Project Summary Repeated drug exposure produces widespread cellular alterations that can manifest in maladaptive behaviors and addiction. These behavioral alterations can be debilitating, and the pathology of addiction is often a life-long affliction. Indeed, even after years of abstinence, relapse can be precipitated by exposure to drug-associated cues. This is a ubiquitous property of drug addiction, and is present across drug class, yet we lack a clear understanding of how these changes can be so long lasting. Dysregulation of many classes of proteins have been implicated in cue-evoked relapse, yet the propensity to relapse persists well past the half-life of these proteins, suggesting that upstream epigenetic changes are permissive to the transcriptional landscape that produces these behavioral aberrations. Understanding the epigenetic alterations that ultimately produce these cellular changes will great expand the number of targets available for potential therapeutic interventions. To approach understanding the epigenetic mechanisms that underlie cue-induced relapse, we must identify the cells that are activated to drive seeking behavior. Our preliminary data show that in any given brain region, only a small percentage of cells are activated to a given stimulus – this group of activated neurons is termed a neural ensemble. Thus, the next frontier of understanding the brain will be defining exactly which cells are activated, when they are activated, and why. Here we combine techniques that allow us to record, manipulate, and sequence neural ensembles during opioid self-administration and subsequent cue-triggered drug seeking to determine how transcriptional activity within each neuronal population dictates which cells are activated. By combining in vivo cellular resolution calcium imaging during cue-induced seeking followed by single cell sequencing - in the same animals - we will define the transcriptional networks that control the neural activity patterns that drive drug seeking. Next, using epigenetic approaches and CRISPR/dCas9 fusion constructs, we will define and manipulate the epigenetic landscape at activity-responsive genes selectively in neurons that are activated by drug-associated cues. This proposal will allow us to define the precise neural ensembles that guide drug seeking and how transcriptional networks within these neurons control the neural activity profiles that guide behavior. By defining these mechanisms, this award will allow research that pushes the boundaries of how we approach understanding information encoding in the brain and expand our understanding of how we can manipulate these processes to reduce relapse across drug classes.