Chronic Ethanol Consumption Alters Glucocorticoid Receptor Isoform Expression in Stress Neurocircuits and Mesocorticolimbic Brain Regions of Alcohol-Preferring Rats

Abstract

Evidence suggests the hypothalamic-pituitary-adrenal (HPA) axis is involved in Alcohol Use Disorders (AUDs), which might be mediated by an imbalance of glucocorticoid receptor (GR), GRα and GRβ, activity. GRβ antagonizes the GRα isoform to cause glucocorticoid (GC) resistance. In the present study, we aimed to investigate the effects of chronic continuous free-choice access to ethanol on GR isoform expression in subregions of the mesocorticolimbic reward circuit. Adult male alcohol-preferring (P) rats had concurrent access to 15% and 30% ethanol solutions, with ad lib access to lab chow and water, for six weeks. Quantitative Real-time PCR (RT-PCR) analysis showed that chronic ethanol consumption reduced GRα expression in the nucleus accumbens shell (NAcsh) and hippocampus, whereas ethanol drinking reduced GRβ in the nucleus accumbens core (NAcc), prefrontal cortex (PFC), and hippocampus. An inhibitor of GRα, microRNA-124-3p (miR124-3p) was significantly higher in the NAcsh, and GC-induced gene, GILZ, as a measure of GC-responsiveness, was significantly lower. These were not changed in the NAcc. Likewise, genes associated with HPA axis activity were not significantly changed by ethanol drinking [i.e., corticotrophin-releasing hormone (Crh), adrenocorticotrophic hormone (Acth), and proopiomelanocortin (Pomc)] in these brain regions. Serum corticosterone levels were not changed by ethanol drinking. These data indicate that the expression of GRα and GRβ isoforms are differentially affected by ethanol drinking despite HPA-associated peptides remaining unchanged, at least at the time of tissue harvesting. Moreover, the results suggest that GR changes may stem from ethanol-induced GC-resistance in the NAcsh. These findings confirm a role for stress in high ethanol drinking, with GRα and GRβ implicated as targets for the treatment of AUDs.

Keywords: GRα; GRβ; P rats; ethanol; nucleus accumbens.

Fig. 1.

Ethanol consumption in P-rats and serum corticosterone level. (A) Chronic ethanol drinking paradigm: Adult male P rats were given free choice, continuous access to water, 15% and 30% ethanol for 6 weeks. The control group was given water only. Both control and ethanol groups were single housed with ad libitum access to food, and animals were euthanized at the end of week 6. (B) Ethanol intake (g/kg) over 6 weeks in P rats (n = 8) with continuous access to 15 and 30% ethanol in water. (C) Analysis of serum corticosterone levels in P rats. Unpaired t-test revealed no significant difference between the ethanol and water-control group (Mean ± SEM, p > 0.05).

Fig. 2.

Expression of total GR, GRα, GRβ, CRH, and GILZ mRNA in the NAc-core and NAc-shell of the ethanol and water-control P rat groups. (A) Left: GRβ mRNA expression was significantly downregulated in the NAc-core of the ethanol group (unpaired t-test, Mean ± SEM, p < 0.05), while total GR was not changed. Right: GRα/GRβ ratio was significantly higher in the ethanol group compared with the water-control group (unpaired t-test, ± SEM, p < 0.05). (B) microRNA-124–3p (rno-miR124–3p) expression was not changed in the NAc-core. (C) Tsc22d3 (GILZ) and Crh mRNA expression was not changed in the NAc-core. (D) Left: GRα mRNA level was significantly downregulated in the NAc-shell of the ethanol group compared with the water-control group (unpaired t-test, Mean ± SEM, p < 0.001), and total GR (Nr3c1) mRNA expression was significantly decreased (unpaired t-test, Mean ± SEM, p < 0.001) in the ethanol group as well. Right: the GRα/GRβ ratio was not changed in the ethanol vs the water-control group. (E) microRNA-124–3p (rno-miR124–3p) expression was significantly (unpaired t-test, Mean ± SEM, p < 0.01) increased in the NAc-shell. (F) Tsc22d3 (GILZ) and Crh mRNA expression was not changed in the NAc-shell. (unpaired t-test, Mean ± SEM, p < 0.05).

Fig. 3.

Expression of total GR, GRα, GRβ, and CRH mRNA in the PL-PFC and IL-PFC in ethanol and water-control P rat groups. (A) Left: GRα and GRβ mRNA expression were not significantly changed in PL-PFC of the ethanol group compared with the water-control group (unpaired t-test, Mean ± SEM, p > 0.05). Right: GRα/GRβ ratio was not significantly changed in the PL-PFC. (B) Crh mRNA expression was also not significantly changed in the PL-PFC of the ethanol vs control group. (C) Left: GRα mRNA levels did not change, whereas GRβ mRNA expression was reduced in the IL-PFC of the ethanol compared with the water-control group (unpaired t-test, Mean ± SEM, p > 0.05, *p < 0.05). Right: GRα/GRβ ratio was not significantly changed. (D) Crh mRNA expression was not significantly changed in IL-PFC of ethanol and control groups.

Fig. 4.

Expression of total GR, GRα, GRβ in the hippocampus and amygdala, in addition to CRH in the amygdala of ethanol-drinking and water-control rats. (A) Left: GRα, GRβ, and total (Nr3c1) GR mRNA expression were significantly downregulated in the hippocampus of the ethanol group as compared with the control group (unpaired t-test, Mean ± SEM, p < 0.05). Right: The GRα/GRβ ratio was not changed in the ethanol group vs the water-control group. (B) GRα, GRβ, and total (Nr3c1) GR mRNA expression were unchanged in the amygdala of the ethanol group compared with the water-control group (unpaired t-test, Mean ± SEM, p > 0.05). (C) Crh mRNA expression was not changed in the amygdala.

Fig. 5.

Expression of total GR, GRα, GRβ, and POMC in the hypothalamus and pituitary gland, as well as CRH in the hypothalamus of the ethanol-drinking and water-control rats. (A) Left: total (Nr3c1) GR, Grα, and GRβ mRNA expression were not significantly changed in the hypothalamus (unpaired t-test, Mean ± SEM, p > 0.05). Right: GRα/GRβ ratio was not changed in the ethanol group compared with the water-control group. (B) Crh mRNA expression was not significantly changed in the hypothalamus following ethanol consumption. (C) Pomc mRNA expression in the hypothalamus was not significantly changed by ethanol drinking. (D) Left: GRα and GRβ mRNA expression were not significantly changed in the pituitary gland of the ethanol-drinking group vs the water-control group (unpaired t-test, ± SEM, p > 0.05). Right: GRα/GRβ ratio remained unchanged in the ethanol group compared with the water-control group. (E) Pomc mRNA expression was not significantly changed in the pituitary gland following chronic ethanol drinking.