Alcohol Dependence in Rats Is Associated with Global Changes in Gene Expression in the Central Amygdala

Abstract

Alcohol dependence is associated with adverse consequences of alcohol (ethanol) use and is evident in most severe cases of alcohol use disorder (AUD). The central nucleus of the amygdala (CeA) plays a critical role in the development of alcohol dependence and escalation of alcohol consumption in dependent subjects. Molecular mechanisms underlying the CeA-driven behavioral changes are not well understood. Here, we examined the effects of alcohol on global gene expression in the CeA using a chronic intermittent ethanol (CIE) vapor model in rats and RNA sequencing (RNA-Seq). The CIE procedure resulted in robust changes in CeA gene expression during intoxication, as the number of differentially expressed genes (DEGs) was significantly greater than those expected by chance. Over-representation analysis of cell types, functional groups and molecular pathways revealed biological categories potentially important for the development of alcohol dependence in our model. Genes specific for astrocytes, myelinating oligodendrocytes, and endothelial cells were over-represented in the DEG category, suggesting that these cell types were particularly affected by the CIE procedure. The majority of the over-represented functional groups and molecular pathways were directly related to the functions of glial and endothelial cells, including extracellular matrix (ECM) organization, myelination, and the regulation of innate immune response. A coordinated regulation of several ECM metalloproteinases (e.g., Mmp2; Mmp14), their substrates (e.g., multiple collagen genes and myelin basic protein; Mbp), and a metalloproteinase inhibitor, Reck, suggests a specific mechanism for ECM re-organization in response to chronic alcohol, which may modulate neuronal activity and result in behavioral changes, such as an escalation of alcohol drinking. Our results highlight the importance of glial and endothelial cells in the effects of chronic alcohol exposure on the CeA, and demonstrate further insight into the molecular mechanisms of alcohol dependence in rats. These molecular targets may be used in future studies to develop therapeutics to treat AUD.

Cell-type specific changes in PKC-delta neurons of the central amygdala during alcohol withdrawal

“…Bulk transcriptomics studies have demonstrated that alcohol exposure can alter gene expression in the CeA [20][21][22][23][24], and it is hypothesized that these changes underlie escalated alcohol consumption in dependent animals. These studies have implicated several novel biological pathways and molecular mechanisms that may serve as targets for treatment or future research.…”

“…These studies have implicated several novel biological pathways and molecular mechanisms that may serve as targets for treatment or future research. For example, alcohol alters the expression of genes that are associated with CRF signaling, cell activation, and synaptic transmission [20,22]. While these bulk transcriptome studies have been critical in identifying molecular targets of alcohol action, they lack the cell-type specific context required to better understand circuitlevel molecular mechanisms.…”

“…For example, microglia are required for escalation of drinking that is associated with alcohol dependence, and eliminating them perturbs GABAergic and glutamatergic gene expression in the CeA [23]. Bulk sequencing experiments have identified affected cells based on deconvolution and enrichment analyses [20]. However, direct, cell-type specific characterization of the CeA has not been reported.…”

Subregional Differences in Alcohol Modulation of Central Amygdala Neurocircuitry

“…Transcriptomic analysis showed that systemic downregulation of microglia does not result in changes in alcohol intake, however, the combination of the downregulation of microglia and chronic alcohol resulted in an upregulation of reactive astrocyte genes (Warden et al, 2021). A recent gene expression study has also shown that astrocyte-specific genes were differentially expressed in the CeA after chronic intermittent alcohol exposure compared to control group (Kisby et al, 2021). Overall, the aforementioned studies suggest that alcohol exposure can increase the expression of astrocyte-specific genes and that CeA astrocytes may be critical regulators of initial alcohol effects on CeA circuit function and behavior.…”

“…Mediates the mechanism of acute and chronic alcohol on glutamatergic transmission in the CeA (Kisby et al, 2021;Melkumyan et al, 2022) Microglia Mediates the mechanism of chronic alcohol on glutamatergic and GABAergic transmission in the CeA with no effect on alcohol drinking (Warden et al, 2020(Warden et al, , 2021Nelson et al, 2021) Pro-inflammatory cytokines Pro-inflammatory cytokines generally increase CeA GABAergic transmission;…”