AKT signaling pathway in the nucleus accumbens mediates excessive alcohol drinking behaviors

Abstract

Background: Neuroadaptations within the nucleus accumbens (NAc) have been implicated in molecular mechanisms underlying the development and/or maintenance of alcohol abuse disorders. We recently reported that the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in the NAc of rodents, after exposure to alcohol, contributes to alcohol drinking behaviors. The kinase AKT is a main upstream activator of the mTORC1 pathway. We therefore hypothesized that the activation of AKT in the NAc in response to alcohol exposure plays an important role in mechanisms that underlie excessive alcohol consumption.

Methods: Western blot analysis was used to assess the phosphorylation levels of enzymes. Acute exposure of mice to alcohol was achieved by the administration of 2 g/kg alcohol intraperitoneally (i.p.). Two-bottle choice and operant self-administration procedures were used to assess drinking behaviors in rats.

Results: We found that acute systemic administration of alcohol and recurring cycles of excessive voluntary consumption of alcohol and withdrawal result in the activation of AKT signaling in the NAc of rodents. We show that inhibition of AKT or its upstream activator, phosphatidylinositol-3-kinase (PI3K), within the NAc of rats attenuates binge drinking as well as alcohol but not sucrose operant self-administration.

Conclusions: Our results suggest that the activation of the AKT pathway in the NAc in response to alcohol exposure is an important contributor to the molecular mechanisms underlying alcohol-drinking behaviors. AKT signaling pathway inhibitors are therefore potential candidates for drug development for the treatment of alcohol use and abuse disorders.

Figure 1. Systemic administration of alcohol to mice results in a rapid activation of the AKT pathway in the NAc

Mice were systemically administered (i.p.) with 2 g/kg of alcohol or saline solution, and the NAc was removed 15 min later. The level of AKT phosphorylation on threonine 308 and serine 473 (A), of ERK1/2 on tyrosine 204/187 (B), as well as the levels of GSK3α and GSK3β phosphorylation on serine 21 and serine 9 respectively (C), were determined by western blot analyses. Optical density quantification of the phosphorylation is expressed as the ratio of the phosphorylated protein to the total protein. n=9 per group. Data are presented as mean ± SEM and expressed as percentage of control. **p< 0.01, ***p<0.001, one-tailed unpaired t-test.

Figure 2. Excessive alcohol consumption results in the activation of AKT but not ERK1/2 signaling in the NAc of rats

Rats experienced 2 months of intermittent-access 20% alcohol 2-bottle choice drinking sessions. Control animals underwent the same paradigm but had access to water only. The NAc were removed after the last 24 hrs of alcohol deprivation session (24 hr of withdrawal). The levels of AKT (A), GSK-3α, GSK-3β (B) and ERK1/2 (C) phosphorylation were determined by western blot analyses. n=16 (A), n=8–10 (B), n=6 (C) per group. Data are presented as mean ± SEM and expressed as percentage of control. **p<0.01, ***p<0.001, one-tailed unpaired t-test.

Figure 3. Intra-NAc infusion of wortmannin reduces binge drinking of alcohol in rats

Vehicle (Veh) or wortmannin (Wort, 0.1 or 0.4 µg/side) were infused into the NAc 1 hr before the beginning of the 24-hr alcohol-drinking session in rats trained to consume a high amount of a 20% solution of alcohol in a 2-bottle choice paradigm. (A, B) Alcohol intake was measured 30 min after the beginning of the session. Alcohol (C, D) and water (E, F) intakes were also measured at the end of the 24-hr drinking session. Consumption is expressed in grams per kilogram (g/kg) of body weight. n=8–9 per group. Data are presented as mean ± SEM. One-way ANOVA with repeated measures showed significant effects of treatment for A [F_(1,7) = 13.84, P = 0.007] and B [F_(1,8) = 8.38, P = 0.02] but not for C [F_(1,7) = 3.28, P = 0.11] and D [F_(1,8) = 4.71, P = 0.062], *p< 0.05 and **p<0.01.

