Leptin Acts via Leptin Receptor-Expressing Lateral
Hypothalamic Neurons to Modulate the Mesolimbic
Dopamine System and Suppress Feeding
Gina M. Leinninger,1Young-Hwan Jo,4Rebecca L. Leshan,1,2Gwendolyn W. Louis,1,2Hongyan Yang,3Jason G. Barrera,5
Hilary Wilson,5Darren M. Opland,1Miro A. Faouzi,1Yusong Gong,1Justin C. Jones,1Christopher J. Rhodes,6
Streamson Chua, Jr.,4Sabrina Diano,7Tamas L. Horvath,7Randy J. Seeley,3Jill B. Becker,3Heike Mu ¨nzberg,1,8
and Martin G. Myers Jr.1,2,*
1Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine
2Department of Molecular and Integrative Physiology
3Molecular and Behavioral Neuroscience Institute
University of Michigan, Ann Arbor, MI 48109, USA
4Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
5Department of Psychiatry and Genome Research Institute, University of Cincinnati, Cincinnati, OH 45237, USA
6Department of Medicine, University of Chicago, Chicago, IL 60637, USA
7Yale University, New Haven, CT 06511, USA
8Present address: Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA
The lateral hypothalamic area (LHA) acts in concert
with the ventral tegmental area (VTA) and other com-
ponents of the mesolimbic dopamine (DA) system to
control motivation, including the incentive to feed.
The anorexigenic hormone leptin modulates the
mesolimbic DA system, although the mechanisms
underlying this control have remained incompletely
understood. We show that leptin directly regulates
a population of leptin receptor (LepRb)-expressing
inhibitory neurons in the LHA and that leptin action
via these LHA LepRb neurons decreases feeding
and body weight. Furthermore, these LHA LepRb
neurons restores VTA expression of the rate-limiting
enzyme in DA production along with mesolimbic DA
content in leptin-deficient animals. Thus, these find-
ings reveal that LHA LepRb neurons link anorexic
leptin action to the mesolimbic DA system.
The processes that regulate feeding and body weight represent
likely points of dysregulation and potential sites for therapeutic
interventionin obesity andmetabolic disease, butremain incom-
pletely understood. The adipose-derived hormone leptin plays
a central role in the regulation of energy homeostasis (Friedman,
2002; Myers, 2004; Morton et al., 2006; Elmquist et al., 2005;
Gao and Horvath, 2007; Berthoud, 2007; Myers et al., 2009).
Leptin activates the long form of the leptin receptor (LepRb) on
CNS neurons to mediate most leptin action, including the
suppression of feeding (Cohen et al., 2001; de Luca et al.,
2005). LepRb-expressing neurons lie in numerous regions
involved in the regulation of energy balance, including medio-
basal hypothalamic (MBH) ‘‘satiety centers’’ (e.g., the arcuate
nucleus [ARC]) and ‘‘feeding centers’’ such as the lateral hypo-
thalamic area (LHA) (Elmquist et al., 1998; Myers et al., 2009).
A number of aspects of leptin action in the MBH are beginning
to be unraveled, including the role of leptin in regulating LepRb/
pro-opiomelanocortin (POMC)-expressing neurons and their
opposing LepRb/agouti-related protein/neuropeptide Y (AgRP/
NPY)-expressing neurons in the ARC (Morton et al., 2006; Elm-
quist et al., 2005; Gao and Horvath, 2007; Berthoud, 2007).
These neurons contribute to satiety and thus mediate an impor-
tant component of the anorectic response to leptin as well as
modulate energy expenditure and aspects of glucose homeo-
stasis. Many data suggest that the action of leptin on these
LepRb-expressing MBH neurons only accounts for a fraction
of leptin action on energy balance, however (Balthasar et al.,
2004; Dhillon et al., 2006; Myers et al., 2009; van de Wall et al.,
2008). Indeed, MBH LepRb neurons represent a minority of
LepRb-expressing neurons in the brain (Elmquist et al., 1998;
Myers et al., 2009). Thus, populations of LepRb neurons in other
brain areas are likely to be crucial for leptin action.
In addition to promoting satiety, leptin suppresses the incen-
tive value of food and other rewards (Figlewicz et al., 2006; Ful-
ton et al., 2000), presumably by modulating components of the
mesolimbic dopamine (DA) system, which arises from dopami-
nergic neurons in the ventral tegmental area (VTA) (Kelley et al.,
2005; Nestler, 2005; DiLeone et al., 2003). Indeed, leptin directly
regulates a population of LepRb-expressing VTA DA neurons
(Figlewicz et al., 2006; Hommel et al., 2006; Fulton et al.,
2006). Furthermore, leptin modulates DA-dependent measures
bic DA content of leptin-deficient animals (Figlewicz et al., 2006;
Fulton et al., 2006; Roseberry et al., 2007).
