Abstract The urgency of the opioid crisis (over 80,000 deaths in the past year) has encouraged research on understudied agents, such as the 5HT2A agonist psilocybin (PSI), for potential clinical benefit. Recently, PSI has been designated as a "breakthrough therapy" for depression and has demonstrated its potential benefits in substance-use disorders, including alcohol and nicotine addiction. Observational data suggest a link between PSI use and reduced odds of developing opioid use disorder (OUD). Emerging preclinical data suggest PSI may have neuroplasticity and greater neurocognitive flexibility as potential mechanisms of action in clinical disorders, but clinical trials have generally proceeded without mechanistic information in either the neurocognitive flexibility (FLEX), or the “classic” motivational (“GO”!) and regulatory (STOP!) domains. The proposed R61/R33 will address this need, examining PSI's impact on these 3 brain-behavioral domains in OUD patients. 72 individuals (R61 = 24; R33 = 48) will be prospectively assigned to receive either 25mg) or 1mg (control) of PSI, with selected pre- and post-PSI assessments that probe brain, cognition, and behavior. Aim 1 ("GO!" motivational domain), will examine brain and behavioral responses to cues and to drug-related videos. The hypothesis is PSI will reduce the brain (motivational circuitry) and behavioral (reduced positive affective bias) response to drug cues. Aim 2 ("STOP!" inhibitory domain) will assess brain responses during a valenced Go-NoGo task and during attempted inhibition of craving to drug videos and during behavioral performance of a standard (motor pre-protency) Go-NoGo task. The hypothesis is PSI will increase recruitment of STOP circuitry in the brain tasks and will reduce errors of commission in the behavioral probe. Aim 3 will investigate the "FLEX" (neurocognitive flexibility) domain using the Wisconsin Card Sort Task (brain) and Penn Conditional Exclusion Task (behavior). The hypothesis is PSI will enhance recruitment in FLEX substrates and reduce perseverative errors. Exploratory measures will include craving, withdrawal, psychedelic effects, depression, and prior adversity. Brain-behavioral probes showing large effect sizes will proceed to the R33 phase, and examinations will expand to include network-level changes in the brain. This proposal is of high importance for understanding PSI's effects on brain, cognition, and behavior in those with OUD – critical for the rational advance of PSI for OUD – and for the many other disorders with unmet need.

NARRATIVE This proposal, the first of its kind, focuses on understanding the brain and behavioral mechanisms of psilocybin treatment in opioid use disorder. The opioid crisis remains a critical public health issue, and novel treatments are needed to reduce overdose risk. Psilocybin may help by reducing barriers to treatment and ongoing illicit drug use, as well as by increasing adherence to life-saving opioid medications.