Histone deacetylases mediate GABAA receptor expression, physiology, and behavioral maladaptations in rat models of alcohol dependence

Abstract

Alcohol use disorders are chronic debilitating diseases characterized by severe withdrawal symptoms that contribute to morbidity and relapse. GABA_A receptor (GABA_AR) adaptations have long been implicated in the chronic effects of alcohol and contribute to many withdrawal symptoms associated with alcohol dependence. In rodents, GABA_AR hypofunction results from decreases in Gabra1 expression, although the underlying mechanism controlling Gabra1 expression after chronic ethanol exposure is still unknown. We found that chronic ethanol exposure using either ethanol gavage or two-bottle choice voluntary access paradigms decreased Gabra1 expression and increased Hdac2 and Hdac3 expression. Administration of the HDAC inhibitor trichostatin A (TSA) after chronic ethanol exposure prevents the decrease in Gabra1 expression and function as well as the increase in Hdac2 and Hdac3 expression in both the cortex and the medial prefrontal cortex (mPFC). Chronic ethanol exposure and withdrawal, but not acute ethanol exposure or acute withdrawal, cause a selective upregulation of HDAC2 and HDAC3 associated with the Gabra1 promoter that accompanies a decrease in H3 acetylation of the Gabra1 promoter and the reduction in GABA_AR α1 subunit expression. TSA administration prevented each of these molecular events as well as behavioral manifestations of ethanol dependence, including tolerance to zolpidem-induced loss of righting reflex, reduced open-arm time in the elevated plus maze, reduced center-time and locomotor activity in the open-field assay, and TSA reduced voluntary ethanol consumption. The results show how chronic ethanol exposure regulates the highly prominent GABA_AR α1 subunit by an epigenetic mechanism that represents a potential treatment modality for alcohol dependence.

Fig. 1

Chronic ethanol exposure-induced changes in GABA_AR expression and H3 acetylation in cerebral cortex are prevented by TSA. a Schematic outlining 14-day gavage procedure (5 g/kg, 25% EtOH) with 24 h withdrawal. TSA (2 mg/kg, i.p.) was administered on the last 3 days of the procedure. b qPCR analysis reveals that chronic ethanol exposure causes decreases in Gabra1 and increases in Gabra4 mRNA transcripts that are prevented by the administration of TSA. A significant interaction between ethanol and TSA exposure was observed for Gabra1 (F_1,20 = 19.42, p = 0.0003, n = 6) and Gabra4 (F_1,20 = 30.11, p < 0.0001, n = 6), using two-way ANOVA. Values are normalized to Gapdh, and expressed as fold control. c Representative western blots of GABA_A subunit membrane expression following chronic 14-day ethanol administration with 24 h withdrawal ± TSA treatment. d Quantification of western blots for GABA_A subunit membrane expression shown in c. A significant interaction between ethanol and TSA exposure for α1 subunit expression was found (F_1,19 = 10.53, p = 0.0043, n = 6, 6, 6, 5) using two-way ANOVA. A significant main effect of ethanol exposure on α4 subunit expression (F₁,₁₈ = 10.09, p = 0.0052, n = 6, 5, 5, 6) was found using two-way ANOVA. Values shown represent % control, normalized to β-actin. e Representative blots of H3 PTMs in the chromatin fraction after chronic ethanol exposure ± TSA treatment. f Quantification of blots shown in e. TSA prevents chronic ethanol-induced reductions in global H3 acetylation and H3K9Ac. A significant interaction was found between ethanol exposure and TSA exposure for pH3Ac (F_1,20 = 18.38, p = 0.0004, n = 6), and H3K9Ac (F_1,20 = 8.212, p = 0.0096, n = 6). There was a significant main effect of TSA exposure for H3K14Ac (F_1,18 = 24.41, p = 0.0001, n = 6, 5, 5, 6) found using two-way ANOVA. Two-way ANOVA’s were followed by Bonferroni’s post hoc tests, *p < 0.05, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM

Fig. 2

Chronic ethanol exposure increases HDAC2 and HDAC3 both in the chromatin fraction and at the Gabra1 promoter likely facilitating decreases in H3 acetylation at the Gabra1 promoter. a qPCR analysis of the 11 Hdac isoforms and the effect of TSA. Chronic ethanol administration selectively upregulates Hdac2 and Hdac3 transcript levels. A significant main effect of ethanol exposure was found for Hdac2 (F_1,20 = 13.10, p = 0.0017, n = 7, 7, 5, 5) while a significant interaction was found for Hdac3 and TSA (F_1,18 = 6.233, p = 0.0225, n = 7, 5, 5, 5) using two-way ANOVA. b Chronic ethanol exposure increases HDACs in the chromatin fraction. HDAC2 (t₁₀ = 5.733, p = 0.0002) and HDAC3 (t₁₀ = 3.605, p = 0.0048) were significantly increased using Student’s unpaired two-tailed t test. c TSA prevents changes in histone acetylation associated with the Gabra1 promoter and TSS. A significant main effect of TSA on acetylation at the Gabra1 promoter (F_1,16 = 8.840, p = 0.0090, n = 4, 5, 5, 6) and a significant interaction between ethanol exposure and TSA exposure at the Gabra1 TSS (F_1,15 = 7.570, p = 0.0148, n = 4, 5, 5, 6) was found using two-way ANOVAs. d Chronic ethanol administration and withdrawal does not change association of the H3K9me3 with the Gabra1 promoter. e ChIP assays reveal that chronic ethanol exposure increases HDAC2 association with the Gabra1 promoter, but not the Gabra4 or Gabrg2 promoters; this effect is blocked by TSA administration. There was a significant interaction between ethanol and TSA exposure for HDAC2 associated with the Gabra1 promoter (F_1,16 = 7.353, p = 0.0154, n = 5) using two-way ANOVA. f Chronic ethanol exposure increases HDAC3 enrichment at the Gabra1 promoter, but not Gabra4 or Gabrg2 promoters, and this effect is blocked by the administration of TSA. There was a significant interaction between ethanol and TSA exposure (F_1,20 = 46.05, p < 0.0001, n = 6) found using two-way ANOVA. Two-way ANOVAs were followed by Bonferroni’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM.