The LHA feeding center also represents a critical component
of the mesolimbic reward circuit and modulates the incentive
salience of food and other rewards (Kelley et al., 2005; Nestler,
Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc. 89
2005; DiLeone et al., 2003; Harris et al., 2005). Within the LHA
there are two known populations of orexigenic neurons: one
expressing the neuropeptide melanin-concentrating hormone
project to the striatum and VTA, respectively, to modulate
feeding and measures of reward (Georgescu et al., 2005; Mieda
and Yanagisawa, 2002). Leptin inhibits the fasting-induced
activity of OX neurons and decreases the expression of MCH
and OX,and deletion of MCHimproves the obesityof leptin-defi-
cient ob/ob mice (Qu et al., 1996; Segal-Lieberman et al., 2003;
Yamanaka et al., 2003). Thus, LHA neurons likely participate in
the regulation of energy homeostasis by leptin. The LHA also
contains a substantial population of LepRb-expressing neurons,
leading us to hypothesize that these neurons might contribute to
energy homeostasis and the regulation of the mesolimbic DA
system. We have thus examined the role of these LHA LepRb
neurons in leptin action.
Leptin Directly Regulates LepRb-Expressing LHA
Neurons to Suppress Feeding
We developed a reporter mouse strain to facilitate the detection
and study of LepRb neurons in the LHA and throughout the brain
by crossing Leprcremice (Leshan et al., 2006, 2009) onto the
background, in which cre-mediated
recombination promotes the expression of enhanced green
fluorescent protein (EGFP) (Mao et al., 1999) (Figure 1A).
Double-homozygous (henceforth referred to as LepRbEGFP)
mice demonstrate CNS EGFP expression consistent with re-
ported patterns of LepRb expression (Elmquist et al., 1998;
Myersetal.,2009),including alargepopulationof EGFP-positive
neurons in the LHA (Figure 1B). Activated LepRb directly recruits
STAT3 to promote its tyrosine phosphorylation (pSTAT3) (Myers,
2004). While STAT3 protein expression is widespread, the de-
tection of pSTAT3 immunoreactivity (IR) during leptin stimulation
thus reveals cell-autonomous LepRb signaling (Mu ¨nzberg et al.,
2003). Examination of pSTAT3-IR in LepRbEGFPmice following
systemic treatment with leptin to stimulate LepRb revealed that
81% ± 4% of LHA pSTAT3-IR neurons detectably express
EGFP, and 87% ± 1% of EGFP-labeled neurons demonstrate
pSTAT3-IR following i.p. leptin treatment (Figures 1C and 1D).
functional LepRb that responds to systemically administered
leptin, and EGFP expression in these animals identifies the
majority of LHA LepRb neurons.
In order to determine the regulation of LHA LepRb neurons by
leptin, we examined c-fos-IR (FosIR, a marker of neuronal
Figure 1. LepRbEGFPMice Reveal a Large Population of Leptin-Responsive LHA LepRb Neurons
(A) Schematic diagram demonstrating cre-mediated EGFP expression in LepRb-expressing cells of LepRbEGFPmice.
(B) Immunofluorescent detection of EGFP (green) in the LHA of LepRbEGFPmice.
LHA LepRb Neurons Modulate Dopamine and Feeding
90 Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc.
activation [Hoffman et al., 1993]) in EGFP-expressing LHA
neurons from LepRbEGFPmice following fasting or systemic lep-
tin treatment (Figures 2A–2F). A modest number of LHA LepRb
neurons from ad libitum-fed, vehicle-treated animals exhibit
FosIR, and fasting tended to decrease this number (ad libitum,
12% ± 3% versus fasted, 5% ± 1%; p = 0.05). In contrast, leptin
treatment of ad libitum-fed animals induces FosIR (PBS, 12% ±
3% versus leptin, = 41% ± 7%; p < 0.05) in EGFP-expressing
LHA neurons of LepRbEGFPmice, demonstrating that systemic
leptin treatment activates a substantial number of LHA LepRb
In order to determine whether leptin regulates the activity of
LHA LepRb neurons directly, we examined the electrophysio-
logic response of LHA LepRb neurons to leptin in acute hy-
pothalamic slice preparations from LepRbEGFPmice (Figures
2G and 2H). EGFP fluorescence identified LepRb-expressing
LHA neurons for recording in current clamp configuration. As
in vivo, where leptin treatment caused an approximately 30%
increase in FosIR colocalization with LHA LepRb neurons, leptin
(100 nM) depolarized 34% of LHA LepRb neurons (p < 0.05)
(Figures 2G and 2H and Table S1). Furthermore, the presence
or absence of inhibitors of synaptic transmission did not alter
the percentage of cells depolarized by leptin, suggesting that
the depolarizing effect of leptin on this subset of LHA LepRb
neurons is direct. We additionally observed that some LHA
LepRb neurons were hyperpolarized by leptin; the presence of
inhibitors did not alter the proportion of cells responding to leptin
(Table S1). Thus, leptin acts directly upon LHA LepRb neurons to
of LHA LepRb neurons display different electrophysiologic
in leptin action.
In order to determine the role for LHA LepRb neurons in leptin
action in vivo, we examined the effect of direct leptin injection
Figure 2. Leptin Directly Regulates the Activity of LHA LepRb Neurons in LepRbEGFPMice
(A–F) Immunohistochemical detection of FosIR (red nuclei, pseudocolored) and immunofluorescent detection of GFP (green) in LepRbEGFPanimals following ad
libitum feeding (A and D) or fasting for 36 hr (C and F) followed by treatment with vehicle or of ad libitum-fed animals following leptin treatment (B and E) (5 mg/kg,
i.p., 4 hr). Scale bars = 10 mm. Panels (D)–(F) show higher magnification images of (A)–(C), respectively.
