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.
- SourceAvailable from: Segev Barak[Show abstract] [Hide abstract]
ABSTRACT: Moderate social consumption of alcohol is common; however, only a small percentage of individuals transit from social to excessive, uncontrolled alcohol drinking. This suggests the existence of protective mechanisms that prevent the development of alcohol addiction. Here, we tested the hypothesis that the glial cell line-derived neurotrophic factor (GDNF) in the mesolimbic system [e.g. the nucleus accumbens (Acb) and ventral tegmental area (VTA)] is part of such a mechanism. We found that GDNF knockdown, by infecting rat Acb neurons with a small hairpin RNA (shRNA) targeting the GDNF gene, produced a rapid escalation to excessive alcohol consumption and enhanced relapse to alcohol drinking. Conversely, viral-mediated overexpression of the growth factor in the mesolimbic system blocked the escalation from moderate to excessive alcohol drinking. To access the mechanism underlying GDNF's actions, we measured the firing rate of dopaminergic (DAergic) neurons in the VTA after a history of excessive alcohol intake with or without elevating GDNF levels. We found that the spontaneous firing rate of DAergic neurons in the VTA was reduced during alcohol withdrawal and that GDNF reversed this alcohol-induced DA deficiency. Together, our results suggest that endogenous GDNF in the mesolimbic system controls the transition from moderate to excessive alcohol drinking and relapse via reversal of alcohol-dependent neuro-adaptations in DAergic VTA neurons.Addiction Biology 06/2014; · 5.93 Impact Factor
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ABSTRACT: Prenatal alcohol exposure can lead to long-lasting changes in functional and genetic programs of the brain, which may underlie behavioral alterations seen in Fetal Alcohol Spectrum Disorder (FASD). Aberrant fetal programming during gestational alcohol exposure is a possible mechanism by which alcohol imparts teratogenic effects on the brain; however, current methods used to investigate the effects of alcohol on development often rely on either direct application of alcohol in vitro or acute high doses in vivo. In this study, we used our established moderate prenatal alcohol exposure (PAE) model, resulting in maternal blood alcohol content of approximately 20 mM, and subsequent ex vivo cell culture to assess expression of genes related to neurogenesis. Proliferating and differentiating neural progenitor cell culture conditions were established from telencephalic tissue derived from embryonic day (E) 15–17 tissue exposed to alcohol via maternal drinking throughout pregnancy. Gene expression analysis on mRNA derived in vitro was performed using a microarray, and quantitative PCR was conducted for genes to validate the microarray. Student’s t tests were performed for statistical comparison of each exposure under each culture condition using a 95% confidence interval. Eleven percent of genes on the array had significantly altered mRNA expression in the prenatal alcohol-exposed neural progenitor culture under proliferating conditions. These include reduced expression of Adora2a, Cxcl1, Dlg4, Hes1, Nptx1, and Vegfa and increased expression of Fgf13, Ndn, and Sox3; bioinformatics analysis indicated that these genes are involved in cell growth and proliferation. Decreased levels of Dnmt1 and Dnmt3a were also found under proliferating conditions. Under differentiating conditions, 7.3% of genes had decreased mRNA expression; these include Cdk5rap3, Gdnf, Hey2, Heyl, Pard6b, and Ptn, which are associated with survival and differentiation as indicated by bioinformatics analysis. This study is the first to use chronic low to moderate PAE, to more accurately reflect maternal alcohol consumption, and subsequent neural progenitor cell culture to demonstrate that PAE throughout gestation alters expression of genes involved in neural development and embryonic neurogenesis.Alcohol 08/2014; · 2.04 Impact Factor
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ABSTRACT: Ethanol and nicotine are frequently co-abused. The biological basis for the high co-morbidity rate is not known. Alcohol-preferring (P) rats will self-administer EtOH or nicotine directly into the posterior ventral tegmental area (pVTA).Psychopharmacology 08/2014; · 3.99 Impact Factor
GDNF is a fast-acting potent inhibitor of alcohol
consumption and relapse
Sebastien Carnicella*, Viktor Kharazia*, Jerome Jeanblanc*, Patricia H. Janak*†, and Dorit Ron*†‡
*The Ernest Gallo Research Center and†Department of Neurology, University of California at San Francisco, Emeryville, CA 94608
Edited by Hans Thoenen, Max Planck Institute of Neurobiology, Martinsried, Germany, and approved April 23, 2008 (received for review December 13, 2007)
Previously, we demonstrated that the action of the natural alka-
loid, ibogaine, to reduce alcohol (ethanol) consumption is medi-
ated 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
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 re-
sults suggest that GDNF, via activation of the MAPK pathway, is a
fast-acting selective agent to reduce the motivation to consume
and seek alcohol.
