Erk1/2 Mediates Leptin Receptor Signaling in the Ventral
Tegmental Area
Richard Trinko1, Geliang Gan2, Xiao-Bing Gao2, Robert M. Sears1, Douglas J. Guarnieri1, Ralph J.
DiLeone1*
1Division of Molecular Psychiatry, Ribicoff Research Facilities, Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, United States of
America, 2Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut, United States of America
Abstract
Leptin acts on the ventral tegmental area (VTA) to modulate neuronal function and feeding behavior in rats and mice. To
identify the intracellular effectors of the leptin receptor (Lepr), downstream signal transduction events were assessed for
regulation by direct leptin infusion. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and
phosphorylated extracellular signal-regulated kinase-1 and -2 (pERK1/2) were increased in the VTA while phospho-AKT
(pAKT) was unaffected. Pretreatment of brain slices with the mitogen-activated protein kinase kinase -1 and -2 (MEK1/2)
inhibitor U0126 blocked the leptin-mediated decrease in firing frequency of VTA dopamine neurons. The anorexigenic
effects of VTA-administered leptin were also blocked by pretreatment with U0126, which effectively blocked
phosphorylation of ERK1/2 but not STAT3. These data demonstrate that pERK1/2 may have a critical role in mediating
both the electrophysiogical and behavioral effects of leptin receptor signaling in the VTA.
Citation: Trinko R, Gan G, Gao X-B, Sears RM, Guarnieri DJ, et al. (2011) Erk1/2 Mediates Leptin Receptor Signaling in the Ventral Tegmental Area. PLoS ONE 6(11):
e27180. doi:10.1371/journal.pone.0027180
Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America
Received July 3, 2011; Accepted October 11, 2011; Published November 4, 2011
Copyright: � 2011 Trinko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported by National Institutes of Health (NIH) grant DK076964 to R.J.D. and the State of Connecticut, Department of Mental Health
and Addiction Services (R.J.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ralph.dileone@yale.edu
Introduction
Leptin is a protein hormone produced by adipocytes that serves
to communicate fat levels to the brain. Within the central nervous
system (CNS), leptin acts on multiple brain regions including the
brainstem, hypothalamus, hippocampus, and ventral tegmental
area (VTA) by activating the cytokine type I leptin receptor
[1,2,3,4,5]. Work on the hypothalamus has shown that Lepr
signaling regulates multiple downstream pathways to modulate
neuronal function, food intake, and body weight homeostasis. Lepr
is coupled to Janus Kinase 2 (JAK2), which is required for all
leptin-mediated signaling, including recruitment and subsequent
activation of STAT3, ERK1/2, and phosphatidylinositol-3-kinase
(PI3-K) [6]. Conditional mutant mice lacking either neural
STAT3 expression, or STAT3 activation, have recapitulated the
obese phenotypes observed in leptin deficient (ob/ob) and the Lepr
deficient (db/db) mice [7,8,9]. While these mutant models suggest
that STAT3 is clearly an important mediator of leptin signaling,
the rapid regulation of neuronal firing in the hypothalamus [10],
and in the VTA [4], is not likely to be mediated by STAT3-
dependent transcriptional events.
In the hypothalamus, Lepr signaling can also activate ERK1/2
and PI3-K [11,12,13,14]. Pretreatment of rats with MEK1/2
inhibitors blocked leptin-induced ERK1/2 phosphorylation in the
hypothalamus, as well as attenuating the homeostatic feeding
effects of Lepr [15]. It has been demonstrated that PI3-K is also
required for the anorexic effects of insulin as well as leptin in the
CNS, thus illustrating potential cross-talk between leptin and
insulin signaling [14,16]. Moreover, in the hypothalamus, it has
been suggested that the ERK1/2 pathway mediates leptin-induced
reduction of firing rates, while PI3-kinase is responsible for the
leptin’s disinhibitory effects on firing [17].
In the VTA, Lepr signaling reduces dopamine neuron firing
and food intake, while RNAi-mediated knockdown of Lepr in the
VTA results in a chronic increase in food intake without an
associated weight gain [4]. These data, complemented with studies
by others [5], supports a physiologic role of leptin signaling to this
brain region. Like the hypothalamus, leptin signaling results in
STAT3 phosphorylation at residue Tyr-705 in the VTA [4]. In
contrast to the hypothalamus however, PI3-kinase appears not to
be a mediator of leptin’s effects in the VTA [18]. This represents
the first observed difference in leptin signaling pathways between
these brain regions, thus highlighting the need for additional
characterization. In the VTA, it remains unclear whether the
ERK1/2 pathway: 1) is regulated by leptin, 2) contributes to
dopamine neuron firing, and 3) has a role in mediating leptin
feeding responses specific to this brain region. Here, we evaluate
potentially important Lepr signaling events using western blotting,
electrophysiology and behavioral pharmacology to identify the
contributions of the ERK1/2 pathway in the VTA.
