Opioid Use Disorder is known to have a heritable component, but the genetics underlying this heritability are not well understood. Inbred laboratory mice provide a stable genetic platform useful for interrogating the genetic component of specific opioid related behaviors, with the goal of identifying novel biology underlying opioid phenotypes. In this work we used inbred mice, leveraging both the near isogenic nature of substrains and the strain-specific inheritance of ancestral haplotypes, to explore the genetics of opioid phenotypes. Through these studies we found 1) Robust, female-specific increases in oxycodone state-dependent reward learning and whole brain concentrations of the oxycodone metabolite oxymorphone in BALB/cJ vs BALB/cByJ substrains. Oxymorphone is a full agonist at the mu opioid receptor, with a higher potency and efficacy than oxycodone, and thus, increased brain oxymorphone could enhance state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. Cis-expression QTL analysis (eQTL), exon-level eQTL analysis, liver and brain proteomics identified Zhx2 as candidate underlying these differences. 2) In a study of ancillary phenotypes, BALB/cByJ mice showed enhanced sensitivity to thermal nociception and mechanical stimulation and decreased brain weight versus BALB/cJ. We identified a QTL on chromosome 13 for hot plate sensitivity and a QTL on chromosome 5 for brain weight. Cis-eQTL mapping identified H2afy and spliceome analysis revealed differential H2afy exon usage. Whole brain proteomics further supported H2afy as a candidate gene for thermal nociception and identified Acads as a candidate for reduced brain weight. 3) Using a panel of 29 inbred mice we assessed behavioral variation and associations within opioid sensitivity, reward, antinociception, and dependence. We identified very little correlation between measures, suggesting distinct genetic components regulating theses facets of OUD. This study is preliminary to GWAS analysis and identification of loci underlying phenotypic variation. In summary, inbred mice, combined with a multi-omic approach, provide a powerful tool to identify genetic loci and candidate genes underlying complex behaviors. Future studies will validate Zhx2 and H2afy through reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains and involve GWAS analysis using oxycodone strain survey data.

The liver is an essential organ for cholesterol homeostasis. If this process becomes dysregulated, cardiovascular disease (CVD) develops. Zinc-fingers and homeoboxes 2 (Zhx2) as an important hepatic gene regulator and contributes to CVD. BALB/cJ mice, with mutant Zhx2 allele, have fewer atherosclerotic plaques compared to other strains on a high fat diet. In my dissertation, I focused on the liver phenotype in BALB/cJ mice on a high-fat diet and found increased liver damage compared to wild-type Zhx2 mice. These data indicates that reduced Zhx2 in the liver leads to CVD resistance, but increases liver damage. Therefore, Zhx2 has an important role in lipid metabolism and liver function. Hepatic alpha-fetoprotein (AFP) is expressed abundantly in the fetal liver and repressed after birth regulated through three enhancers (E1, E2, and E3). E3 activity is restricted to a single layer of hepatocytes surrounding central veins (pericentral region) along with glutamine synthetase (GS). In my dissertation, I explore pericentral gene regulation in the adult liver. A GS enhancer (GSe) also exhibits pericentral activity which, along with E3, is regulated by the β-catenin signaling pathway. Orphan receptors, Rev-erbα, Rev-erbβ, and RORα, contribute to E3 activity elucidating a potential mechanism for zonation.