GDNF is a fast-acting potent inhibitor of alcohol consumption and relapse

Abstract

Previously, we demonstrated that the action of the natural alkaloid, ibogaine, to reduce alcohol (ethanol) consumption is mediated by the glial cell line-derived neurotrophic factor (GDNF) in the ventral tegmental area (VTA). Here we set out to test the actions of GDNF in the VTA on ethanol-drinking behaviors. We found that GDNF infusion very rapidly and dose-dependently reduced rat ethanol, but not sucrose, operant self-administration. A GDNF-mediated decrease in ethanol consumption was also observed in rats with a history of high voluntary ethanol intake. We found that the action of GDNF on ethanol consumption was specific to the VTA as infusion of the growth factor into the neighboring substantia nigra did not affect operant responses for ethanol. We further show that intra-VTA GDNF administration rapidly activated the MAPK signaling pathway in the VTA and that inhibition of the MAPK pathway in the VTA blocked the reduction of ethanol self-administration by GDNF. Importantly, we demonstrate that GDNF infused into the VTA alters rats' responses in a model of relapse. Specifically, GDNF application blocked reacquisition of ethanol self-administration after extinction. Together, these results suggest that GDNF, via activation of the MAPK pathway, is a fast-acting selective agent to reduce the motivation to consume and seek alcohol.

Fig. 1.

Ten minutes and 3 h post infusion of GDNF in the VTA decreases operant ethanol self-administration. (A) Mean ± SEM number of lever presses in 1 h after GDNF microinjection into the VTA (0, 2.5, 5, or 10 μg per side) 10 min before the self-administration session. Two-way ANOVA with repeated measures showed significant effects of lever [F_(1,24) = 27.08, P < 0.001] and treatment [F_(3,24) = 9.96, P < 0.001] and a significant interaction between both factors [F_(3,24) = 9.16, P < 0.001] (n = 10). (B) Mean ± SEM number of lever presses in 1 h after GDNF microinjection into the VTA (0, 5, or 10 μg per side) 3 h before the self-administration session [main effect of lever: F_(1,16) = 13.28, P < 0.001; treatment: F_(2,16) = 26.15, P < 0.001; and a significant interaction: F_(2,16) = 11.80, P < 0.001] (n = 10). (C) Mean ± SEM of ethanol intake during acquisition of voluntary ethanol consumption of a 20% ethanol solution in an intermittent-access (24 h on/24 h off) two-bottle choice paradigm. After 13–14 sessions, rats maintained drinking levels of 5.5 ± 1.5 g/kg in 24 h, with a consumption of 1–1.5 g/kg during the first 30 min of access to ethanol (n = 8). (D) Mean ± SEM number of lever presses in 30 min for a 20% ethanol solution after acquisition of ethanol drinking in the intermittent-access two-bottle choice paradigm following intra-VTA infusion of GDNF (0 or 10 μg per side) before the session [10 min before session, main effect of lever: F_(1,7) = 70.56, P < 0.001; treatment: F_(1,7) = 13.79, P < 0.01; and a significant interaction: F_(1,7) = 12.26, P < 0.01; 3 h before session main effect of lever: F_(1,7) = 37.81, P < 0.001; main effect of treatment: F_(1,7) = 10.85, P < 0.02; significant interaction: F_(1,7) = 7.4, P < 0.05] (n = 8). **, P < 0.01; ***, P < 0.001 (compared with PBS injection).

Fig. 2.

