Excessive alcohol consumption is blocked by glial cell line-derived neurotrophic factor.
ABSTRACT We previously found that activation of the glial cell line-derived neurotrophic factor (GDNF) pathway in the ventral tegmental area (VTA) reduces moderate alcohol (ethanol) intake in a rat operant self-administration paradigm. Here, we set out to assess the effect of GDNF in the VTA on excessive voluntary consumption of ethanol. Long-Evans rats were trained to drink large quantities of a 20% ethanol solution in an intermittent-access two-bottle choice drinking paradigm. The rats were given three 24-h sessions per week, and GDNF's actions were measured when rats achieved a baseline of ethanol consumption of 5.5g/kg/24h. We found that microinjection of GDNF into the VTA 10min before the beginning of an ethanol-drinking session significantly reduced ethanol intake and preference, but did not affect total fluid intake. We further show that GDNF greatly decreased both the first bout of excessive ethanol intake at the beginning of the session, and the later consummatory activity occurring during the dark cycle. These data suggest that GDNF is a rapid and long-lasting inhibitor of "binge-like" ethanol consumption.
- SourceAvailable from: Daniel James Olan Roche[Show abstract] [Hide abstract]
ABSTRACT: Studies have implicated neuroinflammatory processes in the pathophysiology of various psychiatric conditions, including addictive disorders. Neuroimmune signaling represents an important and relatively poorly understood biological process in drug addiction. The objective of this review is to update the field on recent developments in neuroimmune therapies for addiction. First, we review studies of neuroinflammation in relation to alcohol and methamphetamine dependence followed by a section on neuroinflammation and accompanying neurocognitive dysfunction in HIV infection and concomitant substance abuse. Second, we provide a review of pharmacotherapies with neuroimmune properties and their potential development for the treatment of addictions. Pharmacotherapies covered in this review include ibudilast, minocycline, doxycycline, topiramate, indomethacin, rolipram, anakinra (IL-1Ra), peroxisome proliferator-activated receptor agonists, naltrexone, and naloxone. Lastly, summary and future directions are provided with recommendations for how to efficiently translate preclinical findings into clinical studies that can ultimately lead to novel and more effective pharmacotherapies for addiction.International Review of Neurobiology 01/2014; 118C:381-401. · 2.46 Impact Factor
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ABSTRACT: Converging evidence indicates that epigenetic mechanisms are involved in drug addiction, and that enzymes involved in chromatin remodeling may represent interesting targets in addiction treatment. No study has addressed whether histone deacetylase (HDAC) inhibitors (HDACi) can reduce excessive ethanol intake or prevent relapse in alcohol-dependent animals. Here, we assessed the effects of two HDACi, sodium butyrate (NaB) and MS-275, in the operant ethanol self-administration paradigm in dependent and non-dependent rats. To characterize some of the epigenetic mechanisms associated with alcohol dependence and NaB treatment, we measured the levels of histone H3 acetylation in different brain areas of dependent and non-dependent rats, submitted or not to NaB treatment. Our results demonstrated that (1) NaB and MS-275 strongly decreased excessive alcohol intake of dependent rats in the operant ethanol self-administration paradigm but not of non-dependent rats; (2) NaB reduced excessive drinking and prevented the escalation of ethanol intake in the intermittent access to 20% ethanol paradigm; and (3) NaB completely blocked the increase of ethanol consumption induced by an alcohol deprivation, thus demonstrating a preventive effect of NaB on relapse. The mapping of cerebral histone H3 acetylation revealed a hyperacetylation in the amygdala and cortical areas in dependent rats. Interestingly, NaB did not exacerbate the hyperacetylation observed in these regions, but instead restored it, specifically in cortical areas. Altogether, our results clearly demonstrated the efficacy of NaB in preventing excessive ethanol intake and relapse and support the hypothesis that HDACi may have a potential use in alcohol addiction treatment.Addiction Biology 08/2014; · 5.93 Impact Factor
Excessive alcohol consumption is blocked by glial cell lineederived
Sebastien Carnicellaa, Ryoji Amamotoa, Dorit Rona,b,*
aThe Ernest Gallo Research Center, 5858 Horton St, Ste 200, Emeryville, CA 94608; and
bDepartment of Neurology, University of California, San Francisco, Emeryville, CA USA
Received 24 September 2008; received in revised form 26 November 2008; accepted 4 December 2008
We previously found that activation of the glial cell lineederived neurotrophic factor (GDNF) pathway in the ventral tegmental area
(VTA) reduces moderate alcohol (ethanol) intake in a rat operant self-administration paradigm. Here, we set out to assess the effect of
GDNF in the VTA on excessive voluntary consumption of ethanol. LongeEvans rats were trained to drink large quantities of a 20% ethanol
solution in an intermittent-access two-bottle choice drinking paradigm. The rats were given three 24-h sessions per week, and GDNF’s
actions were measured when rats achieved a baseline of ethanol consumption of 5.5 g/kg/24 h. We found that microinjection of GDNF into
the VTA 10 min before the beginning of an ethanol-drinking session significantly reduced ethanol intake and preference, but did not affect
total fluid intake. We further show that GDNF greatly decreased both the first bout of excessive ethanol intake at the beginning of the
session, and the later consummatory activity occurring during the dark cycle. These data suggest that GDNF is a rapid and long-lasting
inhibitor of ‘‘binge-like’’ ethanol consumption. ? 2009 Elsevier Inc. All rights reserved.
