Genome-wide transcriptomics of the amygdala reveals similar oligodendrocyte-related responses to acute and chronic alcohol drinking in female mice

Abstract

Repeated excessive alcohol consumption is a risk factor for alcohol use disorder (AUD). Although AUD has been more common in men than women, women develop more severe behavioral and physical impairments. However, relatively few new therapeutics targeting development of AUD, particularly in women, have been validated. To gain a better understanding of molecular mechanisms underlying alcohol intake, we conducted a genome-wide RNA-sequencing analysis in female mice exposed to different modes (acute vs chronic) of ethanol drinking. We focused on transcriptional profiles in the amygdala including the central and basolateral subnuclei, brain areas previously implicated in alcohol drinking and seeking. Surprisingly, we found that both drinking modes triggered similar changes in gene expression and canonical pathways, including upregulation of ribosome-related/translational pathways and myelination pathways, and downregulation of chromatin binding and histone modification. In addition, analyses of hub genes and upstream regulatory pathways revealed that voluntary ethanol consumption affects epigenetic changes via histone deacetylation pathways, oligodendrocyte and myelin function, and the oligodendrocyte-related transcription factor, Sox17. Furthermore, a viral vector-assisted knockdown of Sox17 gene expression in the amygdala prevented a gradual increase in alcohol consumption during repeated accesses. Overall, these results suggest that the expression of oligodendrocyte-related genes in the amygdala is sensitive to voluntary alcohol drinking in female mice. These findings suggest potential molecular targets for future therapeutic approaches to prevent the development of AUD, due to repeated excessive alcohol consumption, particularly in women.

Fig. 1. Experimental design, fluid consumption levels, and initial assessment of sequencing results.

A Experimental design and timeline. B Ethanol intake over 24 h on water/20% EtOH drinking days. C EtOH preference ratios. D BEC measured in Acute and Chronic Drinking groups of 2nd cohort of mice on Day 28. E Correlation of BEC to the amount of ethanol consumed by mice in Acute and Chronic Drinking groups on Day 28. Data are mean ± SEM. **p < 0.01 and ****p < 0.0001 difference between groups. F Diagram for amygdala tissue collection. G PCA plot showing no separation on alcohol drinking condition over the first two principal components. H Bar graphs showing the estimated cell type abundance for seven relevant cell types as determined by cell type deconvolution analysis. Each bar represents a single sample.

Fig. 2. GO enrichment analysis of DEGs in response to Acute and Chronic alcohol drinking.

The ordinate represents the GO terms, the upper abscissa indicates the number of genes in the GO terms, and the lower abscissa indicates the level of significance of the enrichment (gray bar, FDR = 0.01). A Genes were categorized with the Biological Process domain. B Genes were categorized with the Cellular Component domain.

Fig. 3. Downregulation of Sox17 in the amygdala reduces alcohol consumption.

A Schematic illustrating the surgical strategy with a lentivirus encoding Sox17-shRNA. B Alcohol intake of mice bilaterally injected with either control or Sox17-shRNA lentivirus into the amygdala. The injection was conducted 2 weeks before subjecting the animals to EOD paradigm. C Changes of Sox17 expression were examined by qPCR in Sox17 shRNA- or control lentivector-injected mice. D Downregulation of Sox17 expression does not lead to significant changes in anxiety (time spent in the center of an open field) or locomotion (speed and travel distance). Data are mean ± SEM. **p < 0.01 and ****p < 0.0001 difference between groups.