Results
Direct leptin infusion to the VTA of rats results in multiple
phosphorylation events
To assess Lepr signaling pathways in the VTA, leptin or vehicle
was directly infused to the VTA of rats, which were sacrificed
45 minutes later. Western blot analysis of dissected VTA tissue
PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27180
revealed phosphorylation events within two canonical leptin
signaling pathways. Consistent with our prior study, leptin induced
the phosphorylation of STAT3 (Tyr-705), as well as pERK1/2
(Thr-202, Tyr-204) (Fig. 1A, B). The PI3-K pathway was
evaluated indirectly by measuring a downstream target, AKT.
In contrast with pSTAT3 and pERK1/2, neither of the two
residues known to be involved in regulating AKT activity (Thr-
308, Ser-473) were affected by leptin at this time point (Fig. 1C,
D).
U0126 abolishes the firing response of dopamine
neurons to leptin in the VTA of mice
The role of ERK1/2 in mediating the electrophysiological
response of dopamine neurons to leptin was assessed by blocking
the MEK1/2 kinase needed for phosphorylation and activation of
ERK1/2. Coronal mouse slices containing the VTA were
incubated with or without the MEK1/2 inhibitor U0126 prior
to application of leptin. Spontaneous firing events of dopamine
cells were observed by whole-cell current clamp. Consistent with
our prior study [4], application of leptin to the bath resulted in
decreased frequency of action potentials (48.5627.3% of control,
n = 3) throughout the duration of application and returned to
baseline level (102.768.6% of control, n = 3) after the removal of
leptin. Representative traces are shown in Fig. 2A (right panel),
and a representative timeline in Fig. 2B. This effect was completely
attenuated when U0126 was applied for 20 minutes prior to leptin
administration (Fig. 2A, left panel; Fig. 2B). The action potential
frequency was 100.166.2% of control (P.0.05, n = 6, t-test) in the
presence of leptin and U0126, and 104.263.4% of control (n = 6)
after the removal of leptin, as summarized in Fig. 2C. Notably,
there was no change in action potential frequency as a result of
U0126 during pretreatment (Fig. 2C).
U0126 abolishes the feeding effects of leptin signaling in
the VTA of rats
We have previously demonstrated that leptin infusions to the
VTA results in consistent and robust reductions in intake [4]. To
investigate the role of the ERK1/2 pathway in mediating the
behavioral effects of leptin, U0126 was infused intracerebroven-
tricular (ICV) 1.5–2 hours before leptin infusion in the VTA, and
food intake was assessed. Rats in the U0126ICV/vehicleVTA group
exhibited no change in basal feeding over a 23 hour period when
compared to the vehicleICV/vehicleVTA group (Fig. 3). Rats in the
vehicleICV/leptinVTA group exhibited a significant decrease in
food intake over 23 hours. Finally, rats in the U0126ICV/
leptinVTA group exhibited no changes in feeding, suggesting that
ERK1/2 signaling is required for leptin’s effects on feeding in the
VTA.
U0126 blocks leptin-induced phosphorylation of ERK1/2,
but not STAT3 (Tyr-705) in the VTA of rats
It is possible that the U0126 compound was interfering with
leptin receptor activation of STAT3 in response to leptin. To test
this, pSTAT3 (Tyr-705) was assessed in the presence of the Mek
inhibitor. U0126 was infused 1.5–2 hours before leptin infusion
into the VTA, and the VTA was dissected 45 minutes later to
evaluate pSTAT3. VTA pSTAT3 (Tyr-705) levels in U0126ICV/
vehicleVTA rats remained unchanged relative to vehicleICV/
vehicleVTA animals, while pSTAT3 (Tyr-705) in both vehi-
cleICV/leptinVTA and U0126ICV/leptinVTA rats showed a signif-
icant increase (Fig. 4A). This demonstrates that the behavioral and
electrophysiological effects of U0126 are not due to indirect effects
via STAT3 phosphorylation. In contrast with pSTAT3, VTA
pERK1/2 (Thr-202, Tyr-204) levels were found to be unchanged
in U0126ICV/leptinVTA rats (Fig. 4B), indicating effective blockade
of the MEK-ERK pathway.
Discussion
Lepr signaling studies in the hypothalamus have identified key
components needed for leptin signaling [19,20,21,22,23]. Our
previous work identified a physiologic role for Lepr expression in
the VTA [4]. Direct infusion of leptin to the VTA of naı¨ve rats
resulted in hypophagia, while local knockdown of Lepr resulted in
hyperphagia, thereby demonstrating responses to localized
exogenous, as well as endogenous leptin [4]. The identification
of insulin and leptin receptor coexpression on dopamine neurons
in the VTA [13], and preliminary signaling studies [4,5] suggest
similarities in pathway activities between the VTA and hypothal-
amus. Here, we initially examined these signaling events by
evaluating protein phosphorylation in the VTA after direct leptin
infusion. ERK1/2 is regulated by leptin, and blockade of this
pathway eliminated leptin’s effects on both neuronal firing and
feeding behavior.