(G) Electrophysiologic response of LHA LepRb neurons to leptin. EGFP-expressing LHA neurons were identified under light and fluorescent microscopy for
recording under current clamp conditions and examined for their electrophysiologic response to leptin. One population of neurons was depolarized in response
(H) Graphs of the response of neurons that were depolarized in response to leptin in the absence (?Inhibitors) or presence (+Inhibitors) of inhibitors of synaptic
transmission. Error bars represent the SEM.
LHA LepRb Neurons Modulate Dopamine and Feeding
Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc. 91
into the LHA on food intake and body weight of Long-Evans rats
(Figure 3). Cannulae were placed unilaterally into the LHA of the
rats. Following recovery, we administered saline and 0.1 mg,
and body weight over the subsequent 24 hr. We chose these
doses based upon the similarity of the lower doses to those
used by others for demonstrating site-specific leptin effects in
the rat brain (Hommel et al., 2006). The upper dose was chosen
to lie below the minimum effective i.c.v. dose for decreased
feeding (Seeley et al., 1996). All three doses of unilateral intra-
LHA leptin decreased weight and food intake (p < 0.05 and p <
0.01) over 24 hr compared to saline treatment (Figures 3A and
3B). In addition to examining cannula placement in these
animals, we analyzed pSTAT3-IR in the brains of animals that
received the lowest dose of leptin, to confirm the restriction of
revealed that leptin-induced pSTAT3-IR was mainly confined to
the ipsilateral LHA, with no increase in pSTAT3-IR in the ARC,
VTA, or other distant regions. Thus, these data suggest that lep-
tin action via LHA LepRb neurons is sufficient to suppress food
intake and weight gain in normal rats. While we did not examine
the detailed timing of the leptin response for all doses, the high-
est dose of leptin did not produce a significant reduction in food
intake before 4 hr (data not shown). This delayed timing is
consistent with the role of leptin as a long-term, rather than
acute, signal of energy status.
LHA LepRb Neurons Are Distinct from Known
Orexigenic LHA Neurons
We examined the potential expression of LepRb in known pop-
ulations of orexigenic LHA neurons by examining the colocaliza-
tion of EGFP with MCH and OX in LepRbEGFPmice. This analysis
revealed no detectable expression of LepRb/EGFP in MCH or
OX neurons in untreated (not shown) or colchicine-treated
animals (Figures 4A and 4B). Furthermore, pSTAT3-IR is not
found in MCH or OX neurons in normal mice following treatment
with high-dose i.c.v. leptin (3 mg) to promote LepRb-mediated
pSTAT3 in all neurons containing functional receptor (Figures
4C and 4D).
To understand the neurochemical mediators by which LHA
LepRb neuronsmightcontribute toleptinaction, weinvestigated
their neurotransmitter content. Reasoning that the LHA LepRb
neurons are distinct from OX neurons, which are known to be
glutamatergic/excitatory (Rosin et al., 2003), we analyzed the
potential GABAergic/inhibitory nature of the LHA LepRb neurons
by utilizing Gad1EGFPmice (which express EGFP in neurons that
contain GAD1, an enzyme that synthesizes the inhibitory neuro-
transmitter, GABA) (Abe et al., 2005) to examine the potential
colocalization of leptin-stimulated pSTAT3-IR in EGFP-express-
ing GABA neurons. All LHA pSTAT3-IR/LepRb neurons in the
Gad1EGFPmice contain EGFP, revealing the GABAergic nature
of the LHA LepRb neurons (Figures 4E and 4F). By contrast,
adjacent OX and MCH neurons do not coexpress EGFP/GAD1
(Figures 4G and 4H).
Thus, LHA LepRb neurons represent a unique population of
neurons distinct from previously described OX and MCH
neurons in the LHA. The inhibitory nature of LHA LepRb neurons
contrasts with the excitatory properties of some orexigenic LHA
neurons. Similarly, while leptin activates at least some LHA
LepRb neurons, it suppresses the fasting-induced activity of
OX neurons (Yamanaka et al., 2003). Thus, these findings are
also consistent with opposite roles in the regulation of feeding
for LHA LepRb neurons (which decrease feeding in response
to LHA leptin) compared to the orexigenic OX neurons.
LHA LepRb Neurons Innervate the VTA
Conventional anterograde tracing studies have demonstrated
the connection of LHA neurons with multiple brain centers within
and outside of the hypothalamus, including the VTA, NAc, and
other areas (Swanson et al., 2005). Such studies do not differen-
tiate the projections of LepRb neurons from those of other LHA
neuronal populations, however. To define projections from
LepRb-expressing soma in the LHA, we thus merged the use
of molecular tracers with the cre-inducible system (for LepRb
specificity) and adenoviral stereotaxic injection (for anatomic
specificity) to generate the adenoviral vector Ad-iZ-EGFPf
(Figure 5A) (Leshan et al., 2009). This vector mediates the cre-
inducible expression of farnesylated EGFP (EGFPf), which local-
izes to the membrane, effectively labeling even very long axonal
projections (Zylka et al., 2005; Leshan et al., 2009).
