Genomic and Behavioral Signatures of Selection for Ethanol Preference from the Heterogeneous Stock Collaborative Cross Mice – The Central Nucleus of the Amygdala

Alcohol use disorder (AUD) is a complex disease with heritability of ~0.5, indicating genetic and non-genetic factors contribute to risk. Identifying gene expression networks contributing to risk using post-mortem human brain tissue has the limitation of conflating risk for AUD with consequences of alcohol use. We leveraged mice selectively bred for differential ethanol preference from a highly genetically diverse population to overcome this limitation. Ethanol intake was highly correlated with preference, high-preferring (HP) mice consumed more sweet- but not bitter-tasting solutions compared to low-preferring (LP) mice, and the lines did not differ in rate of ethanol elimination. Adult, ethanol-naïve HP and LP mice contributed tissue from the central nucleus of the amygdala (CeA), a region critical to ethanol preference and intake. Single-nuclei and bulk RNA sequencing data were used to identify cell types and transcriptome changes related to selective breeding for differential risk for ethanol preference. Single nuclei analysis identified populations of inhibitory (~48% of cells) and excitatory (~23%) neurons, and non-neuronal (~29%) cells, but no differences in cell-type composition or gene expression were identified between the lines. Bulk CeA analysis identified differences between the lines for: (1) gene expression (2996 genes), (2) expression variability (426 genes), and (3) wiring (407 significant gene-gene correlations). Overall, lower variance was found in the HP line. Reduced gene-gene correlation, also found in HP mice, suggested that selection for high preference induced changes in transcriptional regulation resulting in reduced connectivity, specific to gene networks enriched in markers for inhibitory neurons expressing Isl1 and Tac1. Overall design: Mice underwent bidirectional selective breeding for ethanol intake (10% ethanol v/v in water, two bottle choice) for 5 generations starting from the Heteroengeous Stock - Collaborative Cross, a strain which emcompasses ~90% of all genetic diversity known in Mus Musculus. After 5 generations, high and low preference mouse had ~10fold different prefernce for ethanol and consumed 7fold differently. Ethanol naive mice from generation 5 were sacrificied at adulthood and brain tissue was collected for the central nucleus of the amygdala, a region important for addiction. bulk RNA sequencing (paired-end) was run on 200 samples (n=50/sex/strain) to a mean depth of 20M, and aligned to the GRCm39 using STAR. Raw counts were normalized using the limma+voom method, and differential expression and weighted gene co-expression analysis were carried out to identify risk vs protection markers for ethanol drinking.