Consistent with published data, pSTAT3 (Tyr-705) was
increased in the VTA after direct leptin infusion [4,5]. ERK1/2
also exhibited increased phosphorylation in the VTA after direct
leptin. These findings highlight a similarity in leptin signaling
between the hypothalamus and the VTA. In contrast, both
regulatory residues on AKT, a known downstream target of
insulin-induced PI3-K activity [24], showed no significant change
after direct leptin. The lack of PI3-K regulation is consistent with
work showing no role of this pathway in mediating the effects of
leptin in the VTA [18]. This illustrates a potential divergence in
Figure 1. Direct leptin to the rat VTA induced phosphorylation
events. Regulation of various Lepr pathways was evaluated 45 minutes
after direct leptin infusion to the VTA of cannulated awake adult male
rats. Ratios of phospho-/total signal were used to calculate the percent
change from control tissue. Blots shown are representative. Open bars
represent vehicle while black bars represent leptin treatment. A.
pSTAT3 (Tyr-705) (n= 4) *, (P = 0.0077). B. pERK1/2 (Thr-202, Tyr-204)
(n= 4) *, (P = 0.0252). C. pAKT (Ser-473) (n= 4). D. pAKT (Thr-308) (n= 4).
doi:10.1371/journal.pone.0027180.g001
Erk1/2 Mediates Leptin Receptor Signaling in VTA
PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27180
leptin signaling between brain regions, as it has been previously
suggested that hypothalamic Lepr activation of PI3-K is crucial for
mediating the feeding effects of leptin [16,18,25,26].
To date, there is little research evaluating the role of ERK1/2 in
mediating the electrophysiological effects of leptin. It has been
demonstrated that leptin can regulate calcium concentrations in
isolated hypothalamic neuropeptide Y (NPY) and proopiomela-
nocortin (POMC) neurons in an opposing manner [27]. More
recently, Wang and colleagues suggested a role for hypothalamic
ERK1/2 in mediating leptin’s effect on calcium regulation in NPY
neurons, but not POMC neurons [17]. In the VTA, leptin reduces
dopamine firing frequency [4] and data presented here suggests
that ERK1/2 mediates this effect in the VTA dopamine neurons,
as with NPY neurons in the hypothalamus [17].
Figure 2. U0126 abolished the firing response of dopamine neurons to leptin in mice. See results section for statistical information. A.
Representative traces of action potentials recorded before, during, and after the application of leptin to mouse VTA DA neurons in slices with (left
panel) or without (right panel) U0126 (10 mM). Box, H-current recorded in VTA DA neurons. Hyperpolarizing voltage steps from 250 to 2120 mV for
2 s generates a large/h current (.100 pA), the presence of which identifies dopamine neurons. B. A representative time course of the response of
action potential frequency to leptin in the presence (open symbol) or absence (solid symbol) of U0126 in VTA DA neurons. The solid horizontal black
line indicates the duration of leptin application. C. A summary of normalized frequency of action potentials before, during, and after the application
of leptin in DA neurons in VTA slices pre-treated with U0126.
doi:10.1371/journal.pone.0027180.g002
Erk1/2 Mediates Leptin Receptor Signaling in VTA
PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27180
To evaluate the behavioral relevance of the biochemical and
electrophysiological findings, ERK1/2 was tested for its role in
mediating leptin’s anorectic effects. Rats treated with leptin to the
VTA exhibited a reduction in food intake, consistent with our
previous data. Pretreating rats with U0126 attenuated this effect,
thus suggesting a role for ERK1/2 as a critical component of Lepr
signaling in the VTA. The similarity with our findings and those of
Rahmouni and colleagues, with the hypothalamus, is notable. In
both cases, treatment with inhibitors alone did not alter basal
feeding, however, they did attenuate effects of leptin. Additionally,
both studies demonstrate successful pharmacological blocking of
ERK1/2 phosphorylation, while pSTAT3 (Tyr-705) levels
remained high. It is important to note that a major difference
between our feeding study and the Rahmouni study involves site-
specific delivery of leptin. Rahmouni and colleagues delivered
both the inhibitors and leptin ICV, thus potentially affecting
multiple feeding circuits in the CNS, including the VTA, which
could theoretically contribute to their behavioral findings. In our
study, we delivered leptin directly to the VTA, thus attempting to
localize its effects and to identify the importance of ERK1/2
signaling within this region.
