Dynamic regulation of CeA gene expression during acute and protracted abstinence from chronic binge drinking of male and female C57BL/6J mice

Abstract

Binge alcohol consumption is a major risk factor for developing Alcohol Use Disorder (AUD) and is associated with alcohol-related problems like accidental injury, acute alcohol poisoning, and black-outs. While there are numerous brain regions that have been shown to play a role in this AUD in humans and animal models, the central nucleus of the amygdala (CeA) has emerged as a critically important locus mediating binge alcohol consumption. In this study, we sought to understand how relative gene expression of key signaling molecules in the CeA changes during different periods of abstinence following bouts of binge drinking. To test this, we performed drinking in the dark (DID) on two separate cohorts of C57BL/6J mice and collected CeA brain tissue at one day (acute) and 7 days (protracted) abstinence after DID. We used qRTPCR to evaluate relative gene expression changes of 25 distinct genes of interest related to G protein-coupled receptors (GPCRs), neuropeptides, ion channel subunits, and enzymes that have been previously implicated in AUD. Our findings show that during acute abstinence CeA punches collected from female mice had upregulated relative mRNA expression of the gamma-aminobutyric acid receptor subunit alpha 2 (Gabra2), and the peptidase, angiotensinase c (Prcp). CeA punches from male mice at the same time point in abstinence had upregulated relative mRNA encoding for neuropeptide-related molecules, neuropeptide Y (Npy) and somatostatin (Sst), as well as the neuropeptide Y receptor Y2 (Npyr2) but downregulated, Glutamate ionotropic receptor NMDA type subunit 1 (Grin1). After protracted abstinence CeA punches collected from female mice had increased mRNA expression of corticotropin releasing hormone (Crh) and Npy. While CeA punches collected from male mice at the same timepoint had upregulated relative mRNA expression of Npy2r and downregulated mRNA expression of Gabra2, Grin1 and opioid receptor kappa 1 (Oprk1). Our findings support that there are differences in how the CeA of male and female respond to binge-alcohol exposure, highlighting the need to understand the implications of such differences in the context of AUD and binge drinking behavior.

Figure 1:. Drinking in the Dark Alcohol Intake.

A) Schematic highlighting the experimental set up where tissue punches from the CeA of C57BL6/J mice were obtained at acute abstinence (1 day of abstinence following DID) or 7 protracted abstinence (7 days of abstinence following DID). B) Weekly alcohol intake of both male and female mice that underwent 3 cycles of DID and acute abstinence (1 day of abstinence following DID), male (n=10), female (n=10). C) Cumulative alcohol intake g/kg during the 3 cycles of DID of male (n=10), and female (n=10), mice that underwent 3 cycles of DID and acute abstinence (1 day of abstinence following DID). D) Weekly alcohol intake of both male and female mice that underwent 3 cycles of DID and protracted abstinence (7 days of abstinence following DID), (male n=10, females n=9-10). E) Cumulative alcohol intake g/kg during the 3 cycles of DID of male (n=10) and female (n=9) mice that underwent 3 cycles of DID and protracted abstinence (7 days of abstinence following DID). Male are represented as filled green triangles; female are represented as open green circles. Error bars are depicted as ±SEM. The following symbols represent, *= p-value < 0.05, **= p-value < 0.01, depicting statistically significance.

Figure 2:. Male relative gene expression relative to housekeeping gene Actin B via qPCR.

(A-D) Statistically significant relative gene expression levels of CeA derived genes from mice that underwent 1 day of abstinence following DID compared to the respective water group. (E-H) Statistically significant relative gene expression levels of CeA derived genes from mice that underwent protracted abstinence (7 days of abstinence following DID) compare to the water group. Error bars are depicted as ±SEM. The following symbols represent, *= p-value < 0.05, **= p-value < 0.01, depicting statistically significance. Water group n=9-10, DID group n=9-10.

Figure 3:. Female relative gene expression relative to housekeeping gene Actin B via qPCR:

(A-B) Statistically significant relative gene expression levels of CeA derived genes from mice that underwent acute abstinence (1 day of abstinence after DID) compared to the water group. (C-D) Statistically significant relative gene expression levels of CeA derived genes from mice that underwent protracted abstinence (7 days of abstinence after DID) compared to the water group. Error bars are depicted as ±SEM. The following symbols represent, *= p-value < 0.05, **= p-value < 0.01, depicting statistically significance. Water group n=9-10, DID group n=9-10.

Figure 4:. Male Pearson correlation of relative gene expression.

A) Pearson correlation matrix of relative gene expression of genes of interest in the CeA of male mice that only received water. The CeA was isolated at the same time as the acute abstinence group (1 day of abstinence after DID after DID). B) Pearson correlation matrix of relative gene expression of CeA genes of male mice that underwent acute abstinence (1 day of abstinence after 3 cycles of DID). C) Pearson correlation matrix of relative gene expression of CeA genes of interest of male mice that only received water. The CeA of these was isolated at the same time as the protracted abstinence group (7 days of abstinence after DID). D) Pearson correlation matrix of relative gene expression of CeA genes of male mice that underwent protracted abstinence (7 days of abstinence after 3 cycles of DID). The following symbols represent, *= p-value < 0.05, ^= p-value < 0.01, @= p-value < 0.001, depicting statistically significant correlation.

Figure 5:. Female Pearson correlation of relative gene expression.

A) Pearson correlation matrix of relative gene expression of genes of interest in the CeA of female mice that only received water. The CeA was isolated at the same time as the acute abstinence group (1 day of abstinence after DID after DID). B) Pearson correlation matrix of relative gene expression of CeA genes of female mice that underwent acute abstinence (1 day of abstinence after 3 cycles of DID). C) Pearson correlation matrix of relative gene expression of CeA genes of interest of female mice that only received water. The CeA of these was isolated at the same time as the protracted abstinence group (7 days of abstinence after DID). D) Pearson correlation matrix of relative gene expression of CeA genes of female mice that underwent protracted abstinence (7 days of abstinence after 3 cycles of DID). The following symbols represent, *= p-value < 0.05, ^= p-value < 0.01, @= p-value < 0.001, depicting statistically significant correlation.