons in layers V and VI were particularly affected, including a specific subtype enriched for the serotonin 2C receptor. Our 2-photon imaging data showed that psilocybin decreased mPFC activity in response to learned, positively salient cues. Ongoing analysis of recently acquired 1-photon imaging data will uncover the acute effects of psilocybin in real-time. Behaviorally, acute psilocybin administration led to decreased rearing and grooming, changes in gait and stance, and preserved overall mobility. Conclusions: We hope that this work will contribute to the understanding of how psychedelics affect the brain on a molecular, behavioral and neural network level both acutely and longer-term. In doing so, we may begin to elucidate how the natural function of the brain utilizes these pathways for neuroplasticity and consciousness.