Dopamine D3 receptor is necessary for ethanol consumption: an approach with buspirone

Abstract

Mesolimbic dopamine (DA) controls drug- and alcohol-seeking behavior, but the role of specific DA receptor subtypes is unclear. We tested the hypothesis that D3R gene deletion or the D3R pharmacological blockade inhibits ethanol preference in mice. D3R-deficient mice (D3R(-/-)) and their wild-type (WT) littermates, treated or not with the D3R antagonists SB277011A and U99194A, were tested in a long-term free choice ethanol-drinking (two-bottle choice) and in a binge-like ethanol-drinking paradigm (drinking in the dark, DID). The selectivity of the D3R antagonists was further assessed by molecular modeling. Ethanol intake was negligible in D3R(-/-) and robust in WT both in the two-bottle choice and DID paradigms. Treatment with D3R antagonists inhibited ethanol intake in WT but was ineffective in D3R(-/-) mice. Ethanol intake increased the expression of RACK1 and brain-derived neurotrophic factor (BDNF) in both WT and D3R(-/-); in WT there was also a robust overexpression of D3R. Thus, increased expression of D3R associated with activation of RACK1/BDNF seems to operate as a reinforcing mechanism in voluntary ethanol intake. Indeed, blockade of the BDNF pathway by the TrkB selective antagonist ANA-12 reversed chronic stable ethanol intake and strongly decreased the striatal expression of D3R. Finally, we evaluated buspirone, an approved drug for anxiety disorders endowed with D3R antagonist activity (confirmed by molecular modeling analysis), that resulted effective in inhibiting ethanol intake. Thus, DA signaling via D3R is essential for ethanol-related reward and consumption and may represent a novel therapeutic target for weaning.

Figure 1

In the two-bottle choice paradigm, D₃^−/− mice show a lower voluntary ethanol intake as compared with wild-type (WT). D₃ pharmacological antagonism inhibits ethanol intake in WT mice. (a, b) Voluntary ethanol intake was measured every 24 h, for 44 days, in WT (n=30) and D₃^−/− (n=30) mice that had free access to water and ethanol solution (10%). (c) Shows total fluid intake that was not different in the two groups. (d, e), Voluntary ethanol intake was measured as in a, but in mice that had received the day before and kept receiving daily i.p. injection of either saline (vehicle, VEH, n=10), U99194A (n=10) or SB277011A (n=10), either drug at 10 mg/kg. (f) Total fluid intake in either group that was not affected by drug treatment. ***p<0.001 vs water or vehicle (VEH). One-way ANOVA and Newman–Keuls post hoc test.

Figure 2

In the drinking in the dark (DID) paradigm, D₃^−/− mice show a lower ethanol intake as compared with their wild-type (WT) littermates. The D₃ antagonist SB277011A inhibits ethanol intake of WT but not in D₃^−/− mice. (a) DID was measured, for 4 days, in WT (n=12) and D₃^−/− (n=12) mice that had limited access (2 h/day for 3 days and 4 h the 4th day) to ethanol solution (20%). (b, c) Voluntary ethanol intake was measured as in a, but in mice that had received the day before and kept receiving daily i.p. injection of either saline (vehicle, VEH, n=10), or SB277011A (n=10), at 10 mg/kg. *p<0.05, **p<0.01, ***p<0.001 vs wild-type (WT) or vehicle (VEH). One-way ANOVA and Newman–Keuls post hoc test.

Figure 3

Chronic ethanol intake induces D₃ upregulation, associated with the activation of BDNF/RACK1 pathway. Abundance of transcripts in striatum was assessed by quantitative RT-PCR after 44 days of free access to water only (white columns), or to both water and ethanol (black columns, upper panels) or forced ethanol intake (black columns lower panels). In the forced alcohol-drinking procedure (d–h), D₃R^−/− and WT received 10% ethanol only, with or without SB277011A or buspirone for 14 days. (a, f) D₃ Expression profile in WT; (b, d, g) brain-derived neurotrophic factor (BDNF) expression profile in WT and D₃^−/−; (c, e, h) RACK1 expression profile in WT and D₃^−/−. Mean fold changes are expressed relative to transcript levels in controls (WT having access to water only). Each column is the mean (± SEM) from five different samples. *p<0.05, **p<0.01 vs water. One-way ANOVA and Newman–Keuls post hoc test.