We administered Ad-iZ/EGFPf into the LHA of Leprcremice
(Figure 5B) and perfused them for immunofluorescent analysis
5 days later. While intra-LHA administration of Ad-iZ/EGFPf
produced copious EGFPf expression in the LHA of Leprcre
mice (Figure 5C), no EGFPf expression was detected in wild-
type animals (data not shown); nor was EGFPf detected in
MCH- or OX-expressing neurons of Leprcremice (Figure S2),
confirming the cre specificity of EGFPf expression.
To determine the brain regions innervated by LHA LepRb
neurons, we analyzed EGFPf-containing projections in Leprcre
Figure 3. Response of Rats to Intra-LHA Leptin Treatment
(A and B) Change in weight (A) and total food intake (B) in Long-Evans rats in
response to 24 hr treatment with intra-LHA saline (white bars) or 0.1 mg (black
bars), 0.5 mg (diagonally hatched bars), or 1 mg leptin (horizontally hatched
bars). Food intake over 24 hr is plotted as percent of intake following saline
injection; weight data are plotted as change in weight over 24 hr. Average
values ± SEM are shown; *p < 0.05, **p < 0.01 compared to saline by one-
way ANOVA with Dunnett’s posttest.
LHA LepRb Neurons Modulate Dopamine and Feeding
92 Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc.
animals (n = 11) where the injection site and EGFP-immunoreac-
(Figures 5B and 5C). While the DMH, ARC, and other hypotha-
lamic areas contained few or no LHA LepRb-originating EGFPf
projections, in addition to the copious number of neurites
observed in the LHA itself, the VTA contained dense EGFPf
projections ipsilateral to the injection site (Figures 5D and 5E).
Other regions caudal to the LHA also received some projections,
but no projections to the striatum (including the NAc) or other
rostral regions were observed (Figure S2).
To verify the results of this anterograde tract-tracing method,
we utilized retrograde tracing with fluorogold (FG) in LepRbEGFP
mice to verify the projection of LHA LepRb neurons to the VTA
(Figure S3). Intra-VTA FG labeled many LHA neurons, including
LepRb-expressing LHA neurons, while LepRb-containing ne-
urons in other hypothalamic regions (such as the ARC) did
not accumulate FG from the VTA. Thus, LHA LepRb neurons
project caudally to innervate the VTA and may play a unique
role among hypothalamic LepRb-expressing neurons in the
regulation of the mesolimbic DA system. We also examined
leptin-induced c-FosIR in the FG-treated LHA LepRb neurons
(Figure S3): Most FG-labeled EGFP neurons did not contain
c-FosIR. In contrast, strong c-FosIR was noted in non-FG-
labeled EGFP neurons, suggesting that most VTA-projecting
LHA LepRb neurons are not leptin-activated LHA LepRb
Figure 4. Neurotransmitter Content of LHA
(A and B) Immunofluorescent detection of EGFP
(green) and MCH (A, red) or OX (B, red) in the
LHA of colchicine-treated LepRbEGFPmice.
(C and D) Immunohistochemical detection of
pSTAT3-IR (green nuclei, pseudocolored) and
MCH (C, red) or OX (D, red) following i.c.v. leptin
treatment for 1 hr.
(E) Immunohistochemical detection of pSTAT3
(red, pseudocolored) and immunofluorescent de-
tection of EGFP (green) in Gad1EGFPmice.
(F) Digital zoom of (E).
(G and H) Immunofluorescent detection of EGFP
(green) and OX (G, red) or MCH (H, red) in mice
that express EGFP in GAD67 neurons. Scale
bars = 10 mm. 3V, third cerebral ventricle; f, fornix.
LHA LepRb Neurons Regulate
Mesolimbic DA Production
While the administration of exogenous
leptin (even at supraphysiologic doses)
often evokes only modest effects in
normal leptin-replete animals, leptin defi-
ciency promotes dramatic alterations in
CNS function and mammalian physiology
that are readily reversed by the normali-
zation of leptin levels. For instance, exog-
enous leptin does not increase hypotha-
lamic Pomc mRNA expression in normal
fed animals, although such treatment
restores the diminished Pomc expres-
sion of leptin-deficient Lepob/obanimals
(Schwartz et al., 1997). Reasoning that Lepob/obanimals have
impaired VTA expression of tyrosine hydroxylase (Th, the rate-
limiting enzyme in DA production) and decreased mesolimbic
DA content that is normalized by systemic leptin treatment
(Figure 6A) (Fulton et al., 2006) and that LHA LepRb neurons
are well placed to regulate the VTA and mesolimbic DA system
to regulate energy balance and normalize these parameters in
Lepob/obmice (Figures 6B–6F). We utilized unilateral intra-LHA
cannulae to deliver a small dose of leptin (250 pg, designed to
approximate circulating leptin levels in the volume of distribution
of the LHA) to these Lepob/obanimals. This leptin treatment stim-
ulated pSTAT3-IR only in the LHA on the same side as the
cannula (and not in the MBH), demonstrating the specificity of
this treatment for the LHA LepRb neurons (Figure S4).