addiction ? growth factor ? self-administration
and spinal cord motoneurons and exerts a wide range of effects
on peripheral and central neurons (1). GDNF is also a potent
the function of GDNF in the development and maintenance of
dopaminergic neurons in vivo remains unclear (3–6). However,
strong evidence supports an important neurorestorative role of
exogenously applied (7, 8) and endogenous (9) GDNF after
lesion of the nigrostriatal system. GDNF acts through a multi-
component receptor system including the glycosyl-phosphatidyl-
inositol-linked GDNF family receptor ?1 (GFR?1) and the
tyrosine kinase receptor Ret (1). Ligation of GDNF to GFR?1
leads to the recruitment and activation of Ret and to the
consequent activation of the MAPK, phosphoinositide 3-kinase
(PI3K), and phospholipase C? (PLC?) pathways (1). In addition,
Src family tyrosine kinases have been implicated in GDNF-
mediated functions mainly via a Ret-independent mechanism (10).
GFR?1 and Ret are highly expressed in the midbrain ventral
tegmental area (VTA) (11, 12), a brain region that is a critical
component of the neural circuitry involved in drug- and alcohol-
seeking behavior (13–15). Moreover, VTA dopaminergic neu-
rons are selectively vulnerable to some neuroadaptations in-
duced by repeated exposure to drugs of abuse and ethanol (16,
17). Interestingly, a role for GDNF in addiction has been
suggested based on evidence acquired from the examination of
a variety of drugs of abuse (18). For example, repeated admin-
istration of cocaine and morphine decreases Ret phosphoryla-
tion (i.e., activity) in the VTA (19), whereas phencyclidine
administration was found to increase GDNF expression in the
VTA and the substantia nigra (20). In addition, administration
of GDNF into the VTA blocks biochemical adaptations to
lial cell line-derived neurotrophic factor (GDNF) is an
essential growth factor for the development of the kidneys
cocaine and morphine exposure (19). Furthermore, heterozy-
gous GDNF knockout mice (Het) are more vulnerable to
morphine- and cocaine-induced psychomotor sensitization than
their wild-type (WT) littermates (19, 21). The GDNF Het mice
also exhibit increased sensitivity to cocaine place conditioning
(19) and to acquisition and reinstatement of methamphetamine
self-administration compared with the WT mice (22). Con-
versely, intra-VTA infusion of GDNF reduces cocaine place
conditioning (19), and sustained administration of GDNF in the
striatum impedes acquisition of cocaine self-administration (23,
24). In addition, Niwa et al. (25) recently reported that increasing
GDNF expression in the brain blocks methamphetamine place
conditioning and psychomotor sensitization. Finally, we previ-
ously showed that the decrease in ethanol self-administration
induced by the natural alkaloid ibogaine is mediated by the
up-regulation of GDNF and activation of its signaling pathway
in the VTA (26). Interestingly, we also found a reduction of
ethanol self-administration after intra-VTA injection of a single
actions of ibogaine are mediated via an autoregulatory positive
feedback loop in which GDNF triggers its own expression (27).
pathway in the mesolimbic system may be a valuable strategy to
combat alcoholism. Therefore, we set out to characterize the
ability of GDNF in the VTA to regulate alcohol-drinking
behavior and to identify a molecular mechanism that mediates
Intra-VTA Microinjection of GDNF Rapidly Decreases Ethanol Self-
Administration. First, we tested the effect of intra-VTA admin-
istration of GDNF on rat operant self-administration of a 10%
ethanol solution (28). We found that GDNF infused into the
VTA 10 min before the test session dose-dependently decreased
responding at the ethanol lever (Fig. 1A). GDNF infused into the
VTA 3 h before the beginning of the test session was also
effective in decreasing lever presses for ethanol (Fig. 1B).
Analysis of cumulative active lever-press responding within the
test session after the microinjection of PBS and the highest dose
of GDNF (10 ?g per side) revealed a reduction in the frequency
no change in the initiation of lever pressing for ethanol [sup-
porting information (SI) Fig. S1 and Table S1]. Next we exam-
ined the effect of GDNF on ethanol self-administration in rats
with a history of high voluntary ethanol consumption (29) (Fig.