Keywords: Alcohol; GDNF; VTA; Addiction; Ethanol; Growth factor
Glial cell lineederived neurotrophic factor (GDNF) is
a secreted protein that was initially identified in a glial-
derived cell line (Lin et al., 1993). GDNF is an essential
growth factor for the development of kidneys and spinal
cord motoneurons (Moore et al., 1996; Pichel et al.,
1996; Sanchez et al., 1996), and plays a critical role in
promoting the survival, regeneration, and maintenance of
the mature phenotype of distinct central and peripheral
neuronal populations, including the midbrain dopaminergic
neurons (Beck et al., 1995; Granholm et al., 2000; Kowsky
et al., 2007; Lin et al., 1993; Pascual et al., 2008; Tomac
et al., 1995a). In the mesolimbic system, GDNF is produced
in striatal neurons (Barroso-Chinea et al., 2005; Pochon
et al., 1997) and is retrogradely transported by dopami-
nergic neurons of the substantia nigra pars compacta
(SNc) and the ventral tegmental area (VTA) in the adult
brain (Ai et al., 2003; Barroso-Chinea et al., 2005;
Kordower et al., 2000; Lapchak et al., 1997; Tomac et al.,
1995b), where the GDNF receptors, GFRa1 and Ret, are
highly expressed (Burazin and Gundlach, 1999; Glazner
et al., 1998; Golden et al., 1998; Matsuo et al., 2000; Trupp
et al., 1997). Ligation of GDNF to GFRa1 leads to the
recruitment and activation of receptor tyrosine kinase Ret,
and to the consequent activation of the mitogen-activated
protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)
and phospholipase Cg (PLCg) pathways (Airaksinen and
Saarma, 2002; Sariola and Saarma, 2003).
In recent years, the role for GDNF as a negative regu-
lator of biochemical and behavioral adaptations to drugs
of abuse has been reported (Ron and Janak, 2005). Specif-
ically, Messer et al. (2000) showed that infusion of GDNF
into the VTA reversed biochemical adaptations to long-
term exposure to cocaine and morphine, and blocked
cocaine conditioned place preference (CPP). Transplanta-
tion of cells engineered to overexpress GDNF or delivery
of nanoparticles conjugated to GDNF into the striatum were
shown to reduce acquisition of rat cocaine self-administra-
tion (Green-Sadan et al., 2003, 2005), and increasing
GDNF expression in the brain reduced the sensitivity of
mice to behavioral responses to methamphetamine (Niwa
et al., 2007b). In addition, heterozygous GDNF knockout
* Corresponding author. Tel.: þ1-510-985-3150; fax: þ1-510-985-
E-mail address: email@example.com (D. Ron).
0741-8329/09/$ e see front matter ? 2009 Elsevier Inc. All rights reserved.
Alcohol 43 (2009) 35e43
(GDNF HET) mice exhibited exaggerated behaviors related
to morphine, cocaine, and methamphetamine exposure
compared to their wild-type (GDNF WT) littermates (Air-
avaara et al., 2007; Messer et al., 2000; Niwa et al.,
2007a, b; Yan et al., 2007). For example, CPP to psychos-
timulants, as well as motivation to self-administer and seek
methamphetamine are potentiated in GDNF HET mice
compared to GDNF WT mice (Messer et al., 2000; Niwa
et al., 2007b; Yan et al., 2007).
