RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release

Abstract

The firing of mesolimbic dopamine neurons is important for drug-induced reinforcement, although underlying genetic factors remain poorly understood. In a recent genome-wide association metaanalysis of alcohol intake, we identified a suggestive association of SNP rs26907 in the ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) gene, encoding a protein that mediates Ca(2+)-dependent activation of the ERK pathway. We performed functional characterization of this gene in relation to alcohol-related phenotypes and mesolimbic dopamine function in both mice and adolescent humans. Ethanol intake and preference were decreased in Rasgrf2(-/-) mice relative to WT controls. Accordingly, ethanol-induced dopamine release in the ventral striatum was blunted in Rasgrf2(-/-) mice. Recording of dopamine neurons in the ventral tegmental area revealed reduced excitability in the absence of Ras-GRF2, likely because of lack of inhibition of the I(A) potassium current by ERK. This deficit provided an explanation for the altered dopamine release, presumably linked to impaired activation of dopamine neurons firing. Functional neuroimaging analysis of a monetary incentive-delay task in 663 adolescent boys revealed significant association of ventral striatal activity during reward anticipation with a RASGRF2 haplotype containing rs26907, the SNP associated with alcohol intake in our previous metaanalysis. This finding suggests a link between the RASGRF2 haplotype and reward sensitivity, a known risk factor for alcohol and drug addiction. Indeed, follow-up of these same boys at age 16 y revealed an association between this haplotype and number of drinking episodes. Together, these combined animal and human data indicate a role for RASGRF2 in the regulation of mesolimbic dopamine neuron activity, reward response, and alcohol use and abuse.

Fig. 1.

Ethanol intake is reduced in Rasgrf2^−/− mice relative to WT controls. (A) Mean (+ SEM) intake of solutions containing increasing concentrations of ethanol in a two-bottle free choice test over a 3-d period (n = 12–13). A two-way ANOVA revealed a significant effect of genotype (F_1,23 = 21.5; P < 0.0001) and ethanol concentration (F_3,69 = 8.4; P < 0.001) as well as a significant genotype × concentration interaction effect (F_3,69 = 8.4; P < 0.0001) on ethanol intake. (B) Mean (± SEM) drinking patterns of a 12% ethanol solution in the home cage over a 3-d period (n = 8–9). A two-way ANOVA revealed a significant genotype (F_1,12 = 70.4; P < 0.00001), circadian (F_17,204 = 9.3; P < 0.00001), and genotype × circadian (F_17,204 = 8.1; P < 0.00001) effect. *P < 0.05.

Fig. 2.

No impairment in acute ethanol-induced ERK activation in the VS of Rasgrf2^−/− mice compared with littermate controls. Acute ethanol-evoked dopamine release in the VS is significantly reduced in Rasgrf2^−/− mice. (A) Mean (+ SEM) phospho-ERK–positive cells in mouse nucleus accumbens (average between shell and core) after an i.p. injection of either saline or ethanol (1 g/kg; n = 5–7 per group). A two-way ANOVA showed a significant effect of treatment (F_1,19 = 14.0, P = 0.0014) but not genotype (F_1,19 = 1.9) and no interaction (F_1,19 = 0.7). (B) Mean (SEM) basal extracellular dopamine levels (n = 6–7). (C) Mean (+ SEM) extracellular dopamine levels after acute ethanol (2 g/kg i.p.; as indicated by arrow) injection. Data are expressed as percentage relative to basal dopamine levels at 20-min intervals (n = 6–7). A two-way ANOVA revealed a significant effect of genotype (F_1,7 = 12.8, P = 0.009) and time (F_12,84 = 2.9, P = 0.002) as well as a significant genotype × time interaction (F_12,84 = 2.8, P = 0.0029) for extracellular dopamine levels. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Reduced excitability of mesolimbic dopaminergic neurons in Rasgrf2^−/− mice relies on ERK-dependent modulation of I_A currents. (A) Sample traces and pooled data of current injection evoked action potentials in mutant and WT mouse brain slices (n = 5–7). A two-way ANOVA revealed a significant effect of genotype (F_1,12 = 5.09, P < 0.05). (B) Sample traces and pooled data of action potentials obtained on current injections in slices pretreated with ERK inhibitors. A two-way ANOVA revealed a significant treatment effects of inhibitors PD098059 (F_1,17 = 6.4, P < 0.05) and SL327 (F_1,15 = 4.9, P < 0.05; n = 7–8). (C) I_A measured in WT brain slices pretreated with the ERK inhibitor or vehicle (t₁₉ = 3.1, P < 0.01; n = 7–10).

Fig. 4.

Whole-brain analysis of reward anticipation and reward feedback. (A) Positive BOLD response (PBR) during reward anticipation and reward feedback (FWE P value < 0.05). The location of the VS (±15, 9, −9; 9-mm radius) is depicted in blue. (B) Whole-brain analysis of the association of RASGRF2 haplotype and BOLD response during reward anticipation. This analysis revealed a significant activation in the precentral gyrus that extended into the anterior cingulate cortex. The figure shows activation in the precentral gyrus (FWE-corrected, P = 0.008; 6, 16, 49; t = 4.37; k = 58) with a subpeak in the anterior cingulate gyrus (−6, −1, 43).