Author contributions: S.C., P.H.J., and D.R. designed research; S.C., V.K., and J.J. performed
research; S.C. and P.H.J. analyzed data; and S.C. and D.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
‡To whom correspondence should be addressed at: 5858 Horton Street, Suite 200, Em-
eryville, CA 94608. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
June 10, 2008 ?
vol. 105 ?
in alcohol-preferring rats (30). After 24 drinking sessions, rats
were trained to lever-press for a 20% ethanol solution. Impor-
tantly, infusion of GDNF (10 ?g per side) into the VTA 10 min
and 3 h before the session also reduced ethanol self-
administration in this paradigm (Fig. 1D). Taken together, these
results suggest that GDNF-mediated reduction of operant eth-
anol self-administration is rapid and persistent.
Microinjection of GDNF into the Substantia Nigra Pars Compacta (SNc)
Does Not Decrease Ethanol Self-Administration. To determine the
site-specificity of GDNF’s action, we infused the growth factor
into the neighboring midbrain dopaminergic region, the SNc. As
shown in Fig. 2A, infusion of GDNF into the SNc did not alter
lever-press responding for ethanol, suggesting that the decrease
in ethanol self-administration observed after microinjection of
GDNF is specific for the VTA.
Intra-VTA Microinjection of GDNF Does Not Decrease Sucrose Self-
Administration. Next we determined the specificity of GDNF’s
actions by examining its ability to attenuate self-administration
of a naturally rewarding substance, sucrose. As shown in Fig. 2B,
intra-VTA injections of GDNF did not affect lever-press re-
sponding for sucrose. Hence, the decrease in ethanol self-
administration induced by intra-VTA infusion of GDNF was not
due to a general attenuation of motivation or to a change in
GDNF Activates the MAPK Extracellular Signal-regulated Kinase 1 and
2 (ERK1/2) in the VTA in Vivo, and Inhibition of ERK1/2 Activation
Blocks GDNF-Induced Decreases in Ethanol Self-Administration. Next
we set out to determine the signaling pathway that mediates the
rapid actions of GDNF on ethanol consumption. The MAPK
signaling pathway is one of the major downstream pathways
activated by GDNF (1), and ex vivo studies suggest that GDNF
rapidly modulates the activity of mesencephalic dopaminergic
neurons via this intracellular pathway (31, 32). Therefore, we
first assessed whether intra-VTA infusion of a behaviorally
effective dose of GDNF (10 ?g) activates ERK1/2, a key enzyme
in the MAPK signaling pathway. As shown in Fig. 3A, GDNF
infusion induced a robust increase in ERK1/2 phosphorylation
(i.e., activation) in the VTA, which was not observed in the
control side infused with PBS. Importantly, a significant fraction
of the phospho-ERK1/2 immunoreactivity was localized to ty-
rosine hydroxylase-positive neurons (Fig. 3), suggesting that
activation of the GDNF pathway in the VTA leads to the
to examine the involvement of the MAPK signaling pathway in
the attenuation of ethanol self-administration by GDNF, we
blocked the activation of ERK1/2 in the VTA by inhibition of
MAPK/ERK kinase (MEK), the upstream kinase that phos-
phorylates and activates ERK1/2 (33). As shown in Fig. 4A,
intra-VTA infusion of the MEK inhibitor U0126 (34) prevented
the decrease in ethanol self-administration induced by GDNF.
However, intra-VTA infusion of the PI3K inhibitor wortmannin
(34) did not alter the GDNF-mediated decrease in ethanol
self-administration (Fig. 4B), suggesting that PI3K is not in-
volved in GDNF’s regulation of ethanol consumption. We could
not assess whether PLC? also contributes to GDNF’s actions, as
intra-VTA infusion of its inhibitor, U73122 (35), alone resulted
in a reduction in ethanol self-administration (Fig. 4C) and
in 1 h after GDNF microinjection into the VTA (0, 2.5, 5, or 10 ?g 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
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 ?g 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
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 ?g 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)? 7.4, P ? 0.05] (n ? 8).**, P ? 0.01;***, P ? 0.001 (compared with PBS
Ten minutes and 3 h post infusion of GDNF in the VTA decreases
ethanol self-administration, and intra-VTA injections of GDNF do not affect
operant sucrose self-administration. (A) Mean ? SEM number of lever presses
or 3 h (0 or 10 ?g 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 ?g per side) or 3 h
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).
Intra-substantia nigra injections of GDNF do not affect operant
Carnicella et al.PNAS ?