We measured food intake and body weight over 24 hr of treat-
ment in these animals (Figures 6C and 6D). As predicted based
upon the response of normal rats to leptin in the LHA, intra-LHA
leptin treatment of Lepob/obanimals attenuated their food intake
over the 24 hr treatment period and suppressed weight gain (p <
0.05), confirming that the absence of leptin action via LHA LepRb
neurons contributes to the dysregulated feeding and energy
balance of leptin-deficient animals. While we examined food
intake at times earlier than 24 hr, intra-LHA leptin did not signifi-
tent with the role of leptin as a long-term signal of energy stores.
LHA LepRb Neurons Modulate Dopamine and Feeding
Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc. 93
We also examined the effect of intra-LHA leptin treatment on
VTA Th expression and NAc DA content (Figures 6E and 6F).
The unilateral nature of both the LHA LepRb / VTA projections
(Figure 5) and the leptin treatment dictates the confinement of
the leptin effect to the side ipsilateral to the cannula; we thus
compared these parameters ipsilateral and contralateral to the
cannula. This approach also increases the power of the experi-
ment by permitting a ‘‘within-mouse’’ comparison, minimizing
the effects of potential between-animal variation in Th mRNA
and DA content. Intra-LHA PBS treatment tended to modestly
decrease ipsilateral compared to contralateral VTA Th expres-
sion (Figure 6E, PBS-treated samples), potentially consistent
with minor disruption of LHA tissue (and thus the LHA / VTA
projections) by the cannula. By contrast, leptin increased VTA
Th mRNA expression ipsilateral to the cannula by approximately
2.5-fold (p < 0.05) (Figure 6E), as well as increasing ipsilateral
NAc DA content by approximately 40% (p < 0.05) (Figure 6F),
indicating that the LHA / VTA circuit is largely intact and that
LHA leptin action regulates these important parameters of mes-
olimbic DA system function. While the approximately 2.5-fold
increase in VTA Th mRNA expression ipsilateral to the cannula
in intra-LHA leptin-treated Lepob/obanimals (Figure 6E) was
similar in magnitude to the increased total Th expression of
Figure 5. Ad-iZ/EGFPf-Mediated Tracing
Reveals Projection of LHA LepRb Neurons
to the VTA
(A) Schematic diagram showing cre-mediated
expression of EGFPf in infected LepRb-express-
ing neurons following stereotaxic injection of
Ad-iZ/EGFPf into Leprcremice.
(B) Summary of Ad-iZ/EGFPf injection sites for the
11 cases utilized for the study.
(C) Immunofluorescent detection of EGFPf (green)
and MCH (red) in the LHA of an example of
a correctly targeted intra-LHA injection of Ad-iZ/
EGFPf in the Leprcremice.
containing projections in the VTA of Leprcremice
following intra-LHA injection of Ad-iZ/EGFPf.
(E)Higher magnification of(D).Scalebars=10mm.
VTA, ventral tegmental area;SNc, substantia nigra
pars compacta; IP, interpeduncular tubercle.
wild-type compared to Lepob/obanimals
(Figure 6A), intra-VTA leptin administra-
tion did not significantly alter VTA Th
mRNA expression (Figure 6G). Collec-
tively, these data indicate that intra-LHA
(but not intra-VTA) leptin modulates the
tin action specifically via LHA LepRb
neurons promotes VTA Th gene expres-
sion and mesolimbic DA content and
regulates the mesolimbic DA system.
Overall, our findings reveal that leptin
actionvia GABAergicLHA LepRb
neurons suppresses feeding. We also show that these neurons
densely innervate the VTA and that leptin action via LHA LepRb
neurons (but not via VTA LepRb neurons) increases VTA Th
expression and mesolimbic DA content of leptin-deficient
animals. Thus, leptin action via LHA LepRb neurons modulates
the mesolimbic DA system and decreases feeding. The role of
the mesolimbic DA system in mediating the action of leptin in
the LHA is an important area for future study.
While the finding that increased VTA Th expression (and mes-
olimbic DA production/content) decreases food intake contra-
venes the widely held notion that NAc DA release promotes
feeding, increased DA does not always correlate with increased
feeding; cocaine and amphetamines promote the accumulation
ofextracellular DA intheNAcbutbluntfeeding. Indeed, DAinthe
mesolimbic DA system regulates the incentive value of many
behaviors, not just feeding, and leptin would be predicted to
differentially regulate the components of the mesolimbic DA
system that encode feeding compared to reproductive behavior,
for instance. Furthermore, while we were not able to examine
activity in these experiments, it is also possible that the regula-
tion of mesolimbic DA content by leptin modulates activity, in
addition to feeding. Also, DA action in the dorsal striatum has
been suggested to regulate feeding (Palmiter, 2007), and the
LHA LepRb Neurons Modulate Dopamine and Feeding
94 Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc.
role of leptin in the modulation of this system has yet to be
It is also important to note that while our data demonstrate
a role for leptin in promoting DA production, it is possible that
DA release is regulated differently. The regulation of DA content
(rather than DA release) by leptin is more consistent with the
of long-term energy stores and does not fluctuate greatly on an
acute basis. This long-term modulation of DA content (rather
than acute release) by LHA LepRb neurons may also serve to
prevent large acute changes in mesolimbic DA input that might
otherwise occur with alterations in nutritional status; such
changes might produce undesirable long-term consequences
in the mesolimbic DA system, as seen with drugs of abuse.
