CIRCUIT- AND SEX-SPECIFIC ROLE FOR DOPAMINE IN FENTANYL RELAPSE - PROJECT SUMMARY/ABSTRACT SUBSTANCE USE DISORDER INCURS A MASSIVE TOLL ON INDIVIDUAL AND ECONOMIC HEALTH, COSTING THE UNITED STATES BILLIONS OF DOLLARS EVERY YEAR AND THOUSANDS OF LOST LIVES DUE TO FATAL OVERDOSE. IN RECENT YEARS, THIS HAS BEEN DRIVEN BY POTENT SYNTHETIC OPIOIDS SUCH AS FENTANYL IN THE ILLICIT DRUG SUPPLY, ALONGSIDE AN INCREASED PREVALENCE OF OPIOID USE DISORDER IN FEMALES, NECESSITATING IMPROVED UNDERSTANDING OF THE NEURAL MECHANISMS THAT PROMOTE FENTANYL MISUSE ACROSS SEX. A GROWING BODY OF CLINICAL AND PRECLINICAL WORK IMPLICATES MALADAPTIVE SIGNALING IN THE PRELIMBIC CORTEX (PL) AND ITS RECIPROCAL CONNECTIONS TO REWARD CENTERS IN DRIVING DRUG SEEKING BEHAVIOR. HOWEVER, THIS WORK HAS BEEN LARGELY RESTRICTED TO NON-OPIOID DRUGS AND MALE SUBJECTS, LIMITING OUR UNDERSTANDING OF WHETHER THESE MECHANISMS EXTEND TO FENTANYL MISUSE OR TO FEMALES. MY PRELIMINARY EXPERIMENTS IN BOTH SEXES INDICATE DOPAMINERGIC SIGNALING IN PL DRIVES FENTANYL-CONTEXT MEMORIES. THE GOAL OF THIS STUDY IS TO EXPAND THIS LINE OF RESEARCH TO THE MORE TRANSLATIONALLY RELEVANT SELF-ADMINISTRATION PARADIGM TO INTERROGATE THE ROLE OF PL CIRCUITS IN FENTANYL RELAPSE. IN THIS STUDY I AIM TO IDENTIFY THE PL INPUTS AND OUTPUTS THAT PROMOTE FENTANYL RELAPSE. THE OVERARCHING HYPOTHESIS IS THAT FENTANYL RELAPSE IS DEPENDENT ON DOPAMINERGIC INPUT FROM VENTRAL TEGMENTAL AREA (VTA) TO PL AND DOWNSTREAM PL PROJECTIONS TO THE NUCLEUS ACCUMBENS (NAC). IN AIM 1, I WILL IDENTIFY THE ROLE OF DOPAMINOCEPTIVE PL NEURONS IN FENTANYL RELAPSE ACROSS SEX. I WILL LEARN TO USE CELL-TYPE- AND PROJECTION-SPECIFIC CHEMOGENETICS TO BIDIRECTIONALLY MANIPULATE ACTIVITY OF DOPAMINOCEPTIVE PL NEURONS, AND THEIR DOWNSTREAM TERMINALS IN THE NAC, DURING BOTH NON-REINFORCED SEEKING AND FENTANYL-INDUCED REINSTATEMENT. IN AIM 2, I WILL DETERMINE THE NECESSITY OF VTA DOPAMINERGIC INPUT TO PL IN FENTANYL SEEKING. I WILL LEARN TO USE IN VIVO IMAGING TECHNIQUES TO DEMONSTRATE THAT DOPAMINE RELEASE IN PL DIFFERENTIALLY ENCODES FENTANYL SEEKING VERSUS NATURAL REWARD. THEN I WILL USE A DUAL- RECOMBINASE STRATEGY TO SPECIFICALLY INHIBIT DOPAMINERGIC INPUT TO PL AND ATTENUATE FENTANYL SEEKING. TOGETHER, THESE EXPERIMENTS WILL PROVIDE NOVEL INSIGHTS INTO THE SEX-SPECIFIC NEURAL MECHANISMS OF FENTANYL USE AND RELAPSE WHICH MAY TRANSLATE TO OTHER ABUSED SUBSTANCES AND INFORM FUTURE DEVELOPMENT OF ADDICTION THERAPEUTICS.