June 10, 2008 ?
vol. 105 ?
no. 23 ?
therefore might have masked the effect of GDNF. Together,
these results suggest a crucial role for the MAPK signaling
pathway in the decrease in ethanol self-administration observed
after microinjection of GDNF into the VTA.
Intra-VTA Microinjection of GDNF Blocks Reacquisition of Ethanol
Self-Administration. Relapse to alcohol use is one of the core
features of alcoholism and is the main problem in the treatment
of alcohol dependence (36, 37). Reacquisition, a rapid return of
responding when the outcome is made available again after a
period of extinction, is a measure of relapse (38, 39) that is
especially relevant for therapies that seek to extinguish drug-
related behaviors (40). We therefore tested whether intra-VTA
infusion of GDNF would alter reacquisition of operant ethanol
self-administration after a period of extinction. To obtain a
one-session reacquisition, the retrieval of operant ethanol self-
administration was triggered with a prime of a noncontingent
session started, as described in Materials and Methods.
As shown in Fig. 5A, this procedure induced a rapid and
effective reacquisition of ethanol self-administration. Impor-
of the session blocked this reacquisition of operant responding
for ethanol as the active lever responding after GDNF treatment
was not significantly different from active lever-press responding
at the end of extinction (P ? 0.41), or from inactive lever
responding during the reacquisition session (P ? 0.20). In
third press, i.e., the first reward, relative to the latency on the last
day of extinction in vehicle-treated rats (Fig. 5B and Table S1),
indicating that this rapid reacquisition was promoted by the
ethanol prime. This result also suggests that the ethanol prime
reinstated interest in the reinforced lever. Interestingly, the
ethanol priming effect was completely blocked by intra-VTA
infusion of GDNF (Fig. 5B), as the latencies to the third press
during the reacquisition test and on the last day of extinction
were similar. This observation was further supported by the
analysis of cumulative active lever-press responding (Fig. S3)
showing a significant delay in the initiation of lever pressing for
ethanol in GDNF-treated rats. Taken together, these data
suggest that the ability of an ethanol prime to induce rapid
reacquisition of operant ethanol self-administration is blocked
by the application of GDNF.
We identified a very rapid effect of GDNF to selectively reduce
ethanol self-administration in two different paradigms; in one
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 ?m.) (B) Enlarged image
both for p-ERK1/2 and tyrosine hydroxylase. (Scale bar: 50 ?m.)
GDNF activates ERK1/2 in midbrain dopaminergic neurons in vivo.
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:
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.
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 (0 or 10 ?g/1 ?l per side) 10 min before the
reacquisition test. Two-way ANOVA with repeated measures showed signif-
icant 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
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 (?2? 12.78, P ? 0.01).
*, P ? 0.05;***, P ? 0.001 (n ? 9).
Intra-VTA injection of GDNF blocks reacquisition of operant ethanol
www.pnas.org?cgi?doi?10.1073?pnas.0711755105Carnicella et al.
paradigm, rats self-administer relatively low doses of ethanol
whereas in the other rats self-administer relatively high levels of
ethanol after a history of high consumption (Fig. S2). This action
of GDNF is specifically mediated by the VTA, a primary site for
the rewarding effects of ethanol (14), via the activation of the
MAPK signaling pathway. We also demonstrate that GDNF
infused into the VTA only 10 min before the operant test session
completely blocked reacquisition of ethanol self-administration.
Together, these findings indicate that GDNF in the VTA rapidly
reduces ethanol drinking and relapse.
To our knowledge, this is the first evidence of a rapid action
(within minutes) of a growth factor on drug consumption and
relapse. As such, it is likely that GDNF’s actions are mediated
via a nontranscriptional mechanism. Several ex vivo studies
suggest that GDNF acutely regulates the function of dopami-
nergic neurons in the midbrain. GDNF treatment of mesence-
phalic dopaminergic neurons increased the activity of tyrosine
hydroxylase within minutes (32), as well as the excitability of the
neurons by inhibition of A-type K?channels (31). GDNF was
also shown to rapidly increase synaptic transmission by poten-
of tyrosine hydroxylase and A-type K?channel activity de-
pended on the activation of MAPK (31, 32). Here we show that
in vivo administration of GDNF results in rapid activation of
the MAPK signaling pathway in the VTA and that blocking the
MAPK signaling pathway in this brain region prevents the
modulation of ethanol self-administration by GDNF. Thus, it is
plausible that the growth factor rapidly modifies neuronal ex-
citability via activation of the MAPK pathway, and by doing so
GDNF alters the incentive value and/or reinforcing strength of
Activation of the MAPK signaling pathway, but not the PI3K
pathway, within the VTA is critical for the rapid action of GDNF
on ethanol self-administration. However, the role of the PLC?/
PKC could not be assessed because administration of the PLC
inhibitor alone reduced ethanol self-administration, suggesting
that PLC is involved in mechanisms that underlie consumption
of ethanol. This result is not entirely surprising because the
involvement of several PKC isozymes in ethanol-drinking be-
haviors has been documented (42).
