Project 2's objective is to understand the role for circular RNAs (circRNAs) in the mechanisms by which cocaine and opioids remodel the nucleus accumbens (NAc) and dorsal striatum (DS) to drive drug self-administration and relapse. circRNAs are highly conserved single-stranded ‘back-spliced’ RNAs in which the 5′ and 3′ ends of the transcript are covalently joined. Emerging evidence suggests that circRNAs are a major class of regulatory noncoding RNAs that are enriched in brain and that play key roles in basic aspects of neuronal function, but their involvement in the molecular, cellular, and behavioral actions of stimulant and opioid drugs of abuse has yet not been investigated. We will characterize patterns of circRNA expression in NAc and DS of mice during cocaine or opioid self-administration using paired-end ribominus RNA-sequencing. We will also assess circRNA expression in postmortem striatal tissues from humans with cocaine or opioid use disorders in collaboration with Project 4. We already have robust evidence for prominent cocaine and opioid regulation of several circRNAs in these brain regions. Those drug-responsive circRNAs that show similar abnormal expression in mice and humans will be prioritized for further investigation. We will determine whether prioritized drug-responsive circRNAs are regulated specifically in different types of NAc and DS neurons. We will then investigate the role played by these prioritized drug-responsive circRNAs in regulating the molecular and cellular responses to cocaine or opioids within these cell types of NAc and DS. This will be accomplished by use of in vivo CRISPR technology or RNAi to knockdown prioritized circRNAs in a cell type-specific manner and assess the influence of these circrRNAs in controlling baseline and drug-induced alterations in the excitability of the neurons as well as their transcriptional responses to drug exposure. Finally, we will investigate the ways in which drug-responsive circRNAs control cocaine and opioid self-administration behavior including relapse in mice. These highly innovative studies promise to yield fundamentally new insights into the molecular and cellular mechanisms of stimulant and opioid addiction.