Here, we report that microinjection of GDNF into the VTA of rats significantly reduces excessive voluntary ethanol consumption in an intermittent-access 20% ethanol two-bottle-choice drinking paradigm. Importantly, we found that this inhibitory action of the growth factor was rapid and long lasting, as GDNF infused 10 min before the session decreased both the first bout of excessive ethanol intake and the later consummatory activity that occurred mainly during the dark cycle.
We observed that repetition of the 24- or 48-h ethanol deprivation periods induced episodes of heavy drinking for short periods of time that is reminiscent of binge-drinking in humans. Interestingly, binge drinking is defined by the National Institute Abuse and Alcoholism of (NIAAA) as a pattern of drinking that results in BEC to 80 mg% or above (NIAAA, 2004
), which is similar to the average of the BEC we measured in the rats. This suggests that the majority of the animals in the intermittent-access 20% ethanol two-bottle choice drinking paradigm are highly motivated to consume ethanol for its pharmacological effects and as such, exhibit “binge-like” drinking behavior. Interestingly, the escalation in ethanol intake is not observed when rats are subjected to a continuous ethanol access (Wise, 1973
and data not shown), supporting a critical role of the repeated cycles of ethanol deprivation and consumption that result in neuroadaptative changes that lead to further increase in the ethanol intake. In line with this possibility, chronic intermittent, but not continuous, exposure to ethanol was found to significantly increase dopamine transporter levels within the nucleus accumbens (Healey et al., 2008
), and voluntary chronic ethanol intake in the same intermittent-access 20% ethanol two-bottle choice drinking paradigm enhances postsynaptic AMPA receptor function in VTA neurons (Stuber et al., 2008
Importantly, we found that GDNF acts as a potent inhibitor of excessive ethanol drinking. It is unlikely that this action of GDNF was due to nonspecific locomotor effects or a general decrease in motivation as the total fluid intake was unaffected by GDNF, and we previously showed that intra-VTA microinjection of GDNF did not alter operant self-administration of sucrose (Carnicella et al., 2008
). Together, our data suggest that GDNF in the VTA acts specifically on ethanol-related behaviors.
Intra-VTA administration of GDNF resulted in a 70% and 50% decrease in ethanol intake during 30 min and 24 h access to ethanol, respectively. Previously, we found that infusion of GDNF into the VTA reduced operant self-administration of a 20% ethanol solution to a lesser extent (45–50%) (Carnicella et al., 2008
). Although we cannot quantitatively compare the two paradigms, the results may suggest that GDNF may reduce free-choice drinking to a greater degree compared to a paradigm in which the animal has to work for alcohol as a reinforcer.
Due to the limited solubility of the growth factor, 1 µL of the solution was infused into the VTA. This volume is relatively high for the VTA, therefore it might be possible that the reduction of ethanol intake by GDNF was due to the diffusion of the growth factor to the neighboring dopaminergic area, the SNc. However, this is unlikely because our previous study shows that GDNF infusion into the SNc did not affect operant ethanol self-administration (Carnicella et al., 2008
). GDNF can undergo anterograde and retrograde axonal transport (Ai et al., 2003
; Kordower et al., 2000
) and therefore, we cannot exclude the possibility that the effect of GDNF on ethanol intake is mediated by the growth factor’s actions in other brain areas. However, this is also unlikely for several reasons. First, VTA is a preferential site of action of GDNF as the GDNF receptors Ret and GFRα1 are highly expressed in this area in the adult brain (Burazin and Gundlach, 1999
; Glazner et al., 1998
; Trupp et al., 1997
). In contrast, very low or negligible levels of GFRα1 and Ret are detected in the nucleus accumbens (Burazin and Gundlach, 1999
; Glazner et al., 1998
; Trupp et al., 1997
). Second, the effect of intra-VTA infusion of GDNF on ethanol intake is very rapid, within 10 min (present study and Carnicella et al., 2008
), and we found that this period of time allowed GDNF to activate the MAPK pathway that is downstream to Ret in the VTA (Carnicella et al., 2008
), but not in the brain areas targeted by VTA dopaminergic neurons, such as the nucleus accumbens or the prefrontal cortex (unpublished data, V. Kharazia, S. Carnicella, and D. Ron). Taken together, these data suggest that the effects of GDNF on ethanol-drinking behaviors are specifically mediated by the VTA. Nevertheless, it might be of great interest for future investigation to test whether GDNF can rapidly modulate ethanol-drinking behaviors when microinjected in other brain limbic regions expressing a high level of GDNF receptors, such as the amygdala or the habenula (Trupp et al., 1997
GDNF, within minutes, dramatically reduced the bout of excessive ethanol intake that occurred at the beginning of the session. We previously showed that infusion of GDNF into the VTA very rapidly decreased operant ethanol self-administration and relapse (Carnicella et al., 2008
). However, animals in the operant self-administration paradigm consumed relatively moderated amounts of ethanol (0.4–0.8 g/kg in 30 min or 1 h). The mechanisms underlying moderate and excessive consumption of ethanol are believed to be different (e.g., Koob, 2003
), and as such, pharmacological substances can affect moderate and excessive drinking differently. For example, corticotropin-releasing factor 1 antagonists were shown to reduce only excessive ethanol self-administration in dependent rats (Funk et al., 2007
), and acamprosate and naltrexone were found to be more efficient to decrease ethanol consumption in rats consuming high levels of ethanol (Simms et al., 2008
). Interestingly, GDNF reduces both moderate levels of ethanol consumption (that resembles social drinkers in humans) and excessive ethanol consumption, a hallmark of alcoholism, suggesting that the growth factor inhibits a pathway common to adaptations that result in moderate and excessive ethanol intake.