Each population of LepRb-expressing neurons throughout the
brain presumably mediates a distinct aspect of overall leptin
Figure 6. Energy Balance, VTA Th mRNA Expression, and NAc DA Content in Lepob/obMice after Intra-LHA Leptin Treatment
(A) Th mRNA levels in the VTA of normal C57BL/6 mice (WT, hatched bar) and Lepob/obmice (solid bar). Th expression for each genotype is shown relative to
Gapdh expression (calculated by the 2?DDCtmethod) ± SEM; *p < 0.05.
(B) Summary of LHA cannulation sites in the Lepob/obmice included in the study.
(C and D)Twenty-four hour foodintake(C) and body-weight change (D)in Lepob/obmicein response to intra-LHA PBS (hatched bars) or leptin (solid bars). All data
are plotted as average ± SEM; *p < 0.05.
(E and F) VTA Th mRNA expression relative to Gapdh (E) and NAc DA content (F) contralateral (hatched bars) and ipsilateral (solid bars) in Lepob/obmice treated
with 24 hr of intra-LHA PBS or leptin. Values are shown relative to the (unperturbed) contralateral side, ± SEM; *p < 0.05, **p < 0.01.
(G) Expression of Th mRNA relative to Gapdh in Lepob/obmice treated with 24 hr of intra-VTA PBS or leptin. Data are plotted as in (E). Significance determined by
one-way ANOVA with Bonferroni posttests.
LHA LepRb Neurons Modulate Dopamine and Feeding
Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc. 95
action (Myers et al., 2009). For instance, MBH LepRb neurons in
the ARC and VMH appear to regulate satiety, some aspects of
energy expenditure, and glucose homeostasis (Morton et al.,
2006; Elmquist et al., 2005), while our present data suggest
that LHA LepRb neurons contribute to the actions of leptin on
the mesolimbic DA system. Leptin also acts directly upon a pop-
ulation of LepRb-expressing neurons in the VTA (Fulton et al.,
2006; Roseberry et al., 2007; Hommel et al., 2006), inhibiting
mel et al., 2006). In contrast, systemic leptin increases VTA Th
expression and NAc DA content in Lepob/obanimals (Fulton
et al., 2006; Roseberry et al., 2007). While VTA LepRb neurons
may contribute to leptin action, our present findings that LHA,
but not VTA, leptin administration in Lepob/obmice increases
VTA Th expression (and NAc DA) toward normal levels suggest
that leptin action via LHA LepRb neurons regulates VTA Th
expression and the mesolimbic DA system.
of the mesolimbic DA system, however, and LHA LepRb neurons
may be divisible into distinct subpopulations (e.g., based on their
electrical response to leptin and/or projection targets). As only
locally within the LHA to modulate the function of OX or MCH
neurons, both of which are inhibited by leptin (Jo et al., 2005;
ulations of LHA LepRb neurons, as well as those played by VTA
for leptin to differentiallyregulatespecificpopulations of midbrain
relative to other behaviors. Going forward, it will be important to
determine how these various populations of mesolimbic DA
system-interacting LepRb neurons differ in terms of their wiring
and the control of different aspects of mesolimbic DA signaling.
Overall, our present observations reveal that the LHA LepRb
neuronal population represents a major link between anorectic
leptin action and the mesolimbic DA system. These findings
reveal important mechanisms that underlie the regulation of the
mesolimbic DA system by a crucial signal of energy stores. In
the future, it will be crucial to address the potential dysregulation
of these neurons in states of obesity.
Leptin was purchased from NHPP (Los Angeles, CA) or was a generous gift
from Amylin Pharmaceuticals, Inc. (San Diego, CA).
The generation of Leprcremice has been described previously (Leshan et al.,
2006). To generate LepRbEGFPmice, we crossed Leprcre/+with Gt(ROSA)26-
Sortm2Shopurchased from Jackson Laboratory and then intercrossed their
double-heterozygous progeny to generate double-homozygous LeprCre/Cre;
Gt(ROSA)26Sortm2Sho/tm2Sho(LepRbEGFP) mice, which were propagated by
intercrossing. C57BL/6 (WT) and Lepob/obmice were purchased from Jackson
Laboratory, and Gad1EGFPmice were propagated at Yale University. All other
mice were housed in a 12 hr light/dark cycle and cared for by ULAM at the
University of Michigan or similar facilities at AECOM. All animals had ad libitum
access to food and water, except in experiments where mice were fasted
before perfusion. All care and procedures for mice were in accordance with
versity’s University Committee on Use and Care of Animals (UCUCA).
Immunohistochemistry and Immunofluorescence
Treatment with i.c.v. colchicine (10 mg) to concentrate neuropeptides in the
soma for some experiments was administered for 2 days prior to perfusion.
Adult mice were anesthetized with an overdose of intraperitoneal (i.p.) pento-
barbital, transcardially perfused with 4% paraformaldehyde or 10% neutral
buffered formalin, and processed for pSTAT3 or c-fos immunoreactivity by
DAB, as described (Mu ¨nzberg et al., 2007). DAB staining was pseudocolored
using Photoshop software to appear colored in images. All other antibodies
were subsequently added and visualized via immunofluorescent secondary
detection using species-specific Alexa 488 or 568 antibodies (1:200, Invitro-
gen) and were processed and imaged as previously (Mu ¨nzberg et al., 2007).
