Histone Deacetylase Inhibitor Suberanilohydroxamic Acid Treatment Reverses Hyposensitivity to γ-Aminobutyric Acid in the Ventral Tegmental Area During Ethanol Withdrawal

Abstract

Background: The ventral tegmental area (VTA) is important for alcohol-related reward and reinforcement. Mouse VTA neurons are hyposensitive to γ-aminobutyric acid (GABA) during ethanol (EtOH) withdrawal, and GABA responsiveness is normalized by in vitro treatment with histone deacetylase inhibitors (HDACi). The present study examined the effect of a systemically administered HDACi, suberanilohydroxamic acid (SAHA) on GABA sensitivity, and related molecular changes in VTA neurons during withdrawal after chronic EtOH intake in rats.

Methods: Sprague Dawley male adult rats were fed with Lieber-DeCarli diet (9% EtOH or control diet) for 16 days. Experimental groups included control diet-fed and EtOH diet-fed (0- or 24-hour withdrawal) rats treated with either SAHA or vehicle injection. Single-unit recordings were used to measure the response of VTA neurons to GABA. Immunohistochemistry was performed to examine levels of HDAC2, acetylated histone H3 lysine 9 (acH3K9), and GABA_A receptor α1 and α5 subunits in the VTA; quantitative polymerase chain reaction was performed to examine the mRNA levels of HDAC2 and GABA_A receptor subunits.

Results: VTA neurons from the withdrawal group exhibited GABA hyposensitivity. In vivo SAHA treatment 2 hours before sacrifice normalized the sensitivity of VTA neurons to GABA. EtOH withdrawal was associated with increased HDAC2 and decreased acH3K9 protein levels; SAHA treatment normalized acH3K9 levels. Interestingly, no significant change was observed in the mRNA levels of HDAC2. The mRNA levels, but not protein levels, of GABA_A receptor α1 and α5 subunits were increased during withdrawal.

Conclusions: Withdrawal from chronic EtOH exposure results in a decrease in GABA-mediated inhibition, and this GABA hyposensitivity is normalized by in vivo SAHA treatment. Disruption of signaling in the VTA produced by alteration of GABA neurotransmission could be 1 neuroadaptive physiological process leading to craving and relapse. These results suggest that HDACi pharmacotherapy with agents like SAHA might be an effective treatment for alcoholism.

Keywords: SAHA; VTA; Dopamine; Epigenetic; Ethanol.

Figure 1

Effects of ethanol (EtOH) withdrawal and SAHA on GABA inhibition of ventral tegmental area (VTA) neurons. (A) Ratemeter graphs of single‐neuron firing rates: Each graph represents the firing rate of a single VTA neuron over time; vertical bars are proportional to the firing rate over 5‐second intervals. The duration of application of each GABA concentration (in μM) is indicated by horizontal bars. Neurons were recorded in brain slices obtained from EtOH‐withdrawn rats after chronic EtOH exposure. Slices were made 2 hours after i.p. injection of SAHA or vehicle. Addition of GABA to the extracellular medium resulted in a dose‐dependent reduction in firing in all of these neurons, although the responsiveness of the VTA neuron from the Withdrawal+Vehicle (WV)–treated rat was less than the responsiveness of VTA neurons from rats in the other treatment groups. (B) Mean concentration–response curves: Rats were randomly assigned into 4 groups as described in the Methods: GABA (50 to 500 μM) inhibited VTA neurons in a concentration‐dependent manner, 3‐way ANOVA, F(2, 111) = 35.0, main GABA effect, p < 0.001. VTA neurons of rats from the WV group (filled triangle) showed significant reduction in sensitivity to GABA, compared to Control+Vehicle (CV) and Withdrawal+Vehicle (WS) groups, 3‐way ANOVA, F(1, 111) = 6.78 for (EtOH diet * SAHA) effect, p < 0.01; Tukey post hoc WV different from CV and WS p < 0.05.

