PROJECT SUMMARY In this new R01 application that is directly responsive to RFA-DA-23-015, our research team proposes to study the cell type specific molecular mechanisms regulated by fentanyl and methamphetamine polysubstance use at both the initiation and withdrawal stages of the substance use trajectory. We propose to overcome current limitations in polysubstance research by utilizing preclinical assays of drug-vs.-food choice procedures in male and female rats, which more fully capture the severity of polysubstance use seen in humans by modeling the behavioral misallocation and decision making between concurrently available addictive drugs and alternative non-drug reinforcers. We will combine these enhanced behavioral models with single nuclei RNA sequencing (snRNAseq) of the medial prefrontal cortex (PFC) and nucleus accumbens (NAc), key brain regions implicated in drug reinforcement and drug-taking, to capture and characterize the exact drug-induced molecular adaptations that occur in specific cell types, including non-neuronal cells. We will directly test the hypothesis that the synergistic action of combined fentanyl and methamphetamine use produces enhanced drug use behaviors and brain molecular adaptations that are distinct from what is achieved by either fentanyl or methamphetamine use alone; that this polysubstance synergy involves unique transcriptional adaptations by brain region, accumulates as a function of drug experience, and contributes to the behavioral misallocation towards drug use over more beneficial rewarding activities that is the hallmark of drug addiction. We will test this overarching hypothesis in two Aims. In Aim 1, we will uncover the impact of fentanyl/methamphetamine polysubstance use during the withdrawal phase of the substance use trajectory. We will use drug-vs.-food self- administration choice procedures for saline, fentanyl alone, methamphetamine alone, and fentanyl/methamphetamine combinations to uncover how an extended history of polysubstance use synergizes to increase somatic withdrawal effects and drug taking behavior while experiencing withdrawal. We will then perform snRNAseq in the PFC and NAc to interrogate the brain cell type specific transcriptional adaptations in these rats. In Aim 2, we will similarly perform drug-vs.-food choice procedures and snRNAseq of these brain regions to explore the emergence of behavioral and transcriptional adaptations at the of initiation of drug use experience. We will go on to compare our snRNAseq data from Aims 1 and 2 to understand how the fentanyl/methamphetamine polysubstance cell type transcriptional profile changes over the substance use trajectory. This project will reveal how fentanyl and methamphetamine synergize to produce maladaptive drug choice behaviors and brain cell type specific transcriptional responses at distinct stages of the substance use trajectory that are common barriers to recovery. Results gained from this project will inform the discovery o...