We also show that the action of GDNF on ethanol self-
administration is specific to the VTA, because infusion of effective
doses of GDNF in the neighboring SNc dopaminergic area did not
because the immunocytochemistry experiment suggests that
SNc and activate the MAPK signaling pathway in this structure as
well. Therefore, these results strongly suggest that activation of the
MAPK signaling pathway by GDNF specifically in the VTA
reduces ethanol self-administration.
Interestingly, the ability of GDNF to reduce ethanol self-
administration was observed 3 h after infusion. As a growth
factor, it is possible that GDNF induces several transcriptional
changes that could sustain its action. For example, it has been
shown that GDNF increases the expression of tyrosine hydrox-
ylase and the dopamine transporter (43), inducing modifications
of the mesolimbic system that persist beyond the activation and
termination of GDNF signaling. Another possible mechanism is
a sustained effect by GDNF itself, as we recently showed that
GDNF up-regulates its own expression, leading to a sustained
activation of the GDNF signaling pathway (27).
Intra-VTA infusion of GDNF did not alter self-administration
of sucrose, a natural reward, suggesting that the reduction in
lever-press responding for ethanol was not due to nonspecific
motor effects of GDNF. Other pharmacological manipulations
have been previously reported to modulate the seeking and
consumption of drugs of abuse and ethanol but not natural
rewards (26, 44–46). Although it has been widely accepted that
drugs of abuse and natural rewards have the mesolimbic dopa-
minergic system as a common substrate, several studies have
suggested that processes involved in the rewarding effects of
sucrose and ethanol are different. For example, both sucrose
self-administration and ethanol self-administration induce an
increase in dopamine concentration in the nucleus accumbens
(47–49); however, in the case of sucrose, this seems to be linked
to locomotor or operant/learning processes because neither the
first ingestion of sucrose nor the unpredicted delivery of sucrose
during operant self-administration changes dopamine levels in
the nucleus accumbens, whereas ethanol does (50, 51). Also,
microinjection of a D2 agonist in the anterior part of the VTA
decreases ethanol but not saccharin self-administration (52), and
microinjection of D1/2 agonists/antagonists produces distinct
effects on cocaine and sucrose self-administration (53). To-
gether, these results suggest a specific involvement of GDNF in
the VTA in ethanol and/or addictive processes but not in general
rewarding and/or motivational mechanisms.
Relapse is one of the main challenges in the treatment of
alcohol abuse (36, 37). A therapeutic approach to treat relapse
is to prevent the initial lapse, or the consequence of the initial
lapse, that spirals into relapse (54–56). In this regard, rapid
reacquisition of an operant response for a drug of abuse after
re-exposure to the drug is a particularly relevant relapse model,
especially in the case of therapies that seek to extinguish
drug-taking behaviors (39, 40). We found that re-exposure to the
exteroceptive properties of ethanol (i.e., the sensory cues of the
ethanol prime, such as the taste and the specific odor) induced
Importantly, intra-VTA infusion of GDNF 10 min before the
operant session blocked this reacquisition of responding for
ethanol. This effect of GDNF is likely mediated by a specific
action on some reacquisition mechanism(s), because the ethanol
reinforced lever and responding for ethanol. This is illustrated by
a representative example of one individual rat’s pattern of
responding during the reacquisition test (Fig. S3B), showing a
significant delay to perform the first ratio in the GDNF condi-
tion. This effect of the ethanol prime suggests that GDNF
reduces not only ethanol consumption but also ethanol seeking.
In conclusion, our results suggest a unique fast-acting effect of
GDNF mediated by the MAPK pathway in the VTA that
selectively reduces ethanol consumption. Moreover, GDNF in
the VTA blocked reacquisition of operant ethanol self-
administration (a model of relapse), suggesting that GDNF is
involved in different aspects of ethanol-drinking and -seeking
behaviors. Our results also put forward the potential use of
targets within the GDNF pathway for the development of
treatment against alcohol abuse and, most importantly, relapse.