During the past several years, we obtained evidence sug-
gesting that GDNF negatively regulates behaviors associ-
ated with exposure to ethanol. We found that systemic
administration of the naturally occurring alkaloid, ibogaine,
to rats increases the expression of GDNF in the VTA,
leading to the reduction of rat operant self-administration
of ethanol (He et al., 2005). We further showed that GDNF
reverses a biochemical adaptation resulting from prolonged
exposure of cells to ethanol. Specifically, we found that
treatment of the dopaminergic-like SH-SY5Y cell line with
ethanol leads to an increase in the protein stability of tyro-
sine hydroxylase, which was reversed by GDNF (He and
Ron, 2008). Importantly, we found that a single infusion
of GDNF into the VTA leads to a rapid and sustained reduc-
tion of rat operant ethanol self-administration (Carnicella
et al., 2008). Interestingly, GDNF did not affect operant
sucrose self-administration, suggesting a selective effect
of GDNF in the VTA on ethanol-mediated actions. We also
found that the actions of GDNF to attenuate the motivation
to consume ethanol are mediated via the activation of the
MAPK pathway (Carnicella et al., 2008). Finally, we
showed that a single infusion of GDNF into the VTA blocks
reacquisition of ethanol self-administration after a period of
extinction (Carnicella et al., 2008).
Our previous study showed that GDNF is a potent inhib-
itor of the motivation of rats to consume ethanol; however,
the level of ethanol intake in the operant self-administration
paradigm was moderate (Carnicella et al., 2008). Therefore,
we were interested in determining the effect of GDNF in
the VTA on excessive ethanol consumption using a model
that resembles ‘‘binge-drinking’’ in humans. To do so, we
used the intermittent-access 20% ethanol two-bottle-choice
drinking paradigm, in which repeated cycles of deprivation
result in the voluntary consumption of large amounts of
ethanol in a short period of time (Carnicella et al., 2008;
Simms et al., 2008; Wise, 1973).
Materials and methods
GDNF was purchased from R&D systems (Minneapolis,
MN). b-Nicotinamide adenine dinucleotide (NAD) and
alcohol dehydrogenase (ADH) were purchased from Sigma
(St. Louis, MO).
Male LongeEvans rats (Harlan; 400e450 g at the time
of the surgery) were housed under a 12-h light/dark cycle
(lights on at 7:00 a.m.) with food and water available ad
libitum, and kept in conditions of constant temperature
(23?C) and humidity (50%). All animal procedures in this
report were approved by the Gallo Center Institutional
Animal Care and Use Committee and were conducted in
agreement with the Guide for the Care and Use of Labora-
tory Animals, National Research Council, 1996.
Intermittent-access 20% ethanol two-bottle-choice
Intermittent-access of 20% ethanol was similar to the
paradigm described previously by Simms et al. (2008)
and Stuber et al. (2008). Specifically, animals were given
24-h concurrent access to one bottle of 20% vol/vol ethanol
in tap water and one bottle of water, starting at 11:00 a.m.
on Monday, Wednesday, and Friday, with 24 or 48-h
drinking sessions. The placement (left or right) of each
solution was alternated between each session to control
for side preference. The water and ethanol bottles were
weighted after 30 min, 4 h, and 24 h of access, according
to the experimental protocol. A bottle containing water in
a cage without rats was used to evaluate the spillage due
to the experimental manipulations during the test sessions.
The spillage was always #0.5 mL (!2.5% of the total fluid
intake). Blood ethanol concentration (BEC) determination,
21 ethanol access sessions, when rats maintained a stable
baseline of ethanol consumption.
Blood ethanol concentration measurements
Thirty minutes after the re-introduction of the ethanol
bottle, rats were briefly anesthetized with isoflurane and
blood was collected from the lateral tail vein with heparin-
ized capillary tubes. Serum was extracted with 3.4%
trichloroacetic acid, centrifuged for 5 min at 2,000 rpm,
and then assayed for ethanol content using the NAD-NADH
enzyme spectrophotometric method (Weiss et al., 1993;
Zapata et al., 2006). BECs were determined using a standard
Surgery and microinjection
Rats were anesthetized continuously with isoflurane
(Baxter Health Care Corporation, Deerfield, IL). Bilateral
guide cannulae (C235G-1.5, 26 ga, Plastics One, Roanoke,
VA) were aimed dorsal to the VTA (5.6 mm posterior to
bregma, 0.75 mm mediolateral, 8.0 mm ventral to the skull
surface), according to Paxinos and Watson (1998). After
3 days of recovery, rats returned to the intermittent-access
20% ethanol procedure and microinjections began after
36 S. Carnicella et al. / Alcohol 43 (2009) 35e43
three ethanol-drinking sessions, when drinking returned to
a stable baseline. GDNF (10 mg/mL) or vehicle phosphate-
buffered saline (PBS) were microinjected 10 min before
the beginning of a 24-h ethanol-access session. A total of
1 mL of GDNF or vehicle was infused over 2 min into the
VTA of gently restrained rats via injection cannulae extend-
ing 0.5 mm beyond the guide cannula tip. Injection
cannulae were left in place for an additional 2 min. The
GDNF microinjection experiment was conducted within 1
week. On Monday, half of the animals were microinjected
with GDNF and the other half with vehicle, 10 min prior to
the beginning of the 24-h ethanol-drinking session. The
second ethanol-access session that started on Wednesday
was conducted without any treatment, allowing ethanol
intake to return to baseline level (vehicle- and GDNF-
treated animals showed similar levels of basal level of
ethanol consumption throughout the experiment; data not
shown). Ten minutes prior to the third ethanol-access
session of the week (on Friday), animals were microin-
jected with GDNF or vehicle, but with the drug treatment
reversed. The parameters used in this study (i.e., site and
time of injection as well as the GDNF concentration) were
previously found to effectively reduce operant ethanol self-
administration (Carnicella et al., 2008).