Intra-substantia nigra injections of GDNF do not affect operant ethanol self-administration, and intra-VTA injections of GDNF do not affect operant sucrose self-administration. (A) Mean ± SEM number of lever presses in 1 h after microinjection of GDNF into the SNc 10 min (0, 5, or 10 μg per side) or 3 h (0 or 10 μg per side) before the self-administration session. Two-way ANOVA with repeated measures showed significant main effects [lever: F_(1,32) = 92.72, P < 0.001; treatment: F_(4,32) = 4.03, P < 0.01] and a significant interaction between both factors [F_(4,32) = 3.80, P < 0.02]. Post hoc analysis showed significant differences in lever presses for ethanol between the 3-h GDNF pretreatment and the 10-min pretreatment (P < 0.05), but not with the PBS control (P = 0.12) (n = 9). (B) Mean ± SEM number of lever presses in 1 h after GDNF microinjection into the VTA 10 min (0, 5, or 10 μg per side) or 3 h (0 or 10 μg per side) before sucrose self-administration. Two-way ANOVA with repeated measures showed a significant effect of lever [F_(1,20) = 13.86, P < 0.01] but no effect of treatment and no interaction [F_(4,20) = 0.13 and F_(4,20) = 0.12, respectively, nonsignificant] (n = 8).

Fig. 3.

GDNF activates ERK1/2 in midbrain dopaminergic neurons in vivo. Shown is dual-channel immunofluorescence for phospho-ERK1/2 (p-ERK1/2, red), tyrosine hydroxylase (TH, green), and overlay (yellow). (A) Images depict ERK1/2 phosphorylation in the midbrain 10 min after GDNF (right brain side) or PBS (left side) infusion into the VTA. Images are representative of results from three rats (nine sections per rat). (Scale bar: 500 μm.) (B) Enlarged image of VTA area infused with GDNF. The arrowheads point to cells immunostained both for p-ERK1/2 and tyrosine hydroxylase. (Scale bar: 50 μm.)

Fig. 4.

Inhibition of the MAPK pathway blocks GDNF-induced decreases in ethanol self-administration. (A–C) Mean ± SEM number of lever presses in 1 h after microinjection into the VTA of kinase inhibitors or the appropriate vehicles and GDNF (10 μg per side) or PBS, 1 h and 10 min before the self-administration session, respectively. (A) The MAPK inhibitor U0126 was injected at a concentration of 500 ng per side. Two-way ANOVA with repeated measures showed significant main effects [lever: F_(1,30) = 249.52, P < 0.001; treatment: F_(3,30) = 3.03, P < 0.05] and a significant interaction between both factors [F_(3,30) = 6.19, P < 0.01) (n = 10). (B) The PI3K inhibitor wortmannin was injected at a dose of 50 ng per side. Two-way ANOVA with repeated measures showed significant main effects [lever: F_(1,24) = 35.61, P < 0.001; treatment: F_(3,24) = 6.06, P < 0.01] and a significant interaction between both factors [F_(3,30) = 4.89, P < 0.01] (n = 9). (C) The PLC inhibitor U73122 was injected at a dose of 100 ng per side. Two-way ANOVA with repeated measures showed significant main effects [lever: F_(1,30) = 95.06, P < 0.001; treatment: F_(3,24) = 12.51, P < 0.001] and a significant interaction between both factors [F_(3,30) = 13.05, P < 0.001] (n = 10). **, P < 0.01; ***, P < 0.001.

Fig. 5.

Intra-VTA injection of GDNF blocks reacquisition of operant ethanol self-administration. (A) Mean ± SEM number of lever presses in 1 h after GDNF microinjection into the VTA (0 or 10 μg/1 μl per side) 10 min before the reacquisition test. Two-way ANOVA with repeated measures showed significant main effects [lever: F_(1,32) = 40.52, P < 0.001; treatment: F_(4,32) = 15.31, P < 0.001] and a significant interaction between both factors [F_(4,32) = 20.97, P < 0.001]. Baseline represents the mean responding for the last 4 days of self-administration training, and Extinction represents the mean lever presses during the final extinction session. (B) Mean ± SEM latency in seconds to the third press (first reward) during the baseline, the final extinction session, and the reacquisition test for the PBS and GDNF conditions (χ² = 12.78, P < 0.01). *, P < 0.05; ***, P < 0.001 (n = 9).