Materials and Methods
Reagents. Reagents are detailed in SI Materials and Methods.
housed under a 12-h light/dark cycle (lights on at 0700 hours) with food and
by the Gallo Center Institutional Animal Care and Use Committee and were
conducted in agreement with the Guide for the Care and Use of Laboratory
Animals, National Research Council, 1996.
Operant Ethanol Self-Administration. Rats were habituated to drinking etha-
nol in their home cages by exposure to 10% ethanol in tap water (vol/vol)
mixed with a decreasing concentration of sucrose (10%, 5%, and 0%, wt/vol).
After 3 weeks, operant ethanol self-administration training commenced. The
which presses result in delivery of 0.1 ml of a 10% ethanol solution, and an
inactive lever, for which responses are counted as a measure of nonspecific
Carnicella et al.PNAS ?
June 10, 2008 ?
vol. 105 ?
no. 23 ?
sessions were conducted 5 days per week, with the schedule requirement
increased to FR3 over the first week. Because the level of presses on the
inactive lever was low after acquisition of the self-administration paradigm
(?10 presses), and the activity on this lever was not affected by any of the
experimental treatments, this measure was removed from the figures for
better clarity but taken into account in the statistical analyses. After 2 months
of training, surgery to implant cannulae was conducted.
Operant Self-Administration After Intermittent Access to Ethanol. Intermittent
access to 20% ethanol in tap water (vol/vol) was provided according to
above, with the reinforcer being 0.1 ml of a 20% ethanol solution and the
session length shortened to 30 min after 2 weeks of training. Surgery to
Sucrose Self-Administration. Rats were initially trained under FR1 by using 8%
sucrose (wt/vol) as the reinforcer during two overnight sessions. The FR
schedule was then progressively increased to FR3, and sucrose concentration
was progressively decreased to 2%. The rats were trained under this final
schedule 5 days per week in 60-min sessions. As for the ethanol experiments,
the inactive lever measure was removed from the figures but not from the
the cannulae was conducted before the behavioral procedure.
Surgery and Microinjection. Bilateral guide cannulae (C235G-2.0, 26 gauge;
Plastics One) were aimed dorsal to the VTA (5.6 mm posterior to bregma, 1.0
mm mediolateral, 8.0 mm ventral to the skull surface) or the SNc (5.4 mm
according to Paxinos and Watson (57). Drug or vehicle was infused into the
VTA or the SNc of gently restrained rats via injection cannulae extending 0.5
mm beyond the guide cannula tip. All subjects received each treatment in a
counterbalanced manner, with one injection per week (see SI Materials and
Methods for details).
Intra-VTA Microinjection of MAPK, PI3K, and PLC Inhibitors. A total of 0.5 ?l of
U0126 (1 ?g/?l), wortmannin (0.1 ?g/?l), U73122 (0.2 ?g/?l), or the appropri-
of GDNF (0.5 ?l of a 10 ?g/?l solution per side) (see SI Materials and Methods
Immunohistochemistry. GDNF or PBS was infused in the VTA 10 min before
perfusion and removal of the brain as described in SI Materials and Methods.
Perfusion and immunocytochemistry procedures are as described in ref. 58
and in SI Materials and Methods.
Intra-VTA Microinjection of GDNF and Reacquisition of Ethanol Self-Adminis-
tration. After 2 months of ethanol self-administration training, rats under-
the reacquisition test session, in which a 0.2-ml drop of 10% ethanol was
delivered into the reward port noncontingently to the lever response when
the session started (the ethanol prime). Subsequently, three lever presses
the self-administration procedure. After 2 weeks of reacquisition of ethanol
self-administration followed by nine further extinction sessions, a second
reinstatement test session was conducted with the drug treatment reversed.
Histology. Locations of cannulae were verified in 60-?m coronal sections
stained with thionin. Only data from subjects with injectors located in the
region of interest (Fig. S4) were included in the analysis.
Statistical Analyses. Each experiment was conducted in a within-subjects
design. The number of lever presses was analyzed by using ANOVAs with
repeated measures. Significant main effects and interactions of the ANOVAs
were further investigated by using the Student–Newman–Keuls test. Because
the data did not conform to a normal distribution, the latency to the first
Significant effects of these ANOVAs were further investigated by using the
nonparametric variant of the Student–Newman–Keuls test (Sigmastat 2004;
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health–National Institute on Alcohol Abuse and Alcoholism Grant R01
AA014366-02 (to D.R. and P.H.J.) and the State of California for Medical
Research on Alcohol and Substance Abuse through the University of Califor-
nia, San Francisco (D.R. and P.H.J.).
