Binge Ethanol Drinking Produces Sexually Divergent and Distinct Changes in Nucleus Accumbens Signaling Cascades and Pathways in Adult C57BL/6J Mice

Abstract

We previously determined that repeated binge ethanol drinking produced sex differences in the regulation of signaling downstream of Group 1 metabotropic glutamate receptors in the nucleus accumbens (NAc) of adult C57BL/6J mice. The purpose of the present study was to characterize RNA expression differences in the NAc of adult male and female C57BL/6J mice following 7 binge ethanol drinking sessions, when compared with controls consuming water. This binge drinking procedure produced high intakes (average >2.2 g/kg/30 min) and blood ethanol concentrations (average >1.3 mg/ml). Mice were euthanized at 24 h after the 7th binge session, and focused qPCR array analysis was employed on NAc tissue to quantify expression levels of 384 genes in a customized Mouse Mood Disorder array, with a focus on glutamatergic signaling (3 arrays/group). We identified significant regulation of 50 genes in male mice and 70 genes in female mice after 7 ethanol binges. Notably, 14 genes were regulated in both males and females, representing common targets to binge ethanol drinking. However, expression of 10 of these 14 genes was strongly dimorphic (e.g., opposite regulation for genes such as Crhr2, Fos, Nos1, and Star), and only 4 of the 14 genes were regulated in the same direction (Drd5, Grm4, Ranbp9, and Reln). Interestingly, the top 30 regulated genes by binge ethanol drinking for each sex differed markedly in the male and female mice, and this divergent neuroadaptive response in the NAc could result in dysregulation of distinct biological pathways between the sexes. Characterization of the expression differences with Ingenuity Pathway Analysis was used to identify Canonical Pathways, Upstream Regulators, and significant Biological Functions. Expression differences suggested that hormone signaling and immune function were altered by binge drinking in female mice, whereas neurotransmitter metabolism was a central target of binge ethanol drinking in male mice. Thus, these results indicate that the transcriptional response to repeated binge ethanol drinking was strongly influenced by sex, and they emphasize the importance of considering sex in the development of potential pharmacotherapeutic targets for the treatment of alcohol use disorder.

Keywords: C57BL/6J mice; alcohol; hormone signaling; immune function; neurotransmitter metabolism; qPCR arrays; sex differences.

FIGURE 1

Binge ethanol intake (A,B) and blood ethanol concentration (BEC, C) in male and female mice. Mice in the binge groups had a total of seven binge drinking sessions, with a binge session every 3rd day (A). BEC was measured at the end of the 3rd (day 9) and 7th (day 21) binge sessions. Although overall binge ethanol intake, averaged across the seven sessions, did not differ in the female (2.34 g/kg) and male (2.35 g/kg) mice, ethanol intake and corresponding BECs were lower in the male vs. female mice on the final binge session (day 21, B,C). However, BECs greatly exceed the criteria for binge drinking on all days (0.80 mg/mL; depicted by dashed line in C). Shown are mean ± SEM for all mice in the binge groups (n = 9/sex), which included the mice in the subgroup that were used for the qRT-PCR analysis (n = 4/sex), and for the mice in the subgroup that were used for the qPCR arrays (n = 3/sex). ⁺p = 0.06, ^∗p < 0.05, ^∗∗p < 0.01 vs. respective female all or female array group.

FIGURE 2

Heat map and hierarchical cluster analysis of genes significantly regulated by repeated binge drinking in male and female mice. All significantly regulated genes (106 total) were used to generate the heat map and to perform the cluster analysis to visualize the transcriptional response at 24 h after the 7th binge ethanol drinking session. Each column represents the combined data from six arrays (three binge, three control), with the binge ethanol-induced change in expression shown for males and females in separate columns. Shades of color indicate up-regulation (red) or down-regulation (blue) for a particular gene following binge drinking. Hierarchical cluster analysis was performed on the ethanol regulated genes. Genes (represented by rows, names at the right of the image) were clustered according to the similarity of expression profile as the result of repeated binge drinking. Clustering of genes emphasizes the sexually dimorphic response to repeated binge drinking experience. ^∗p < 0.05 for significantly regulated genes (binge vs. control).