Locations of cannulae were verified in 60-mm coronal
sections of paraformaldehyde-fixed tissue stained with
The correlation between BEC (mg%) and ethanol intake
(g/kg/30 min) was analyzed by linear regression. Experi-
ments were conducted in a within-subjects design and
analyzed by paired t-test or one-way analysis of variance
with repeated measures. Preference was calculated as the
percentage of ethanol solution consumed relative to total
fluid intake (ethanol þ water).
Intermittent-access of LongeEvans rats to 20% ethanol
results in heavy drinking and high concentration of ethanol
in the blood
et al., 2008), rats showed a significant escalation of ethanol
intake [F(20, 440)524.05, P!.001] and preference [F(20,
440)528.49, P!.001] across sessions of access to 20%
ethanol (Fig. 1A, B) and reached a stable baseline of ethanol
consumption(5.3960.37 g/kg/24 h)
(49.564.10%) after 15 sessions. Interestingly, we observed
that at the beginning of the session, rats consumed large
quantities of ethanol very rapidly and the average ethanol
intake was 1.3960.11 g/kg in the first 30 min of the session,
which corresponded to |25% of the total ethanol consumed
the 30-min period of access to ethanol, and the amount of
ethanol consumed significantly correlated with the measured
BEC (r250.63, P!.001, Fig. 1D). The BEC ranged from
7.1 to 158.6 mg% (1.5e34 mM) with an average of
80.967.2 mg% (17.561.5 mM), and in about 50% of the
rats the BEC was above average (Fig. 1D). Approximately
60% of the total ethanol intake (3.2860.31 g/kg) was
consumed during the second period (4e24 h) (Fig. 1C), and
cycle on a subset of animals, we observed that the majority of
ethanol was consumed during this time (3.65 g/kg ethanol
during the dark cycle out of a 4.65 g/kg intake during the
4e24 h period).
Intra-VTA microinjection of GDNF reduces excessive
voluntary ethanol intake
Next, we assessed the effect of intra-VTA administration
of GDNF on excessive voluntary ethanol intake. Fig. 2
displays the placement of the injector tips of the subjects
included in the behavioral analysis. The injection sites were
confined within the posterior region of the VTA. Rats were
infused with a dose of 10 mg/side of GDNF because it was
the most effective dose to reduce operant ethanol self-
administration (Carnicella et al., 2008).
As shown in Fig. 3A, intra-VTA microinjection of
GDNF 10 min before the beginning of the session signifi-
cantly decreased ethanol intake over the 24-h access period
[T(13) 5 2.80, P ! .02]. Infusion of GDNF into the VTA
also reduced the preference for ethanol [T(13) 5 2.46,
P ! .05; Fig. 3B]. Total fluid intake was unchanged
[T(13) 5 0.76, P 5 .48; Fig. 3C], and the decrease in
ethanol intake was accompanied by an increase in water
intake [T(13) 5 2.66, P ! .05; Fig. 3D]. Importantly, we
observed that GDNF application into the VTA 10 min
before the beginning of the session prevented the ‘‘binge-
like’’ drinking behavior observed during the first 30 min
of drinking [T(13) 5 8.49, P ! .001; Fig. 4A]. In addition,
GDNF was effective in reducing ethanol intake during the
second period of consumption [T(13) 5 2.19, P ! .05;
Fig. 4B]. Together, these data suggest a rapid and sustained
inhibitory effect of GDNF on excessive ethanol consump-
tion over the 24-h ethanol access session.
