Extrasynaptic delta-containing GABAA receptors in the nucleus accumbens dorsomedial shell contribute to alcohol intake

Abstract

Recent findings suggest that extrasynaptic δ-subunit-containing GABA(A) receptors are sensitive to low-to-moderate concentrations of alcohol, raising the possibility that these receptors mediate the reinforcing effects of alcohol after consumption of one or a few drinks. We used the technique of viral-mediated RNAi to reduce expression of the GABA(A) receptor δ-subunit in adult rats in localized regions of the nucleus accumbens (NAc) to test the hypothesis that δ-subunit-containing GABA(A) receptors in the NAc are necessary for oral alcohol consumption. We found that knockdown of the δ-subunit in the medial shell region of the NAc, but not in the ventral or lateral shell or in the core, reduced alcohol intake. In contrast, δ-subunit knockdown in the medial shell did not affect intake of a 2% sucrose solution, suggesting that the effects of GABA(A) receptor δ-subunit reduction are specific to alcohol. These results provide strong evidence that extrasynaptic δ-subunit-containing GABA(A) receptors in the medial shell of the NAc are critical for the reinforcing effects of oral ethanol.

Fig. 1.

Confirmation of infection and δ-subunit knockdown after infusion of the Ad-shδ virus into the NAc. Rats were infused with viruses expressing shRNA to target the GABA_AR δ-subunit (Ad-shδ) or a control virus expressing a nonspecific sequence (Ad-NSS) into the NAc shell. At 18 d after infusion, brains were removed and prepared for histology (A and B), subunit expression quantification with TaqMan quantitative PCR (C), or protein quantification by Western blotting (D). (A) Presence of GFP after virus infusion into the medial, ventral, or lateral subregions of the NAc shell. (B) Sections of NAc shell after microinfusion stained with anti-GFP (green) antibodies and anti-NeuN (red; Upper) or anti-GFAP (red; Lower). (Upper) Overlap of NeuN and GFP staining (yellow) indicates infected neurons. Arrows point toward examples of Ad-shδ–infected neurons. (Lower) Little overlap of GFAP and GFP staining (yellow) indicates low level of glial infection. (C) Ad-shδ infusion reduces δ-subunit mRNA in NAc. Histogram depicts the mean ratio of δ to GAPDH ± SEM; n = 3 per group. (D) Ad-shδ infusion reduces δ-subunit protein levels. Histogram depicts the mean ratio of δ to actin ± SEM; n = 4 per group. *P < 0.05 compared with Ad-NSS.

Fig. 2.

Viral-mediated GABA_AR δ-subunit knockdown in NAc shell decreases oral alcohol intake. Rats were infused with Ad-shδ (n = 12) to reduce expression of the δ-subunit or the control Ad-NSS (n = 12) into the NAc shell, and drinking within an intermittent-access procedure was assessed. Time course of ethanol intake expressed as grams per kilogram of body weight before (B) and after (days 5–30) virus infusion. (In Figs. 2–4, “B” refers to baseline, which is defined as the average of the last 3 d before virus infusion). *P < 0.02,**P < 0.01, and ***P < 0.005 compared with Ad-NSS. Values depict mean ± SEM.

Fig. 3.

Reduction of alcohol intake by viral-mediated GABA_A R δ knockdown is localized to the medial zone of the NAc shell. Rats were infused with Ad-shδ or Ad-NSS into the medial (A and B), ventral (C and D), or lateral (E and F) NAc shell, and drinking within an intermittent-access procedure was assessed. (A) Time course of intake expressed as grams per kilogram of body weight before (B) and after (days 5–17) virus infusion into the medial NAc. Ad-shδ, n = 9; Ad-NSS, n = 8. **P < 0.005, ***P < 0.001 compared with Ad-NSS. (B) Alcohol preference [(gram of ethanol consumed/ gram of ethanol + gram of water) × 100] for the same Ad-shδ–treated rats as in A. **P < 0.005 compared with baseline. ^###P < 0.001 compared with Ad-NSS within day 17. (C) Time course of alcohol intake before (B) and after (days 5–17) virus infusion into the ventral NAc. Ad-shδ, n = 13; Ad-NSS, n = 5. (D) Alcohol preference for same Ad-shδ–treated rats as in C. (E) Time course of alcohol intake before (B) and after (days 5–17) virus infusion into the lateral NAc. Ad-shδ, n = 9; Ad-NSS, n = 5. (F) Alcohol preference for the same Ad-shδ–treated rats as in E. Values depict mean ± SEM.

Fig. 4.

GABA_AR δ knockdown in the NAc core does not affect alcohol intake and knockdown in the medial shell does not affect sucrose intake. (A) Rats were infused with Ad-shδ (n = 9) or Ad-NSS (n = 11) into the NAc core, and drinking within an intermittent-access procedure was assessed. Time course of alcohol intake before (B) and after (days 5–17) virus infusion into the NAc core. (B) Alcohol preference for same Ad-shδ–treated rats as in A. (C) Rats were infused with Ad-shδ (n = 9) or Ad-NSS (n = 7) into the NAc medial shell, and sucrose intake within an intermittent-access procedure was assessed. Time course of sucrose intake before (B) and after (days 5–17) virus infusion into the NAc medial shell. (D) Sucrose preference for same Ad-shδ–treated rats as in C. Values depict mean ± SEM.