Antibodies used were GFP (1:1000, Abcam Inc.; Cambridge, MA), Orexins
(1:2000 Orexin A + 1:30 Orexin B, Calbiochem; San Diego, CA), and MCH
(1:200, Phoenix Pharmaceuticals, Inc.; Burlingame, CA).
Electrophysiology Slice Preparation
Transverse brain slices were prepared from LepRbEGFPmice at postnatal age
28–35 days. Animals were anesthetized with a mixture of ketamine and
xylazine. After decapitation, the brain was transferred into a sucrose-based
solution bubbled with 95% O2/5% CO2and maintained at ?3?C. This solution
NaHCO3, and 10 mM glucose. Transverse coronal brain slices (200 mM) were
prepared using a Vibratome (VT1000S, Leica Microsystems, Inc.; Bannock-
burn, IL). Slices were equilibrated with an oxygenated artificial cerebrospinal
fluid (aCSF) for > 1 hr prior to transfer to the recording chamber. The slices
were continuously superfused at a rate of 2 ml/min with aCSF containing
113 mM NaCl, 3 mM KCl, 1 mM NaH2PO4, 26 mM NaHCO3, 2.5 mM CaCl2,
1 mM MgCl2, and 5 mM glucose in 95% O2/5% CO2at RT. Membrane poten-
tials of LepRbEGFPneurons were measured 5 min after the onset of recordings
and at the maximum response to leptin. Student’s t test was used to test for
significance between control and leptin-treated samples. Data were consid-
ered significant for p < 0.05.
Rat Cannulation, Microdissection, and Analysis
Male Long-Evans rats (250–300 g, Harlan; Indianapolis, IN) were individually
were in accordance with the guidelines and approval of the University of Cin-
cinnati IACUC. Under ketamine/xylazine anesthesia, rats were placed in
a stereotaxic frame (David Kopf Instruments; Tujunga, CA) with the incisor
bar set at ?0.3 mm. The skull was exposed via a 2 mm midline incision, and
the periosteum was gently scraped to the side. A 26G guide cannula (Plastics
One; Roanoke, VA) was lowered toward the left lateral hypothalamus at the
following coordinates: AP, ?2.30 mm; ML, ?1.80 mm; and DV, ?7.80 mm.
and fitted withanobturatorthatextended 1.0 mmbelow theguide cannula. On
the day of the experiment, rats were weighed, and food was removed from
cages and weighed at 8:00 a.m. At 9:00 a.m., rats received a 0.2 ml intra-
LHA infusion of saline or either 0.1 mg, 0.5 mg, or 1.0 mg recombinant mouse
leptin (Calbiochem) over 2 min. The 1 mg dose of leptin was chosen because
it is below the threshold dose required to observe an anorectic response to
i.c.v. leptin (Seeley et al., 1996), while the lower 0.1 mg and 0.5 mg leptin doses
are similar to those utilized for the analysis of leptin action in the VTA (Hommel
et al., 2006). Infusions were made using a microinfusion pump (Harvard Appa-
ratus; Holliston, MA) holding a 25 ml Hamilton syringe (Hamilton Company;
Reno, NV) attached via saline-filled polyethylene tubing to a 33G internal
cannula (Plastics One) that extended 1.0 mm below the guide cannula. Food
was returned to cages at 1:00 p.m., and food intake and body weight change
were measured at 24 hr. After 72 hr (by which time the leptin effects on food
intake and body weight are extinguished), the treatment groups were
switched, and data was collected as above. To verify cannulation site, rats
were anesthetized and their brains removed to a rat coronal brain matrix
(1 mm divisions) to isolate the hypothalamus, which was then placed in 4%
paraformaldehyde for fixation at 4?C for 72 hr, followed by cryoprotection in
30% sucrose and microtome sectioning. A few rats were perfused with 4%
paraformaldehyde and immunostained for pSTAT3 to visualize the spread of
leptin-induced pSTAT3-IR within the brain. Hypothalamic sections from
each animal were immunostained for OX to verify the cannulation site relative
to the LHA OX/LepRb-rich region, and rats were included in the study if
LHA LepRb Neurons Modulate Dopamine and Feeding
96 Cell Metabolism 10, 89–98, August 6, 2009 ª2009 Elsevier Inc.
cannulae were placed in the LHA. Exclusion by these criteria and utilization of
the crossover treatment paradigm yielded the following treatment groups:
50 rats received saline, 16 rats received 0.1 mg leptin, 16 rats received 0.5
mg leptin, and 18 rats were treated with 1 mg leptin.
LepRb Projection Tracing
For the generation of Ad-iZ/EGFPf, the floxed b-geo cassette from pCALL
(kindly provided by Corrine Lobe; Toronto, ON) was excised and inserted
upstream of the MCS from pShuttleCMV (He et al., 1998) to generate pShut-
tle-iZ. The coding sequence of EGFP from pEGFP (Invitrogen) was amplified
with the addition of sequences encoding a COOH-terminal farnesylation by
standard PCR and subcloned into the MCS of pShuttle-iZ to generate pShut-
tle-iZ/EGFPf, which was purified, linearized, and utilized to generatethe Ad-iZ/
EGFPf adenovirus as previously described. Concentrated adenoviral stocks
were generated and purified as previously described (Morton et al., 2003).
