Phenotypic and gene expression features associated with variation in chronic ethanol consumption in heterogeneous stock collaborative cross mice

Abstract

Of the more than 100 studies that have examined relationships between excessive ethanol consumption and the brain transcriptome, few rodent studies have examined chronic consumption. Heterogeneous stock collaborative cross mice freely consumed ethanol vs. water for 3 months. Transcriptional differences were examined for the central nucleus of the amygdala, a brain region known to impact ethanol preference. Early preference was modestly predictive of final preference and there was significant escalation of preference in females only. Genes significantly correlated with female preference were enriched in annotations for the primary cilium and extracellular matrix. A single module in the gene co-expression network was enriched in genes with an astrocyte annotation. The key hub node was the master regulator, orthodenticle homeobox 2 (Otx2). These data support an important role for the extracellular matrix, primary cilium and astrocytes in ethanol preference and consumption differences among individual female mice of a genetically diverse population.

Keywords: Addiction; Alcohol; Drinking; Ethanol preference; RNA-Seq; Sex differences.

Figure 1.. Ethanol preference during the first week of a chronic 3-month drinking trial modestly predicts ethanol preference in the final week.

Correlations are shown for (A) females and (B) males for the first (Week 1) and last (Week 13) weeks of the ethanol drinking trial. Pearsons correlations were significant (ps < 0.001). The solid line is the regression with the dotted lines representing the 95% confidence intervals. N=58 for females; N=56 for males

Figure 2.. In females, there was a significant increase in preference from week 1 to week 13, whereas there was no significant change in males.

Shown are means ± SEM for ethanol preference ratio of all 58 females and 56 males during the first (week1) and final (week 13) weeks of the 3-month ethanol drinking trial. N=58 for females; N=56 for males. ***p < 0.0001

Figure 3.. Initial ethanol preference greater than or equal to 0.5 is partially predictive of higher preference in final week of drinking.

Shown are individual preference ratios for (A) females and (B) males that had preference ratios ≥ 0.5 during the first week of drinking. Six out of 7 females had a preference ratio at this level in week 13, compared to 3 of 5 males. Assigned subject numbers are given in the figure legends.

Figure 4.. For some individual mice, ethanol preference escalated across the weeks of the 3-month trial.

Shown are individual preference ratios for (A) females and (B) males that had initial preference ratios < 0.5 during the first week of drinking that increased to ratios ≥ 0.5 in the final week. Assigned subject numbers are given in the figure legends.

Figure 5.. There was considerable individual animal variation in total amount of ethanol consumed during the 3-month drinking period, with females consuming more than males.

Frequency distributions for females and males representing the number of mice with levels of ethanol intake summed across 3 months. The range of values was 41 – 1508 g/kg (537 ± 45 g/kg for females; 313 ± 30 g/kg for males). N=58 for females; N=56 for males

Figure 6.. Principal components analysis separates RNA-Seq data for the central nucleus of the amygdala into two components for mice with varying levels of ethanol preference.

Component 1 (PC1) accounted for 21% of the variance and was formed from 33 female and 31 male samples. Component 2 (PC2) accounted for 19% of the variance and was formed from 10 female and 10 male samples.

Figure 7.. Composite gene-gene interaction network for differential expression in the CeA, associated with differences in ethanol preference in female HS-CC mice.

This network was created using GeneMANIA [80] and derived from 43 hub nodes (inner circle) found within the brown module, enriched in genes with an astrocyte annotation. The genes in the outer circle were identified by GeneMANIA [80] as functional associates, with symbol size, based on number of network connections. Otx2 (circled in red) was a top hub node and 19 other top hub nodes (circled in green) were down-regulated when Otx2 was absent. Blue lines represent known colocalization (5.47%) and tan lines represent predicted interactions (9.11%). The majority of network interactions are accounted for by co-expression (84.97%), which is the basis for the network. Since representation of co-expression obstructs visualization of colocalization and predicted interactions, co-expression is shown in Supplementary Information Figure S1.