Figure 4

The selective TrKB antagonist, ANA-12 reverses ethanol intake of WT mice and induces D₃ receptor downregulation but does not change ethanol intake of D₃^−/− mice. (a, b and c) Voluntary ethanol intake was measured every 24 h, for 34 days, in WT (n=30) and D₃^−/− (n=20) mice that had free access to water and ethanol solution (10%). At day 31, mice received daily i.p. injection of either vehicle (VEH), or ANA-12 at 0.5 mg/kg. (d, e) Drinking in the dark (DID) was measured, for 4 days, in WT (n=9) and D₃^−/− (n=9) mice that had limited access (2 h/day for 3 days and 4 h the 4th day) to ethanol solution (20%), daily injected with vehicle or ANA-12 1 h before the test. *p<0.05, **p<0.01, ***p<0.001 vs VEH, one-way ANOVA and Newman–Keuls post hoc test. (f) The abundance of transcripts of D₃ receptor in striatum was assessed by quantitative RT-PCR in WT mice exposed to chronic voluntary ethanol intake. Mean fold changes are expressed relative to transcript levels in controls. The abundance of phosphorylated TrkB was assessed by immunoblot, in the striatum WT treated with ANA-12 and exposed to the voluntary ethanol intake. Bars show mean (± SEM). **p<0.01, ***p<0.001 vs vehicle. One-way ANOVA and Newman–Keuls post hoc test.

Figure 5

Buspirone inhibits ethanol intake in WT mice both in the two bottle choice and DID paradigm. (a, b) Voluntary ethanol intake was measured every 24 h, for 44 days, in WT (n=20) and D₃^−/− (n=20) mice that had free access to water and ethanol solution (10%). Mice received for 14 days, from day 31, daily i.p. injection of either vehicle (VEH) or buspirone at 1 mg/kg. (c) Total fluid intake that was not changed by buspirone. *p<0.05, ***p<0.01 vs VEH. One-way ANOVA and Newman–Keuls post hoc test. (d) The dose ranging of buspirone (0.1, 1, 3, and 10 mg/kg) in WT mice exposed to the drinking in the dark (DID) paradigm. DID was measured, for 4 days, in WT (n=33) that had limited access (2 h/day for 3 days and 4 h the 4th day) to ethanol solution (20%). *p<0.05, **p<0.01 vs VEH. One-way ANOVA and Newman–Keuls post hoc test. (e) The effect of the selective 5-HT_1A agonist, 8-OH-DPAT in DID paradigm. 8-OH-DPAT at 1 mg/kg did not change ethanol intake. (f) The action on 5-HT_1A of 3 mg/kg buspirone was compared with 1 mg/kg 8-OH-DPAT by assessing the pharmacologically induced hypothermia. ***p<0.001 vs VEH. One-way ANOVA and Newman–Keuls post hoc test.

Figure 6

DA receptor signaling is enhanced in striatum of D₃R^−/− mice and of SB277011A-treated WT mice. The abundance of phosphorylated DARPP-32 (Thr 34) (a) and phosphorylated GSK3β (Ser 9) (b) was assessed by immunoblot, in the striatum of WT mice exposed to the long-term voluntary ethanol intake (white columns) and injected i.p. for 14 days with either vehicle or 10 mg/kg SB277011A and in D₃^−/− (black columns). Brain tissues were taken 1 h after the last administration of either vehicle or SB277011A. Bar graphs show mean (± SEM) of intensities normalized against the respective non-phosphorylated protein. Each column is the mean (± SEM) from five different samples. *p<0.05 vs control (vehicle-injected WT). Two-way ANOVA and Newman–Keuls post hoc test.