The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors

Abstract

Uncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders.

Figure 1.

Systemic and self-administration of alcohol increases H-Ras activity in the NAc of rodents. A, B, Mice were systemically treated with alcohol (2.5 g/kg, i.p.) or saline, and the NAc was removed 15 min (A) or 24 h (B) later. C, The NAc of rats lever pressing for 20% alcohol and control rats that were never exposed to alcohol were collected at the end of the last 30 min operant session. Active GTP-bound H-Ras was isolated by a pull-down assay using Raf-1 RBD agarose beads. The levels of H-Ras-GTP and total H-Ras were determined by Western blot analysis. Optical density quantification of H-Ras is expressed as the ratio of H-Ras-GTP to the total H-Ras protein. Data are presented as mean ± SEM, and expressed as percentage of control. Two-tailed unpaired t test, *p < 0.05, **p < 0.01; n = 9 (A), n = 6 (B), and n = 4 (C) per group.

Figure 2.

Systemic and self-administration of alcohol increase GRF1 phosphorylation in the NAc of rodents. Mice (A, B) and rats (C) were exposed to alcohol as described in Figure 1, and the levels of pSer⁹¹⁶GRF1 as well as total GRF1 were determined by Western blot analysis. The level of GRF1 phosphorylation is expressed as the ratio of the phosphorylated protein to the total protein and presented as percentage of control. B, A correlation between the level of H-Ras-GTP (y-axis; Fig. 1A) and phosphor-Ser⁹¹⁶-GRF1 (x-axis; A) in the NAc of mice following systemic administration of saline (left) or alcohol (right). Correlation was analyzed by linear regression, the effect size (r² value) was calculated, and the p value was determined. Two-tailed unpaired t test, **p < 0.01; n = 9 per group (A) and one-tailed unpaired t test, *p < 0.05; n = 4 per group (B).

Figure 3.

Infection of mice NAc with lentivirus expressing shRNA that targets H-Ras produces a downregulation of the protein. Ltv-shH-Ras1 (A, B), Ltv-shH-Ras2 (C), Ltv-NS (A, B) or Ltv-SCR (C) were bilaterally infused at a titer of 4 × 10⁷ pg/ml into the NAc of mice. Analyses were conducted at the end of the behavioral experiments (8 weeks after the surgeries) described in the Figure 4 legend. A, Ltv-shH-Ras1 infects NAc neurons. Left, Depicts the specificity of the site of infection, slices were costained with anti-GFP and anti-NeuN antibodies. Scale bar, 1 mm. Right, Depicts costaining of GFP with NeuN (top) or GFAP (bottom). Scale bar, 50 μm. B, C, Ltv-shH-Ras1 (B) or Ltv-shH-Ras2 (C) decreases the protein level of H-Ras in the NAc. The level of H-Ras was determined by Western blot analysis using anti-H-Ras antibody (left, top), and GAPDH immunoreactivity was used as an internal loading control (left, bottom). Right, Histogram depicts the mean ratio of H-Ras/GAPDH ± SEM and data are expressed as the percentage of control (Ltv-NS infected mice). Two-tailed unpaired t test, *p < 0.05, **p < 0.01; n = 7–8 (B), n = 7 (C) per group.

Figure 4.

Downregulation of H-Ras expression in the NAc of mice reduces alcohol intake. A, B, Knockdown of H-Ras in the NAc of mice reduces alcohol intake. Mice were infused with Ltv-shH-Ras1 (A), Ltv-shH-Ras2 (B), or the control viruses Ltv-NS (A) or Ltv-SCR (B) in the NAc. Four weeks later, animals were tested in a continuous-access 20% alcohol two-bottle choice drinking protocol. Mice were allowed access to two bottles, one containing a 20% alcohol solution (v/v) and one containing tap water for 2 weeks. Aa, Ba, Average of daily alcohol intake per week. Ab, Bb, Average of daily water intake per week. Ac, Bc, Average of daily alcohol preference per week. Alcohol preference was calculated as the percentage of alcohol solution consumed relative to total fluid intake (alcohol + water). C, Knockdown of H-Ras in the NAc of mice does not alter intake of saccharin or quinine. Saccharin or quinine intake was each provided for 3 successive days. D, Knockdown of H-Ras in the NAc of mice attenuates binge drinking of alcohol. Mice were infused with Ltv-shH-Ras1 or Ltv-SCR into the NAc. Four weeks later, mice had access to a 20% alcohol solution (v/v) for 4 h every other day for 2 weeks. Alcohol intake was measured at the end of each 4 h session, and presented as average of daily alcohol intake per week. Data are presented as mean ± SEM (A, B, D) Newman–Keuls post hoc test and method of contrasts (C). Two-tailed unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001; n = 12 per group (A, C, D), n = 13–14 per group (B).