The mechanism by which GDNF decreases ethanol-drinking behaviors is unknown. However, GDNF was reported to rapidly increase the excitability and function of mesencephalic neurons in vitro (Kobori et al., 2004
; Wang et al., 2003
; Yang et al., 2001
), and acute injection of GDNF into the midbrain enhances dopaminergic transmission (Hebert et al., 1996
; Hudson et al., 1995
). The mesolimbic dopaminergic system is believed to play an important role in the reinforcing effects of ethanol and in the development of alcohol addiction (Gonzales et al., 2004
; Weiss and Porrino, 2002
). For example, ethanol can directly excite the dopaminergic neurons of the VTA (Brodie et al., 1999
) and increase DA levels in the nucleus accumbens (Gonzales et al., 2004
). In addition, pharmacological manipulations of the dopaminergic transmission within the mesolimbic system alter ethanol self-administration (Gonzales et al., 2004
). Chronic exposure to high levels of ethanol results in substantial modifications of VTA dopaminergic neurons activity (reviewed in Diana et al., 2003
) and a decrease in DA levels in the nucleus accumbens (Darden and Hunt, 1977
; Weiss et al., 1996
) during withdrawal. This DA hypofunction has been suggested to be a part of an allostatic process that leads to compulsive ethanol intake, prolongation of craving, and propensity to relapse to compensate for these DA deficits and the negative emotional state (e.g., dysphoric symptoms) associated with them (Fadda and Rossetti, 1998
; Koob and Le Moal, 2001
; Weiss et al., 1996
; Weiss and Porrino, 2002
). Therefore, it might be plausible that GDNF reduces ethanol consumption by adjusting the activity of VTA dopaminergic neurons and as a consequence, the accumbal DA levels.
We also observed that GDNF infusion into the VTA decreased the ethanol drinking that occurred over 20 h, mainly during the dark cycle, suggesting a long-lasting effect of the protein. As a growth factor, GDNF induces several transcriptional modifications (Airaksinen and Saarma, 2002
). We previously showed that GDNF activates a positive feedback loop in which the growth factor increases its own expression, leading to a sustained activation of the GDNF signaling pathway (He and Ron, 2006
). Therefore, upregulation of the GDNF
gene could account for its long-lasting action to reduce voluntary ethanol intake. However, as a fraction of exogenous GDNF was reported to be detected in the brain 24 h after the infusion (Lapchak et al., 1997
; Tomac et al., 1995b
), we cannot exclude the possibility that the long-lasting effect we observed is due to the presence of a portion of the exogenous GDNF throughout session.
Importantly, GDNF was highly effective in reducing heavy drinking after a short period of deprivation. These results are in line with our previous studies showing that GDNF in the VTA reduces reacquisition to lever presses for ethanol after a 2-week period of extinction (Carnicella et al., 2008
). Curiously, we found that GDNF in the VTA was much more effective in inhibiting reacquisition of ethanol self-administration after the extinction period, than in reducing ethanol self-administration before the period of abstinence (Carnicella et al., 2008
). Taken together, these data suggest an important protective role of GDNF in abstinence and relapse processes.
In summary, we have shown that GDNF in the VTA selectively reduces high levels of consumption and “binge-like” ethanol drinking that followed a short period of deprivation. Binge-drinking is becoming more prevalent during school and college years in North America and Europe, and is a strong predictor of future alcohol-related problems (e.g., Bloomfield et al., 2003
). Moreover, heavy drinking is a factor that largely contributes to the onset and development of several major chronic diseases and alcohol use disorders (Lancaster, 1994
; Rehm et al., 2003
). Furthermore, lapse and relapse to alcohol drinking, which in many cases results in binge-drinking (Rösner et al., 2008
), are fundamental issues in alcoholism treatment. The estimated rate of relapse is in the range of 70–80% within one year post-treatment (Dawson et al., 2007
), and evidence suggests that approximately 90% of alcoholics are likely to experience at least one relapse episode over a 4-year period following treatment (NIAAA, 1989
). Our data support the possibility that upregulation of GDNF expression and/or activation of the GDNF pathway may be valuable strategies for the development of treatment of excessive alcohol consumption and the prevention of relapse.