Figure 4. Intra-NAc infusion of triciribine reduces binge drinking of alcohol in rats

Vehicle (Veh) or triciribine (0.05 or 0.5 µg/side) were infused into the NAc 3 hrs before the beginning of the 24-hr alcohol-drinking session in rats trained to consume a high amount of a 20% solution of alcohol in a 2-bottle choice paradigm. (A) Alcohol intake was measured 30 min after the beginning of the session. Alcohol (B) and water (C) intakes were also measured at the end of the 24-hr drinking session. Alcohol and water intakes are expressed in grams per kilogram (g/kg) of body weight. n=9 per group. Data are presented as mean ± SEM. One-way ANOVA with repeated measures showed significant effects of treatment for (A) [F_(2,16) = 6.62, P = 0.008] and (B) [F_(2,16) = 5.71, P = 0.013]; *p<0.05 and **p<0.01 compared to vehicle (Newman-Keuls post-hoc test).

Figure 5. Intra-NAc infusion of wortmannin reduces operant self-administration of alcohol in rats

(A–F) Rats with a history of high levels of alcohol consumption were trained to self-administer a solution of 20% alcohol in an operant procedure on a fixed ratio 3 (FR3) schedule. One hr before the beginning of a 30-min session, wortmannin (0.1 µg/side) was infused into the NAc of rats. (A) Number of active-lever presses during the 30-min operant alcohol self-administration session. (B) Number of alcohol deliveries (0.1 ml of 20% alcohol per delivery) obtained during the 30-min session. (C) Cumulative mean presses in bins of 2 min, indicative of the rate of presses for alcohol during the session. Two-way ANOVA with repeated measures showed a significant main effect of time [F_(14,112) = 98.52, P < 0.001] and treatment [F_(1,112) = 7.32, P = 0.027] and an interaction between time and treatment [F(14,112) = 2.87, P = 0.001]. Subsequent analysis using the method of contrasts detected a significant difference between vehicle and wortmannin for all time intervals except for the first interval 0–2 min (all other p’s < 0.05). (D) Time of the last alcohol delivery. (E) Time of the first alcohol delivery. (F) Distribution of inter-response intervals. n=9 per group. Data are represented as mean ± SEM. One-way ANOVA with repeated measures showed main effects of treatment for (A) [F_(1,8) = 29.21, P < 0.001]; (B) [F_(1,8) = 27.03, P < 0.001] and (E) [F_(1,8) = 10.45, P = 0.012] but not for (D) [F_(1,8) = 4.65, P = 0.063], *p<0.05 and ***p<0.001.

Figure 6. Intra-NAc infusion of triciribine reduces operant self-administration of alcohol in rats

(A–F) Rats with a history of high levels of alcohol consumption were trained to self-administer a solution of 20% alcohol. Three hrs before the beginning of a 30-min session, triciribine (0.5 µg/side) was infused into the NAc of rats. (A) Number of active-lever presses during the 30-min operant alcohol self-administration session. (B) Number of alcohol deliveries (0.1 ml of alcohol 20% per delivery) obtained during the 30-min session. (C) Cumulative mean presses in bins of 2 min, indicative of the rate of presses for alcohol during the session. Two-way ANOVA with repeated measures showed a significant main effect of time [F_(14,112) = 27.44, P < 0.001] and treatment [F_(1,112) = 5.93, P = 0.041] and an interaction between time and treatment [F_(14,112) = 3.34, P < 0.001]. Subsequent analysis using the method of contrasts detected a significant difference between vehicle and triciribine for all time intervals (all p’s < 0.05). (D) Time of the last alcohol delivery. (E) Time of the first alcohol delivery. (F) Distribution of inter-response intervals. n=9 per group. Data are represented as mean ± SEM. One-way ANOVA with repeated measures showed main effects of treatment for (A) [F_(1,8) = 10.37, P = 0.012]; (B) [F_(1,8) = 11.22, P = 0.01] and (D) [F_(1,8) = 8.57, P = 0.019], *p<0.05.

Figure 7. Intra-NAc infusion of wortmannin and triciribine does not alter operant self-administration of sucrose in rats

Rats were trained to self-administer a solution of 1.5% sucrose in an operant procedure. One hr or 3 hrs before the beginning of a 30-min session, 0.1 µg/side of wortmannin (A) or 0.5 µg/side of triciribine (B) respectively were infused into the NAc of rats and the total number of presses on the sucrose lever was recorded. n=8 per group. Data are represented as mean ± SEM.