Figure 5.

Inhibition of farnesyl transferase activity in the rat NAc reduces excessive drinking of alcohol. A–D, Intra-NAc infusion of FTI-276 reduces binge and sustained drinking of alcohol. Rats were trained to consume a 20% alcohol solution in the intermittent-access two-bottle choice drinking paradigm. Vehicle (Veh) or FTI-276 (10 or 100 ng/side) was infused into the NAc 3 h before the beginning of the drinking session, and alcohol intake was measured after 30 min (A) and 24 h (B). Water consumption was measured at the end of the 24 h session (C), and alcohol preference (D) was calculated as described in the legend of Figure 4. E, Schematic drawings of coronal sections of the rat brain showing the placement of bilateral injection sites in the NAc. Histologically reconstructed sites of infusion localized in the NAc of rats that were included in the analysis of experiments. White circles refer to vehicle-treated group, gray circles refer to rats infused with FTI-276 (10 ng/side), and black circles refer to rats infused with FTI-276 (100 ng/side). Data are presented as mean ± SEM. Newman–Keuls post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle; n = 7–8 per group.

Figure 6.

Inhibition of H-Ras activity in the NAc reduces operant self-administration of alcohol in rats. Rats with a history of high levels of alcohol consumption in an intermittent-access 20% alcohol two-bottle choice drinking paradigm were trained to orally self-administer a solution of 20% alcohol in an operant procedure on an FR3 schedule. Three hours before the beginning of a 30 min session, FTI-276 (100 ng/side) was infused into the NAc of rats. A, Number of active lever presses during the 30 min operant alcohol self-administration session. B, Cumulative mean presses in bins of 2 min, indicative of the rate of presses for alcohol during the session. C, Latency to the first alcohol delivery (0.1 ml of 20% alcohol/delivery) obtained during the 30 min session. D, Latency to the last alcohol delivery. E, Schematic drawings of coronal sections of the rat brain showing the placement of bilateral injection sites in the NAc. Histologically reconstructed sites of infusion localized in the NAc of rats that were included in the analysis of experiments. Data are represented as mean ± SEM. Newman–Keuls post hoc test, *p < 0.05, **p < 0.01 ***p < 0.001; n = 10 per group.

Figure 7.

Intra-NAc infusion of FTI-276 does not alter self-administration of sucrose. A, Rats were trained to self-administer a solution of 1.5% sucrose in an operant procedure. Three hours before the beginning of the 30 min session, rats were infused in the NAc with FTI-276 (100 ng/side) or vehicle (Veh) and the number of presses on the sucrose lever during the 30 min operant self-administration session was recorded. B, Schematic drawings of coronal sections of the rat brain showing the placement of bilateral injection sites in the NAc. Histologically reconstructed sites of infusion localized in the NAc of rats were included in the analysis of experiments. Data are presented as mean ± SEM. n = 8 per group.

Figure 8.

Inhibition of H-Ras activity reduces alcohol-seeking behavior in rats. Rats with a history of high levels of alcohol consumption in an intermittent-access 20% alcohol two-bottle choice drinking paradigm were trained to orally self-administer a solution of 20% alcohol in an operant procedure on an FR3 schedule. Three hours before the beginning of a 30 min extinction session (alcohol was not delivered), FTI-276 (100 ng/side) or vehicle (Veh) was infused into the NAc. A, Graph of the number of active lever presses during the 30 min session. B, Shows latency to the third press. C, Cumulative mean presses in bins of 2 min; data are presented as mean ± SEM. Newman–Keuls post hoc test, *p < 0.05; n = 9 per group.