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Carnicella et al. PNAS ?
June 10, 2008 ?
vol. 105 ?
no. 23 ?
Carnicella et al. 10.1073/pnas.0711755105
SI Materials and Methods
Reagents. GDNF was purchased from R & D Systems. The MEK
inhbitor U0126 was purchased form Calbiochem; the PI3K
inhibitor wortmannin and the PLC inhibitor U73122 were from
Sigma. The monoclonal anti-[pThr202/Tyr-204]-p44/42 MAPK
(p-ERK1/2) rabbit antibody was purchased from Cell Signaling
Technology, and the monoclonal mouse anti-tyrosine hydroxy-
lase antibody was purchased from Sigma. The secondary anti-
bodies Cy3-labeled donkey anti-rabbit and Alexa Fluor 488-
labeled donkey anti-mouse were purchased from Jackson
ImmunoResearch and Invitrogen, respectively.
Surgery and Microinjection. Rats were anesthetized continuously
with isoflurane (Baxter Health Care). Bilateral guide cannulae
(C235G-2.0, 26 gauge; Plastics One) were aimed dorsal to the
VTA (5.6 mm posterior to bregma, 1.0 mm mediolateral, 8.0 mm
ventral to the skull surface) or the SNc (5.4 mm posterior to
bregma, 2.6 mm mediolateral, 6.8 mm ventral to the skull
surface), according to Paxinos and Watson (1). The coordinates
for the VTA were identical to those used in a previous study (2),
allowing us to target mainly the posterior part of this structure,
which is preferentially involved in rewarding processes and
mediation of the reinforcing effects of ethanol (3, 4). One week
after recovery, subjects returned to self-administration training,
and microinjections began when responding was stable.
Drug or vehicle was infused over 2 min into the VTA or the
SNc of gently restrained rats via injection cannulae extending 0.5
mm beyond the guide cannula tip. Injection cannulae were left
in place for an additional 2 min. All subjects received each
treatment in a counterbalanced manner, with one injection per
week, allowing the lever-press responding for ethanol to return
to baseline between treatments.
Intra-VTA Microinjection of MAPK, PI3K, and PLC Inhibitors. U0126
was diluted to a concentration of 1 ?g/?l in 5% DMSO and 6%
Tween 80 in PBS. Wortmannin was diluted to a concentration of
0.1 ?g/?l in 25% DMSO in PBS. U73122 was diluted to a
concentration of 0.2 ?g/?l in DMSO. Concentrations and prep-
aration of compounds were based on previous in vivo studies
(5–8). A total of 0.5 ?l of the inhibitor or the appropriate vehicle
per side was infused into the VTA 1 h before the intra-VTA
infusion of GDNF (0.5 ?l of a 10 ?g/?l solution per side).
Immunohistochemistry. Rats were anesthetized continuously with
isoflurane. Two holes were drilled above the injection sites to
allow the introduction of the injection cannulae targeting the
VTA (5.6 mm posterior to bregma, 1.0 mm mediolateral, 8.5 mm
ventral to the skull surface). After stereotaxic placement of the
injection cannulae, GDNF (10 ?g/1 ?l per side over 2 min) was
infused into one side and PBS was infused into the other side of
paraformaldehyde and brains were removed. Free-floating coro-
nal 40-?m-thick sections from the VTA were incubated with
anti-p-ERK1/2 antibody (1:200) and anti-tyrosine hydroxylase
(1:2,000) antibody. Perfusion and immunocytochemistry fol-
lowed a procedure described previously (9). Images were ac-
quired by using a Zeiss LSM 510 Meta confocal microscope.
Immunocytochemistry and image acquisition were done by an
investigator blind to treatment conditions.
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Carnicella et al. www.pnas.org/cgi/content/short/0711755105 1 of 6
in bins of 5 min, indicative of the rate of presses for ethanol during the session, 10 min (A) or 3 h (B) after intra-VTA injection of PBS and GDNF (10 ?g per side).
Two-way ANOVA with repeated measures (time as the first factor and treatment as the second factor) showed significant main effects of, and a significant
interaction between, both factors at both time points (F ? 9.42, P ? 0.01). Subsequent analysis using the method of contrasts detected the first significant
difference between PBS and GDNF treatment at 10–15 min [T(8)? 4.90, P ? 0.001, black arrow] for the 10-min pretreatment, and at 20–25 min [T(8)? 2.04, P ?
