GDNF is a novel ethanol-responsive gene in the VTA: implications for the development and persistence of excessive drinking

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a potent inhibitor of ethanol consumption and relapse, and GDNF heterozygous knockout mice display increased reward sensitivity to ethanol and consume more ethanol after a period of abstinence than their wild-type littermates. Here, we tested whether ethanol alters GDNF expression in the ventral tegmental area (VTA; GDNF's site of action) and/or the nucleus accumbens (NAc; the main source of GDNF), and if so, determine the role of the endogenous growth factor in the regulation of ethanol consumption. Systemic administration of ethanol increased GDNF expression and protein levels in the VTA, but not the NAc. Additionally, GDNF levels were elevated after an ethanol-drinking session in rats that consumed ethanol in the intermittent-access two-bottle choice procedure for 1 week, but not 7 weeks. Deprivation following 7 weeks of excessive ethanol intake reduced GDNF levels, while a short ethanol binge drinking period following deprivation upregulated GDNF expression. Importantly, knockdown of GDNF within the VTA using adenovirus expressing short hairpin RNA facilitated the escalation of ethanol drinking by ethanol-naïve rats, but not by rats with a history of excessive ethanol consumption. These results suggest that during initial ethanol-drinking experiences, GDNF in the VTA is increased and protects against the development of excessive ethanol intake. However, the growth factor's protective response to ethanol breaks down after protracted excessive ethanol intake and withdrawal, resulting in persistent, excessive ethanol consumption.

Keywords: Addiction; GDNF; VTA; ethanol; growth factor.

Figure 1. Systemic administration of ethanol increases GDNF expression in the VTA

Rats were injected i.p. with 1.8 g/kg ethanol (20% v/v solution; black squares), or saline (white squares). The VTA and NAc were collected 0.5, 4, 10 and 24 hours after the injection. GDNF and GAPDH expression in the VTA (A) and the NAc (B) were measured by qRT-PCR. Each square represents the GDNF/GAPDH ratio of an individual subject, with the group mean depicted by a bar. n = 10 animals for the saline group and 4 for each ethanol time point. ** p < 0.01, as compared with the saline group. (C) GDNF protein levels in the VTA were assessed by western blot 10 hours after a single administration of 1.8 g/kg ethanol. Data are expressed as the mean GDNF/GAPDH ± SEM, n = 4 per group. **p < 0.01, as compared with the water control group.

Figure 2. GDNF is increased in the VTA, but not NAc, following a short training period of ethanol intake but not after a long period of intermittent excessive ethanol consumption

Rats consumed ethanol in the intermittent-access to 20% ethanol two-bottle choice paradigm for 1 week (black bars) or 7 weeks (grey bars). Controls consumed water only for 7 weeks (white bars). The VTA and NAc were collected immediately following the end of the last ethanol drinking session. GDNF (upper panels in gel images) and GAPDH (lower panels in gel images) expression in the VTA (A) and the NAc (B) were determined by RT-PCR. Bar graphs represent the mean GDNF/GAPDH ratio ± SEM, n = 7–8 per group. ** p < 0.01, as compared with water control.

Figure 3. GDNF is reduced in the VTA after a 24-hour period of deprivation following 7 weeks of excessive ethanol intake

Rats consumed ethanol in the intermittent-access to 20% ethanol two-bottle choice paradigm 7 weeks (black bars). Controls consumed water only for 7 weeks (white bars). The VTA and NAc were dissected 24 hours after the end of the last ethanol-drinking session. GDNF (upper panels of gel images) and GAPDH (lower panels of gel images) in expression levels in the VTA (A) and the NAc (B) were determined by RT-PCR. Bar graph represents the mean GDNF/GAPDH ratio, ± SEM. ** p < 0.01, n = 6–8 (A) and 4–5 (B) per group.

Figure 4. A binge drinking session elevates GDNF levels in the VTA

Rats consumed ethanol in the intermittent-access to 20% ethanol two-bottle choice paradigm 7 weeks. Controls consumed water only for 7 weeks. (A) Rats’ drinking patterns segregated into groups of excessive- (black squares) and low-ethanol- drinking (grey squares) subjects. (B) The VTAs of excessive (black bar) and low (grey bar) drinkers, as well as water-only controls (white bar), were collected 30 minutes after the start of the last ethanol-drinking session. GDNF (upper panels of gel image) and GAPDH (lower panels of gel image) expression levels were measured by RT-PCR. Bar graph represents the average GDNF/GAPDH ratio ± SEM, n = 5–10 per group. ** p < 0.01, ***p < 0.001, as compared with the water control group. # p < 0.05, as compared with the excessive-drinking group.

Figure 5. Knockdown of VTA GDNF facilitates ethanol drinking escalation by ethanol-naïve rats

Recombinant adenoviruses containing shRNA targeting GDNF (shGDNF; black squares) or a scrambled control sequence (SCR; white squares) were bilaterally infused into the VTA of rats. Ethanol (A) and water (B) consumption in the intermittent-access to 20% ethanol two-bottle choice paradigm was measured beginning 10 days after the virus infusion. Graph represents amount of ethanol (A) or water (B) consumed ± SEM, n = 9 per group. **p < 0.01, as compared with the SCR control group.

Figure 6. Knockdown of GDNF does not alter ethanol consumption by rats that previously consumed excessive amounts of ethanol for 7 weeks

Rats consumed ethanol in the intermittent-access to 20% ethanol two-bottle choice paradigm 7 weeks. Controls consumed water only for 7 weeks. Recombinant adenovirus expressing shRNA targeting GDNF (shGDNF; black bars) or a scrambled control sequence (SCR; white bars) was then infused into the VTA. Ethanol (A) and water (B) consumption in the intermittent-access to 20% ethanol two-bottle choice paradigm was assessed starting 10 days after the virus infusion. Bar graphs represent the mean ethanol (A) or water (B) intake ± SEM on Day 11 post-virus infusion, n = 7 per group.