Project Summary Alcohol use disorder (AUD) and pain are major public health problems affecting millions of people in the United States. More than 14 million Americans met the diagnostic criteria for AUD in 2019, and 50 million people in the United States were estimated to have a chronic pain condition. These public health crises are related, as people experiencing pain often report using alcohol to manage pain symptoms, and people with chronic pain are at an elevated risk for developing AUD. Drinking alcohol to alleviate pain is a maladaptive strategy and may lead to withdrawal-induced hyperalgesia, peripheral neuropathy, and AUD. While alcohol confers analgesic effects in a dose-dependent manner, genetic differences mean that not every person experiences this analgesia to the same degree. Previous research has revealed moderate-to-strong heritability (h2=0.42) for ethanol-induced analgesia in C57BL/6J and DBA/2J mice, but the genes underlying this relationship are still poorly characterized. Studies in rodent models have revealed evidence that opioid receptor systems, GIRK2 potassium channels, and GABAergic signaling pathways all have roles in ethanol analgesia, but no whole- genome interrogations have been conducted. This project aims to identify candidate genes modulating alcohol- induced analgesia using complementary genetic, behavioral and transcriptomic experiments in genetically informative BXD mouse populations. By mapping quantitative trait loci (QTL) for both basal and post-ethanol hot plate latency with BXD strains, we will identify genomic loci linked with variation in ethanol-induced analgesia. As an additional genome-wide approach, prefrontal cortex and periaqueductal gray samples from high- and low-ethanol analgesia BXD strains will be studied by RNA-seq. We will perform weighted gene co- expression network analysis (WGCNA) to identify differentially expressed genes in these two analgesia groups. By combining evidence from behavioral, statistical, and molecular methodologies, we will select the strongest ethanol analgesia candidate gene for experimental verification using either overexpression or knockout mouse models. This proposal comprises an investigative progression from identifying candidate genes by QTL mapping, to elucidating gene expression patterns in critical brain regions, to validating ethanol analgesia candidate genes. It represents an outstanding training opportunity for the candidate to design and perform a suite of systems genetics and animal behavioral experiments and contribute to the scientific understanding of substance-induced analgesia.