PROJECT SUMMARY Stimulant use disorders (e.g., cocaine, methamphetamine) are a major public health concern. Despite a heritability of ~40-50%, genome-wide association studies (GWAS) have identified very few loci, including one hit for cocaine (COC) dependence that maps to FAM53B, a gene also identified via expression quantitative trait locus (QTL) analysis to be associated with COC self-administration in mice. The primary objective is to rapidly identify novel genetic factors in rats that contribute to premorbid risk (compulsivity, impulsivity) and cocaine use traits in a spontaneously hypertensive rat (SHR) reduced complexity cross (RCC). A rodent systems genetics approach triangulates on discovery-based genetic and multi-level functional genomic analysis and can provide a more rapid genetic and neurobiological insight into drug action and neuroplasticity underlying addiction. For several years, the contact PI has been employing mouse reduced complexity crosses (RCCs) between near-isogenic inbred substrains to facilitate gene mapping, validation, and mechanisms. Because rodent substrains are > 99% genetically identical and contain several orders of magnitude fewer variants compared to classical inbred strains, mapping quantitative trait loci (QTLs) in RCCs yields orders of magnitude fewer causal candidate genes to consider. When combined with functional genomics, RCCs can rapidly lead to causal gene and variant identification. Our preliminary studies establish robust, heritable differences in premorbid impulsivity and compulsivity, sucrose reward sensitivity, and multiple COC use traits between SHR/NCrl and SHR/NHsd substrains, including COC-induced locomotor activity, COC IVSA taking, seeking, and intake cycles, demonstrating feasibility for gene mapping in an RCC. In Aims 1 and 2, we will pioneer the use of a rat RCC where we will conduct whole genome sequencing (WGS) and map behavioral QTLs and expression QTLs (eQTLs) from nucleus accumbens (NAC) and prefrontal cortex (PFC) at the whole transcript and exon levels in an F2 cross comprising COC-trained versus yoked saline (SAL)-trained rats. In Aim 3, we will conduct proteomic analysis of PFC and NAC from COC vs. yoked SAL-trained rats to triangulate on high confidence candidate quantitative trait genes (QTGs) and variants (QTVs) as we build functional connections between DNA variants, transcriptional regulation, protein translation, and cell signaling adaptations underlying premorbid and cocaine use traits. These studies pioneer the use of a rat RCC combined with deep behavioral phenotyping to rapidly identify high-confidence candidate novel genetic factors and molecular mechanisms influencing premorbid risk factors and cocaine use traits. Future gene editing of candidate causal gene variants will be modeled on the two near-isogenic SHR backgrounds to demonstrate necessity (mutation correction; “rescue”) and sufficiency (mutation induction). Deliverables include WGS’s of SHR substrains for fut...