Phenotypic and Gene Expression Features Associated with Variation in Chronic Ethanol Consumption in Heterogeneous Stock Collaborative Cross Mice
Of the more than 100 studies that have examined some aspect of the relationships between excessive ethanol consumption and the brain transcriptome, relatively few rodent studies have examined the effects of chronic consumption. The current study allowed 114 heterogeneous stock collaborative cross mice to freely consume 10% ethanol for 3 months (13 weeks), with a water choice. RNA-Seq data were then used to identify transcriptional differences within the central nucleus of the amygdala, a brain region known to impact ethanol preference. Average week 1 preference for consuming ethanol over water was modestly correlated with average preference for the final week of the study, and over the course of the 3-month trial, a sex-difference emerged. In females but not males, there was a significant escalation of preference from week 1 to week 13 and significant alignment of the transcriptome with average week 13 ethanol preference and consumption was found for female, but not male mice. The genes significantly correlated with preference were enriched in annotations associated with cilium movement, cilium organization, extracellular region and collagen-containing extracellular matrix. For the females, 376 of the total 415 genes that correlated with ethanol preference in the gene co-expression network were associated with a single network module that was enriched in genes with an astrocyte annotation. The key hub node in the module was the master regulator, orthodenticle homeobox 2 (Otx2). These data support an important role for the extracellular matrix and primary cilium in individual differences in ethanol preference and consumption in a genetically diverse population of mice. Overall design: Ninety-six samples were chosen for RNA-Seq analysis, balanced for sex and quartile of ethanol preference. Samples were lost for several reasons: failed dissection, low RNA yield, poor RNA quality, failed library preparation and poor sequencing. The final group included 84 samples: 12 high preference (= 0.5) females, 8 high preference males, and  the remaining 64 were distributed across the preference range from 0 to < 0.5. The analysis of the RNA-Seq data focused on preference phenotype x expression correlations, beginning with the data for all 84 animals, then moving to the 43 females and 41 males in separate analyses. Correlations were calculated for week 1 average, week 13 average and the average across all weeks.