PROJECT SUMMARY Opioid use and dependence prevalence have skyrocketed in the United States. A majority of patients with opioid use disorder (OUD) relapse within months despite treatment. Recent human neuroimaging and postmortem brain studies in OUD reveal the degree of dysfunction within cortical and striatal brain circuits, particularly within dorsolateral prefrontal cortical (DLPFC) and nucleus accumbens (NAc) regions, strongly relates to the opioid use and dependence risk. The PFC provides top-down inhibitory cognitive and emotional control to the NAc, which mediates goal-directed and reward behaviors. Relapse vulnerability in OUD is strongly associated with the severity and persistency of disruptions to sleep and circadian rhythms, raising the possibility that therapeutic interventions which mitigate these disruptions during abstinence may be effective for reducing opioid craving and relapse. However, our understanding of the biological mechanisms underlying the relationships between circadian rhythms and OUD is limited, especially at the molecular level in the brains of people with OUD. We and others have developed novel, innovative approaches using time of death (TOD) to measure molecular rhythms in the human postmortem brain to investigate the mechanistic links between substance use and molecular brain rhythms. Using TOD approaches, we recently found a marked loss of molecular rhythms in the prefrontal cortex associated with normal aging and psychiatric disorders. Notably, we also discovered a gain of rhythmicity in genes within disease-specific molecular pathways, providing novel insights into the biology of brain aging and psychiatric pathology. Preliminary TOD analyses on large-scale gene expression in human subjects with OUD revealed enrichment for pathways related to circadian rhythms in the PFC and NAc. In our proposal, we will directly investigate the relationship between molecular rhythm disruption and opioid use and relapse using both human postmortem brains from subjects with OUD and mouse models of circuit-specific targeting and opioid self-administration. Specifically, we will investigate molecular rhythms in postmortem DLPFC and NAc using RNA-sequencing from a large cohort of subjects with OUD (Aim 1A). We will also examine the impact of specific clinical features (e.g., toxicology reports and overdoses, comorbid psychiatric disorders, history of use, polysubstance use, illness duration) on molecular rhythms in OUD (Aim 1B). We will then directly test the functional relevance of molecular rhythm disruptions in specific brain regions (PFC and NAc; Aim 2A) and circuits (PFC projections to NAc; Aim 2B) during opioid self-administration behavior in mice. Our studies will identify molecular rhythm abnormalities in the brains of subjects with OUD and begin to determine the mechanisms linking circadian rhythms and addiction, which will provide important insight into disease-related pathways and also potential treatment strategies.