The observation that leptin increased pSTAT3 (Tyr-705)
despite pretreatment with U0126 suggests that STAT3 may not
be sufficient for the cellular and behavioral effects of leptin in the
VTA. It is important to emphasize that pTyr-705 assessment may
not always reflect STAT3 activity [28]. Moreover, even if STAT3
activation is not sufficient for the rapid effects of leptin, it is still
possible that long-term modulation of neuronal function is
mediated by transcriptional changes downstream of STAT3.
Conversely, other transcription factors may be regulated by
ERK1/2 to mediate leptin signaling in the VTA. It remains
unknown if the rapid change in dopamine firing caused by leptin is
responsible for the long-term feeding changes, or whether these
are dissociable. However, it is notable that the data presented here
suggest that ERK1/2 is important for the observed neuronal and
behavioral effects. These findings have a potential impact on
future research on behaviors regulated by leptin activity in the
VTA. Further studies are required to determine any potential role
of VTA STAT3, and to further characterize the downstream
effectors that respond to ERK1/2 activation in dopamine neurons
of the VTA.
Materials and Methods
Antibodies
The following antibodies were purchased from Cell Signaling
Technology (Beverly, Ma.): phospho-STAT3 (Tyr-705), STAT3,
phospho-ERK1/2 (Thr-202, Tyr203), phospho-AKT (Ser-473),
phospho-AKT (Thr-308), and AKT. Pan ERK antibody was
purchased from BD Biosciences (San Jose, Ca.).
Animals
Animal experiments were done in accordance with Yale
University School of Medicine and IACUC animal care
guidelines. Sprague Dawley rats were purchased from Charles
River Laboratories, and given access to ad libitum chow and
water. Standard rat chow used was RMH-3000 from Lab Diet
(Richmond, In.). Rats were housed in multiples prior to surgeries,
after which they were singly housed. Mice used for electrophys-
iology were C57BL/6J (Jackson Labs, Bar Harbor, Maine). The
environment was a controlled 12 hr light, 12 hr dark cycle.
Figure 3. ERK1/2 mediates the anorexigenic effect of leptin in
the VTA of rats. Direct leptin infusion to the VTA of rats caused a
significant decrease in food intake, while pretreatment with U0126 ICV
blocked this effect. Treatment groups include: vehicleICV/vehicleVTA
(open bars; n = 13), U0126ICV/vehicleVTA (open-striped bars; n = 11),
vehicleICV/LeptinVTA (gray bars; n= 12), U0126ICV/LeptinVTA (gray-striped
bars; n= 13). * Represents significant effect of leptin (F(1, 45) = 7.78,
P,0.008). # Represents significant Leptin-U0126 interaction (F(1, 45) = 4.17,
P,0.047).
doi:10.1371/journal.pone.0027180.g003
Figure 4. U0126 selectively blocks phosphorylation of the ERK1/2 pathway in the VTA of rats. Pretreatment of rats with U0126 prevented
leptin-induced phosphorylation of ERK1/2 without affecting STAT3 phosphorylation. Treatment groups include: vehicleICV/vehicleVTA (open bars;
n = 5), U0126ICV/vehicleVTA (open-striped bars; n = 4), vehicleICV/LeptinVTA (gray bars; n = 4), U0126ICV/LeptinVTA (gray-striped bars; n = 4). Ratios of
phospho-/total signal were used to calculate the percent change from control tissue. A. Evaluation of pSTAT3 (Tyr-705) phosphorylation across
groups. * Represents significant main effect of leptin (F(1, 15) = 41.9, P,0.001). B. Evaluation of ERK1/2 (Thr-202, Tyr-204) phosphorylation across
groups. # Represents significant interaction of Leptin-U0126 (F(1, 15) = 14.4, P,0.002); significant leptin effect (F(1, 15) = 7.9, P,0.013); significant U0126
effect (F(1, 15) = 9.3, P,0.008).
doi:10.1371/journal.pone.0027180.g004
Erk1/2 Mediates Leptin Receptor Signaling in VTA
PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27180
Rat Cannulation Surgery
Rat VTA cannulations consisted of double-barreled cannulae,
while intracerebral ventricular (ICV) cannulations consisted of
single barrel cannulae. All cannulae were purchased from Plastics1
(Roanoke, Va.). All stereotaxic coordinates are based on the
standard rat atlas [29]. All animals weighed at least 300 g at the
time of surgeries, and were anesthetized with Nembutal. The
following coordinates were used: 1) VTA: anterior-posterior (A/P)
from bregma 25.8 mm; dorsal-ventral (D/V) from skull surface
27.8 mm, 2) ICV: A/P from bregma 20.8 mm; D/V from dura
23.4 mm; and medial-lateral from midline 21.5 mm. All animals
were single housed for the remainder of the experiment, and were
allowed 1 week of recovery prior to subsequent manipulations.
Infusions for Leptin Biochemistry
All infusions w