Fig. 3

Chronic ethanol exposure-induced changes in Gabra1 expression and GABA_A-R function in prelimbic medial PFC are prevented by TSA. a Schematic showing experimental paradigm of ethanol exposure and TSA administration, followed by PFC microdissection or mPFC slice electrophysiology. b qPCR analysis reveals that changes in Gabra1 expression caused by chronic ethanol gavage are prevented by the administration of TSA. There was a significant interaction between ethanol and TSA exposure for Gabra1 expression (F_1,20 = 12.67, p = 0.0020, n = 6). c qPCR analysis reveals that increased Hdac2 expression caused by chronic ethanol gavage is prevented by the administration of TSA. There was a significant interaction between ethanol and TSA exposure for Hdac2 expression (F_1,19 = 9.096, p = 0.0071, n = 6, 6, 6, 5). d qPCR analysis reveals that increased Hdac3 expression caused by chronic ethanol gavage is prevented by the administration of TSA. There was a significant interaction between ethanol and TSA exposure for Hdac3 expression (F_1,19 = 30.88, p < 0.0001, n = 6, 6, 6, 5). e Representative composite traces for sIPSCs measured in layer V and VI in prelimbic mPFC slices. f Changes in sIPSC decay τ1 recorded in the mPFC caused by chronic ethanol exposure are prevented by the administration of TSA. There was a significant effect of TSA on decay τ1 (F_1,48 = 12.68, p = 0.0008, n = 14, 13, 16, 9). Two-way ANOVAs were followed by Bonferroni’s post hoc tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM.

Fig. 4

Voluntary ethanol consumption alters Gabra1, Hdac2, and Hdac3 in the mPFC, and TSA prevents ethanol-induced changes in voluntary consumption. a Schematic demonstrating two-bottle intermittent ethanol access paradigm. Wistar rats were given free access to bottles of EtOH (15% w/v) and water for 24 h on Monday, Wednesday, and Friday in a reverse light-cycle room and allowed to reach baseline drinking levels (approximately eight sessions). Before session 14, rats received either a vehicle or TSA injection; on session 18, rats were given access to EPM and evaluated for anxiety-like behavior. b TSA administration decreased the amount of ethanol consumed. Each session was analyzed individually using a Student’s t test. n = 13, 14. c qPCR analysis reveals that voluntary ethanol consumption decreases Gabra1 expression and TSA administration prevents this effect. One-way ANOVA analysis found that there was a significant difference between ethanol/vehicle- and ethanol/TSA-treated animals for Gabra1 expression (F_2,18 = 43.24, p < 0.0001, n = 7). d qPCR analysis reveals that increased Hdac2 expression caused by voluntary ethanol consumption is prevented by the administration of TSA. One-way ANOVA analysis showed a significant difference between ethanol/vehicle- and ethanol/TSA-treated animals for Hdac2 expression. (F_2,15 = 1264, p < 0.0001, n = 6). e qPCR analysis reveals that increased Hdac3 expression caused by voluntary ethanol consumption is prevented by the administration of TSA. One-way ANOVA analysis found that there was a significant difference between ethanol/vehicle- and ethanol/TSA-treated animals for Hdac3 expression (F_2,15 = 454.1, p < 0.0001, n = 6). *p < 0.05, ****p < 0.0001. Data are represented as mean ± SEM.

Fig. 5

TSA prevents chronic alcohol-induced changes in zolpidem hypnosis and open-field center time, locomotor behavior, and elevated plus maze. a Schematic showing the experimental timeline of zolpidem LORR and open-field assay during gavage paradigm. b Zolpidem-induced loss of righting reflex, expressed as % control. A significant interaction between ethanol and TSA exposure (F_1,25 = 6.740, p = 0.0156, n = 8, 7, 7, 7) was found using two-way ANOVA. c Center time in open-field assay, expressed as % total locomotor activity time. A significant interaction was found between ethanol and TSA exposure (F_1,34 = 12.41, p = 0.0012, n = 10, 9, 9, 10) using two-way ANOVA. d Total locomotor activity in open-field assay. A significant interaction was found between ethanol and TSA exposure (F_1,34 = 11.05, p = 0.0021, n = 10, 9, 9, 10) using two-way ANOVA. e Schematic demonstrating two-bottle intermittent ethanol access paradigm and elevated plus maze testing-day. f On session 18, rats were tested on the elevated plus maze in lieu of ethanol access. Open-arm time was reduced in rats that consumed ethanol for 17 sessions compared to rats that never had ethanol access. TSA administration prevented decreases in open-arm time in the EPM in rats that had alcohol access. Significance was determined using one-way ANOVA (F₂,₂₄ = 7.022, p = 0.0042, n = 9, 9, 8). Results are expressed as % open-arm time out of total time. g There was no change in total locomotor activity in elevated plus maze for any exposure group. One and two-way ANOVAs were followed by Bonferroni’s post hoc tests, *p < 0.05, **p < 0.01, ****p < 0.0001. Data are represented as mean ± SEM.