GIRK3 gates activation of the mesolimbic dopaminergic pathway by ethanol

Abstract

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.

Keywords: Kcnj9; Kir3.3; alcohol; reward; ventral midbrain.

Fig. 1.

Ethanol-induced ataxia, sedation, hypothermia, and withdrawal hyperexcitability in WT and GIRK3 KO mice. (A) Testing timeline for results presented in B–F. (B) Mice were injected with ethanol (2 g/kg i.p.), and the time course of ethanol clearance was determined by repeatedly measuring BAL. (C). Mice were injected with saline or ethanol (1.5 g/kg i.p.) and placed on an accelerating Rotarod 30 min later. Black stars indicate the effect of ethanol. P ≤ 0.001, two-way ANOVA. (D) Mice were injected with ethanol (4 g/kg i.p.) and monitored until they regained their righting reflex, at which time BAL was measured. (E) Body temperature was measured before and after the injection of saline (triangles) or ethanol (4 g/kg i.p., circles). Black stars indicate the effect of treatment. P ≤ 0.001, repeated-measures ANOVA. (F) HICs were scored before and after the injection of saline (triangles) or ethanol (4 g/kg i.p., circles). White stars indicate the effect of genotype. P ≤ 0.05, Mann–Whitney U test. The effect of ethanol is described in the text. The number of mice in each subgroup is indicated on the graphs; the numbers shown in F apply to E as well. Error bars represent SEM.

Fig. 2.

Ethanol binge-like drinking on constitutive GIRK3 deletion and virally mediated expression in the VTA. (A) Testing timeline for results presented in B–E. (B) WT and GIRK3 KO mice were subjected to 2-h sessions of 2BC ethanol drinking. Average ethanol intake and preference for ethanol over the final three sessions are shown. White stars indicate the effect of genotype. P ≤ 0.001, t test. (C) WT and GIRK3 KO mice were subjected to continuous 2BC ethanol drinking. Average ethanol intake and preference over the last 3 d are shown. (D) WT and GIRK3 KO mice were subjected to 4-h sessions of single-bottle ethanol drinking, and intake was measured at 2 h into the session and again at the end of the session. Average intake over the last three sessions before LV vector infusion is shown. White stars indicate the effect of genotype. P ≤ 0.01, t test. (E) WT and GIRK3 KO mice injected with an LV vector expressing YFP-GIRK3 or GFP (negative control) in the VTA were subjected to 4-h sessions of single-bottle ethanol drinking. Average ethanol intake during the first 2 h of the session and over the last three sessions before (preoperative) and three sessions after (postoperative) LV vector infusion were analyzed separately. White stars indicate the effect of genotype. P ≤ 0.05, two-way ANOVA. Gray stars indicate the effect of vector. P ≤ 0.05, two-way ANOVA. The number of mice in each subgroup is indicated on the graphs. Error bars represent SEM. (F and G) Transduction of VTA neurons by the YFP-GIRK3 LV vector. (F) Single chromogenic in situ hybridization. GIRK3 and YFP labeling shows the area of virally mediated overexpression, which overlaps with the anatomic boundaries of the VTA highlighted by TH labeling. (Scale bar: 400 μm.) (G) Double-fluorescent in situ hybridization. The TH probe (green signal) labels DA neurons, and the YFP probe (red signal) labels cells expressing the transgene. The LV vector transduced both DA (white arrows) and non-DA (open arrows) cells within the VTA. (Scale bar: 50 μm.)

Fig. 3.

Ethanol-induced excitation of VTA neurons in WT and GIRK3 KO mice. (A) Representative cell-attached recordings from WT (Left) and GIRK3 KO (Right) VTA neurons before (Upper) and during (Lower) superfusion of ethanol (100 mM). (Scale bar: 500 ms.) (B) Baseline firing frequency of WT and GIRK3 KO VTA neurons. (C) Ethanol-induced change in firing frequency in WT and GIRK3 KO VTA neurons. The interaction between genotype and ethanol dose was significant (F_2,38 = 3.9; P ≤ 0.05, two-way ANOVA). Post hoc analysis: black stars indicate the effect of ethanol dose (compared with 25 mM); white stars indicate the effect of genotype (one star, P ≤ 0.05; three stars, P ≤ 0.001). The number of mice in each group is indicated on the graph. Error bars represent SEM.

Fig. 4.

Ethanol-induced release of DA in the NAc of WT and GIRK3 KO mice. (A) Baseline extracellular DA levels in the NAc of WT and GIRK3 KO mice. (B) Following a 40-min baseline period, mice were injected with ethanol (2 g/kg i.p.), and DA levels in the NAc were measured over 90 min postinjection. Samples were collected in 10-min fractions. Post hoc analysis: black stars indicate the effect of ethanol (compared with baseline); white stars indicate the effect of genotype (one star, P ≤ 0.05). The number of mice in each group is indicated on the graph. Error bars represent SEM. (C) Placement of microdialysis probes targeted to the NAc of WT mice (black bars) and GIRK3 KO mice (white bars).