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
37S. Carnicella et al. / Alcohol 43 (2009) 35e43
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 Abuseand 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%
vated toconsume ethanol for itspharmacological effects and
as such, exhibit ‘‘binge-like’’ drinking behavior. Interest-
ingly, 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, butnot 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 intermit-
tent-access 20% ethanol two-bottle choice drinking para-
digm enhances postsynaptic AMPA receptor function in
VTA neurons (Stuber et al., 2008).
Importantly, we found that GDNF acts as a potent inhib-
itor 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
Ethanol intake (g/kg/30 min)
Ethanol Intake (g/kg/24hrs)
Ethanol preference (%)
20% ethanol access session
Ethanol Intake (g/kg)
20% ethanol access session
25811 20 14 17
r2 = 0.63
Fig. 1. High level of ethanol consumption is obtained in an intermittent access two-bottle-choice drinking paradigm. Mean 6standard error of the mean
(S.E.M.) of ethanol intake (A) and preference (B) during acquisition of voluntary ethanol consumption of a 20% ethanol solution, n524. (C) Mean6S.E.M.
of ethanol intake of the last three drinking sessions, during the first 30 min, the last 20 h (that includes the dark period of the cycle) and over the 24 h of the
session, n524. (D) Correlation between the ethanol consumed during the first 30 min of the drinking session and the blood ethanol concentration (BEC)
(r25 0.63, P ! .001). The average ethanol intake was 1.41 60.12 g/kg/30 min, ranging from 0.37 to 2.38 g/kg. The BEC measurements include the 24
animals used in the present study and eight animals from an earlier work (Carnicella et al., 2008), n532.
38S. Carnicella et al. / Alcohol 43 (2009) 35e43
that infusion of GDNF into the VTA reduced operant self-
administration of a 20% ethanol solution to a lesser extent
(45e50%) (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 mL
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 dopa-
minergic 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 (Car-
nicella 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 possi-
bility 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 GFRa1 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 GFRa1 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
Fig. 2. Schematic representation of the injection cannulae placements in
coronal sections (Paxinos and Watson, 1998). The location of the injector
tips is represented by gray circles. Numbers indicate the distance posterior
to bregma in millimeters. One rat was discarded due to cannulae
Ethanol Intake (g/kg/24hrs)
Ethanol preference (%)
Total fluid intake (ml/kg)
Water intake (ml/kg)
Fig. 3. Intraventral tegmental area microinjection of glial cell lineederived neurotrophic factor (GDNF) reduces high levels of voluntary ethanol consump-
tion in rats. GDNF (10 mg/side) or vehicle was microinjected into the ventral tegmental area 10 min before a 24-h ethanol-drinking session in rats trained to
consume a 20% solution of ethanol. Mean6standard error of the mean of ethanol intake (A), ethanol preference (B), total fluid intake (C), and water intake
(D) during the 24-h session, n 57; *P!.05; **P! .02 compared to vehicle.
39 S. Carnicella et al. / Alcohol 43 (2009) 35e43
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. Neverthe-
less, 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 para-
digm consumed relatively moderated amounts of ethanol
(0.4e0.8 g/kg in 30 min or 1 h). The mechanisms under-
lying moderate and excessive consumption of ethanol are
believed to be different (e.g., Koob, 2003), and as such,
pharmacological substances can affect moderate and exces-
sive 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 trans-
mission (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, pharma-
cological manipulations of the dopaminergic transmission
within the mesolimbic system alter ethanol self-administra-
tion (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 sug-
gested 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 symp-
toms) 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 Saar-
ma, 2002). We previously showed that GDNF activates
a positive feedback loop in which the growth factor
increases its own expression, leading to a sustained activa-
tion 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
Ethanol Intake (g/kg/30min)
Ethanol Intake (g/kg/4-24hrs)
Fig. 4. Intra-ventral tegmental area microinjection of glial cell lineederived neurotrophic factor (GDNF) induces a rapid and sustained reduction of ethanol
consumption. GDNF (10 mg/side) or vehicle was microinjected into the ventral tegmental area 10 min before a 24-h ethanol-drinking session. Mean6stan-
dard error of the mean of ethanol consumed during the first 30 min (A) and the last 20 h (B) of the drinking session, n57; *P! .05; ***P !.001 compared
40S. Carnicella et al. / Alcohol 43 (2009) 35e43
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 exoge-
nous 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 absti-
nence 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 Eu-
rope, 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 (R€ osner
et al., 2008), are fundamental issues in alcoholism treat-
ment. The estimated rate of relapse is in the range of
70e80% 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
Thiswork wassupported byNIH-NIAAA R01
AA014366e02 (D.R.) and the State of California for
Medical Research on Alcohol and Substance Abuse
through the University of California, San Francisco
(D.R.). The authors thank Quinn Yowell for technical
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