Figure 2

Effects of SAHA treatment on acetylated histone H3 lysine 9 (acH3K9) protein expression in ventral tegmental area (VTA) during ethanol (EtOH) withdrawal using gold immunolabeling. (A) Representative photomicrographs (scale bar = 50 μm) showing acH3K9 gold immunolabeling in the VTA. Sprague Dawley rats were randomly assigned to 5 groups: control Lieber‐DeCarli diet with SAHA (CS) or vehicle (CV) injections, or 9% EtOH Lieber‐DeCarli diet for 16 days with vehicle injection (EV), or EtOH diet for 15 days followed by 24‐hour withdrawal and either SAHA (WS) or vehicle (WV) injections. Brains were collected 2 hours after i.p. injection of SAHA or vehicle. (B) Bar diagram showing mean (± SEM) acH3K9 immunolabeling in the VTA. There was a significant decrease, 1‐way ANOVA, F(4, 20) = 6.85, p < 0.002, Tukey post hoc comparison *p < 0.05, n = 5, of acH3K9 protein in the VTA of rats withdrawn from chronic EtOH (WV), compared with all the other groups (CV, CS, EV, and WS).

Figure 3

Changes in HDAC2 expression in ventral tegmental area (VTA) during ethanol (EtOH) withdrawal. (A) Representative photomicrographs (scale bar = 50 μm) showing HDAC2 gold immunolabeling in the VTA. The same treatment groups were used for HDAC2 protein measurement as those for the acetylated acH3K9. Sprague Dawley rats were randomly grouped to CV, CS, EV, WV, and WS. Brains were collected 2 hours after i.p. injection of SAHA (6.25 mg/ml, 50 mg/kg) or vehicle. (B) Bar diagram showing mean (± SEM) HDAC2 immunolabeling in the VTA. There was a significant increase in HDAC2, 1‐way ANOVA, F(4, 20) = 7.891, p < 0.002, Tukey post hoc comparison *p < 0.05, n = 5, in the VTA of rats withdrawn from chronic EtOH (WV and WS) compared to controls (CV and CS) or to EtOH diet‐fed (EV) rats. (C) Bar diagram showing mean HDAC2 mRNA expression in the VTA. Three treatment groups were used: control diet fed (C), EtOH diet fed for 16 days (E), and 24 hours withdrawal from 15 days of EtOH diet (W). No significant difference between treatment groups was observed (1‐way ANOVA; p > 0.05, n = 10).

Figure 4

GABA_A receptor subunits α1 and α5 mRNA and protein level expression in the ventral tegmental area (VTA). Five treatment groups were used: control fed with vehicle injection (CV), control fed with SAHA injection (CS), ethanol (EtOH) fed with vehicle injection (EV), EtOH fed with 24‐hour withdrawal and vehicle injection (WV), EtOH fed with 24‐hour withdrawal and SAHA injection (WS), as described in the Methods section. (A) Bar diagram showing mean GABA_A receptor α1 subunit (Gabra1) mRNA expression in the VTA. There was a significant increase, 1‐way ANOVA, F(4, 17) = 4.478, p < 0.05; Tukey's post hoc test *p < 0.05, n = 4 to 5, of Gabra1 mRNA in the VTA of rats withdrawn from chronic EtOH (WV), compared to CS, EV, and WS groups. (B) Bar diagram showing mean GABA_A receptor α5 subunit (Gabra5) mRNA expression in the VTA. There was a significant increase, 1‐way ANOVA, F(4, 17) = 4.823, p < 0.01; Tukey's post hoc test *p < 0.05, n = 4 to 5, of Gabra5 mRNA in the VTA of rats withdrawn from chronic EtOH (WV), compared to CS and EV treatment groups. (C) Bar diagram showing mean GABA_A receptor α1 subunit protein levels in the VTA. No significant difference between treatment groups was observed (1‐way ANOVA, p > 0.05, n = 5). (D) Mean GABA_A receptor α5 subunit protein levels in the VTA. No significant difference between treatment groups was observed (1‐way ANOVA p > 0.05, n = 5).