FIGURE 3

Simplified corticotropin releasing hormone (CRH) signaling pathway highlights genes influenced by repeated binge drinking in male and female mice. This canonical pathway was identified by IPA as regulated by binge drinking in both males and females. (A) Depicts the CRH signaling pathway and highlights genes regulated by binge drinking in males (pink for up-regulation, green for down-regulation). (B,C) Depict qRT-PCR results and show that expression of Gnaq (B which encodes Gαq) tended to be higher in males vs. females and to be decreased by binge ethanol drinking in males. Expression of Mapk1 (C which encodes Erk2) was significantly higher in males vs. females and was significantly decreased by binge drinking in both sexes. Values are the mean ± SEM for 4/sex/treatment. ⁺p < 0.07 vs. respective control in males or sex difference (over horizontal line); ^∗p < 0.05 for main effect of sex (over horizontal line), ^∗∗p < 0.01 for main effect of treatment. (D) Shows significant regulation by binge drinking of select genes from the qPCR array analysis that are pertinent to the CRH signaling cascade depicted in (A) for male and female mice (↑ for up-regulation, ↓ for down regulation; p < 0.05 at a minimum). For statistical trends, the p-values are provided. Gnaq (guanine nucleotide binding protein, alpha q polypeptide) encodes the protein Gαq. Mapk1 (mitogen-activated protein kinase 1) encodes the protein ERK2 (extracellular signal-regulated kinase).

FIGURE 4

Simplified neuropathic pain signaling pathway highlights genes influenced by repeated binge drinking in male and female mice. This canonical pathway was identified by IPA as regulated by binge drinking in both males and females. (A) Depicts the neuropathic pain signaling pathway and highlights genes regulated by binge drinking in males (pink for up-regulation, green for down-regulation). (B,C) Depict qRT-PCR results and show that expression of Ntrk2 (B which encodes TrkB) was significantly higher in males than in females. Expression of Elk1 (C which encodes transcription factor Elk1) was significantly decreased by binge drinking in both sexes. Values are the mean ± SEM for 4/sex/treatment. ^∗p < 0.05 for main effect of treatment, ^∗∗p = 0.01 for main effect of sex (over horizontal line). (D) Shows significant regulation by binge drinking of select genes from the qPCR array analysis in male and female mice that are pertinent to the neuropathic signaling cascade depicted in (A) (top 10 genes) or that are pertinent to effects on other receptor systems (↑ for up-regulation, ↓ for down regulation; p ≤ 0.05 at a minimum). For statistical trends, the p-values are provided. Ntrk2 (neurotrophic tyrosine kinase, receptor, type 2) encodes the protein TrkB (tropomyosin receptor kinase B). Elk1 (ELK1, member of ETS oncogene family) encodes the transcription factor Elk1.

FIGURE 5

Simplified tumor necrosis factor receptor 2 (TNFR2) signaling pathway highlights genes influenced by repeated binge drinking in male and female mice. This canonical pathway was identified by IPA as regulated by binge drinking in both males and females. (A) Depicts the TNFR2 signaling pathway and highlights genes regulated by binge drinking in females (pink for up-regulation, green for down-regulation). (B–F) Depict qRT-PCR results. Expression of Tnfrsf1a (B which encodes TNFR1 and forms a heterocomplex with TNFR2) was significantly higher in males vs. females. However, Map3k14 (D encodes NIK) and Ikbkap (F encodes IKAP) expression was significantly decreased by binge drinking in both males and females, whereas expression of Traf2 (C encodes TRAF2) and Ikbkg (E encodes IKK-γ) trended toward a decrease by binge drinking in both sexes. Values are the mean ± SEM for 4/sex/treatment. ⁺p < 0.09, ^∗p < 0.05, ^∗∗∗p < 0.001 for main effect of treatment, ^∗∗p < 0.01 for main effect of sex (over horizontal line). (G) Shows significant regulation by binge drinking of select genes from the qPCR array analysis in male and female mice that are pertinent to the TNFR2 signaling cascade depicted in (A) (↑ for up-regulation, ↓ for down regulation; p < 0.05 at a minimum). For statistical trends, the p-values are provided. Tnfrsf1a (tumor necrosis factor receptor superfamily, member 1a) encodes TNFR1, which is a member of the TNF receptor superfamily of proteins. Traf2 (TNF receptor-associated factor 2) encodes TRAF2. Map3k14 (mitogen-activated protein kinase kinase kinase 14) encodes NIK. Ikbkg (inhibitor of kappaB kinase gamma) encodes IKK-γ or NEMO, which is one of three subunits that forms the IκB kinase (IKK) enzyme complex. Ikbkap (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) encodes IKAP.