0.05, black arrow] for the 3-h pretreatment (n ? 10). (C–F) Examples of two different individual representative patterns of presses for the 10 min (C and E) and
3 h (D and F) pretreatment times.
Ten minutes and 3 h post infusion of GDNF change the within-session pattern of responding for ethanol. (A and B) Cumulative mean ? SEM presses
Carnicella et al. www.pnas.org/cgi/content/short/0711755105 2 of 6
for a 20% ethanol solution (20% self-adm) after a period of high voluntary ethanol drinking (n ? 8). (B) Examples of individual representative patterns for the
10% self-administration and the 20% self-administration conditions. Compared with the standard 10% self-administration, rats with a history of high intake
pressed at a higher rate, mainly during the first half of the self-administration session, and for a more concentrated ethanol solution (20%). As such, subsequent
20% ethanol self-administration sessions were reduced to 30 min. These results suggest a higher motivation for ethanol in rats trained to self-administer 20%
ethanol after a period of high intake.
History of elevated ethanol consumption induces sustained self-administration of 20% ethanol. (A) Cumulative mean ? SEM presses in bins of 5 min
Carnicella et al. www.pnas.org/cgi/content/short/07117551053 of 6
in bins of 5 min, indicative of the rate of presses during the session, for PBS and GDNF treatment (F ? 7.08, P ? 0.001). Subsequent analysis using the method
of contrasts detected the first significant difference between PBS and GDNF treatment at 0–5 min [T(8)? 2.14, P ? 0.05] (n ? 9). (B) Example of a representative
pattern of presses from an individual subject.
Carnicella et al. www.pnas.org/cgi/content/short/07117551054 of 6
tip. (B and D) Histological reconstruction of cannulae sites into the VTA (C) and the SNc (D) on sagittal and horizontal diagrams. All of the sites were localized
in the gray area. Diagrams were adapted from Paxinos and Watson (1).
(A and C) Representative guide cannula placements in the VTA (A) and the SNc (C). Note that the injection site was 0.5 mm below the guide cannula
Carnicella et al. www.pnas.org/cgi/content/short/07117551055 of 6
Table S1. Latencies (in seconds) to the first and third (first reward) lever presses
First press, sFirst reward, s
ExperimentMean ? SEM Median (minimum, maximum) Mean ? SEM Median (minimum, maximum)
Intra-VTA PBS, after 10 min
Intra-VTA GDNF, after 10 min
Intra-VTA PBS, after 3 h
Intra-VTA GDNF, after 3 h
Extinction before PBS
Extinction before GDNF
Intra-VTA PBS, after 10 min
Intra-VTA GDNF, after 10 min
86.2 ? 20.5
303.6 ? 132.4
58.8 ? 14.2
113.4 ? 36.0
76.7 (2.4, 228.2)
146.8 (17.0, 466.3)
48.3 (15.9, 166.8)
91.7 (18.9, 376.3)
109.6 ? 17.9
331.2 ? 119.7
128.0 ? 28.7
252.2 ? 99.6
96.4 (45.5, 234)
165.3 (40.7, 876.8)*
96.4 (48.0, 312.6)
108.5 (54.0, 849.4)
90.7 ? 24.2
730.1 ? 200.9
697.8 ? 291.5
127.5 ? 37.5
131.4 ? 67.4
76.3 (14.5, 205.5)
662.3 (23.1, 2,137.3)
376.9 (19.4, 3,145.6)
75.1 (12.7, 406.0)
67.5 (8.1, 720.4)
133.4 ? 37.1
1,523.6 ? 369.2
1,794.2 ? 374.8
342.0 ? 120.9
2,006.2 ? 356.9
101.2 (46.9, 424.2)
1,311.4 (304.5, 3,600)
1,892.4 (20.38, 3,600)
155.3 (76.5, 1,365.7)*
2,182.0 (116.0, 3,600)
For ethanol self-administration experiments:*, P ? 0.05 (Wilcoxon matched-pairs test). For ethanol reacquisition experiment: the Friedman repeated-
measures ANOVA on ranks found significant effects of the latencies to the first press (?2? 8.33, P ?0.05) and the third press (first reward, ?2? 12.8, P ? 0.01).
Post hoc analysis showed significant differences only for the latency to the third press.*, P ? 0.05 for PBS condition compared with the other three conditions.
Carnicella et al. www.pnas.org/cgi/content/short/0711755105 6 of 6