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. Subject terms: Molecular neuroscience, Addiction Introduction Alcohol use disorder (AUD) is a chronic relapsing brain disorder and a major public health concern in the United States, where the lifetime prevalence of AUD among adults is nearly 30% [[44]1]. Notably, although AUD has been more common in men than women, women show a faster transition to dependence and suffer severe behavioral and physical impairments [[45]2, [46]3]. Despite the disorder’s prevalence and severity, our understanding of the molecular and behavioral mechanisms that drive alcohol abuse, specifically from a gender perspective, is fragmented and there are few effective treatments for alcohol abuse. One of the hallmarks of AUD is a gradual increase in alcohol consumption over time [[47]4]. This increase in alcohol intake is thought to result from neurobiological adaptation induced by repeated episodes of alcohol drinking [[48]5]. Moreover, prolonged heavy alcohol exposure appears to cause progressive dysfunction in multiple brain areas, most notably changes in neuronal plasticity in the brain’s reward and stress systems, such as in the amygdala [[49]6]. The amygdala is comprised of multiple interconnected nuclei nested deep in the temporal lobe in humans, and its structures and functions are well-conserved across species. It has been associated with both emotion and motivation, playing an essential role in processing aversive and appetitive valence [[50]7–[51]9]. Previous neuroimaging studies demonstrated that alcohol cues trigger amygdala activation which correlates with craving for alcohol in humans with AUD [[52]10, [53]11]. In animal models, chronic alcohol exposure alters neuronal transmission in the central nucleus of the amygdala (CeA), and the neural activity of the CeA during alcohol withdrawal is associated with levels of alcohol drinking in alcohol-dependent rats [[54]12, [55]13]. Furthermore, the activation of the basolateral amygdala (BLA) and its projections to the nucleus accumbens is necessary for cue-induced alcohol seeking behaviors [[56]14]. As alcohol has broad systemic and molecular targets, identifying and characterizing transcriptional responses to alcohol in a brain region-specific manner is vital to our understanding of the molecular mechanisms underlying alcohol-related behaviors and AUD development and susceptibility [[57]15–[58]18]. Several studies have applied genomics to examine alcohol-induced transcriptional effects using chronic models of voluntary ethanol consumption and forced exposure through ethanol vapor in rodents [[59]19–[60]23]. The results from these studies included molecular targets, such as alterations in neuronal function and signal transduction, indicating that chronic ethanol exposure and withdrawal have prominent actions on gene expression in multiple brain areas including the prefrontal cortex. However, these prior studies using microarrays with pre-determined numbers of genes and forced alcohol exposure have not directly addressed the genome-wide transcriptional responses to repeated voluntary alcohol drinking that leads to the escalation of alcohol intake over time. Furthermore, these studies primarily examined male animals, resulting in an incomplete understanding of the molecular and behavioral mechanisms that drive higher alcohol intake in females. In addition, few studies investigated gene networks in the amygdala that are targeted by voluntary alcohol drinking, where molecular processes may underlie the development and maintenance of alcohol-drinking and seeking behaviors [[61]24, [62]25]. To gain a better insight into gene expression alterations impacted by acute and chronic/repeated voluntary oral ethanol consumption, we subjected C57Bl/6J (B6) mice, an inbred strain that shows high alcohol consumption and preference. Specifically, we used B6 females that are known to self-administer higher amounts of alcohol than males under most conditions in order to parallel the findings from the current study with the existing literature with male mice but to differentially address alcohol-drinking driven transcriptional changes [[63]26, [64]27]. We employed a 2-bottle choice ethanol drinking procedure, in which either a single bout or chronic intermittent access that has been shown to escalate ethanol intake over weeks in mice [[65]27]. We then explored transcriptional changes in the amygdala that may underly a progressive increase in ethanol intake. We found that acute and chronic ethanol drinking induced similar network-level changes in gene expression, suggesting that a single episode of ethanol consumption substantially alters amygdala transcriptomes that may be long-lasting. Furthermore, we identified expression networks that correlated with the level of ethanol consumption and ethanol preference, suggesting mechanistic relationships between amygdala gene expression and behavioral readout. Our analyses also revealed that some of the most strongly correlated genes, including Sox17, are associated with myelination and oligodendrocyte differentiation. In addition, we used a viral vector-assisted knockdown of Sox17 gene expression in the amygdala and confirmed that Sox17 is involved in escalating alcohol intake over time. Together, our findings provide systems-level evidence of the relationships between voluntary alcohol drinking and oligodendrocyte-related gene networks within the amygdala. Materials and methods For more detail, see Supplemental Material: Supplementary Methods. Animals Two separate batches (N = 12 for the first and N = 18 for the second batches) of adult female C57BL/6J mice at 7 weeks of age were purchased from Jackson Laboratories (Bar Harbor, ME) and kept under standard conditions with 12:12 h light/dark cycle (lights on: 07:00). Animals were group housed upon arrival and acclimated for 1–2 weeks. Then, mice were individually housed and allowed access to tap water and free (ad libitum) access to standard laboratory chow during the entire experimental period. All experiments were approved by and carried out in accordance with the Institutional Animal Care and Use Committee at McLean Hospital. All experimental and animal care procedures met the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize distress and the number of animals used. Alcohol drinking procedures Twenty percent of ethanol solution (v/v) was prepared in tap water from 95% ethyl alcohol (Pharmaco-AAPER, Brookfield, CT). Mice were changed to individual housing at least 24 h before the presentation of two 50-ml plastic centrifuge tubes of water on the metal wire cage lid for 2 days for acclimation to drinking from no. 6 rubber stoppers containing stainless steel ball-bearing sippers (Ancare, Bellmore, NY). Centrifuge tubes were securely held through the metal wire cage lid and presented to mice 2 h before the dark cycle and weighed to the nearest hundredth of a gram, 24 h after the fluids were given, and the left/right position of the tubes were alternated before each ethanol drinking session to avoid side preferences. To control for spillage and