For tracing studies, Leprcremice were anesthetized with an isofluorane
to Bregma, were A/P, ?1.34; M/L, ?1.13; and D/V, ?5.20; in accordance with
Franklin and Paxinos, 1997. An access hole was drilled in this spot, through
which a guide cannula with a stylet was inserted and lowered to the D/V coor-
dinate. The stylet was then replaced by an injector, and 250–500 nl of Ad-iZ/
EGFPf was injected into the tissue via a 0.5 ml Hamilton syringe at a rate of
100 nl per min. After 10 min to allow for tissue absorption, the injector and
cannula were removed and the skull and the incision closed with wound clips.
Mice received postsurgical analgesia (Buprenex) and were housed for 5 days
in the animal facility, after which they were perfused and processed for immu-
Lepob/obCannulation, Microdissection, and qPCR and DA Analysis
Twenty male Lepob/obmice (12 weeks, 49–61 g) were anesthetized, and
a stereotaxic frame was utilized to locate the LHA, as described above. A
26G guide cannula with a 4.4 mm projection below the pedestal was lowered
into the LHA site, the pedestal was affixed to the skull with surgical adhesive
and cemented in place, and then the wound was closed around the cannula
with sutures. A dummy injector with a 5.2 mm projection was placed in the
guide cannula and allowed to remain through the recovery period. Mice
received postsurgical analgesia (Buprenex) and were single-housed for 9
days to recover, during which daily food intake and weight were monitored.
On the day before treatment, mice were mock treated, which consisted of
replacing the dummy with an unloaded injector and tubing to simulate the
treatment conditions—this was called the baseline day. On the following day
(treatment day), the dummy was replaced with an injector with a 5.2 mm
projection used to deliver either 250 nl of sterile PBS or leptin (0.001 ng/nl,
thus each dose = 0.25 ng) every 12 hr for 24 hr, during which food intake
and weight were monitored. Mice were then treated once more and 2 hr later
were anesthetized and their brains removed to a rodent coronal brain matrix
(1 mm divisions) for microdissection. The ipsilateral and contralateral VTA
and NAc were microdissected and frozen on dry ice. The hypothalamus was
removed, frozen on dry ice, and then placed in 4% paraformaldehyde for fixa-
tion at 4?C for 72 hr, followed by cryoprotection in 30% sucrose and micro-
tome sectioning. Hypothalamic sections were immunostained for OX to verify
the cannulation site relative to the LHA OX/LepRb-rich region and for pSTAT3
to verify that leptin-induced pSTAT3 activation was confined to the LHA. Mice
were excluded from analysis if cannulae were misplaced, if they failed to gain
weight and eat normally prior to the study, or if mice exhibited significant
pSTAT3 outside of the LHA. Sample sizes for the feeding and weight
measures, following the exclusion of mice by the above criteria, were: PBS,
n = 5; and leptin, n = 6 mice. Food intake is plotted as grams of food ingested
at the end of the 24 hr treatment period minus the weight at the beginning of
treatment ± SEM.
RNA was prepared from microdissected VTA using TRIzol (Invitrogen), and
1 mg samples were converted to cDNA using the SuperScript First-Strand
Synthesis System for RT-PCR (Invitrogen). Sample cDNAs were analyzed in
triplicate via quantitative RT-PCR for Gapdh and Th (Applied Biosystems;
valuesarecalculatedby the2?DDCtmethod, withnormalization ofeachsample
DCt value to the contralateral side DCt; thus, Th expression in the contralateral
sideis set to1,and theipsilateral Th expression of eachsample isrelative to its
contralateral side. Data are plotted as the average fold expression ± SEM. A
sample was lost in processing; thus, sample sizes were: PBS, n = 5; leptin,
n = 6.
For determination of DA content, ipsilateral and contralateral NAc tissues
were weighed, sonicated in 88 ml of 10% trichloroacetic acid/0.1% sodium
bisulfite, and centrifuged for 10 min at 50003 g at 4?C. The resulting superna-
tant was shaken for 10 min with 75 ml iso-octane and centrifuged for 5 min at
13,0003 g, and the aqueous layer was removed and frozen at ?80?C until
further processing. The same volume (10–40 ml) of ipsilateral and contralateral
sample for each mouse was diluted with 0.05 N HClO4containing 200 mg/l
dihydroxybenzylamine as an internal standard with 1.6 mM ethylenediamine-
tetra-acetic acid and 8 mM sodium metabisulfite in a final volume of 200 ml.
Samples were then filtered (0.22 mm filters) and DA concentrations were deter-
mined using HPLC with electrochemical detection (Coulochem II, ESA; Wal-
tham, MA), as described (Hu and Becker, 2003). For each sample, the contra-
lateral and ipsilateral values were normalized to the contralateral value. Thus,
DA expression in the contralateral side is set to 1, and the ipsilateral DA
expression of each sample is relative to its contralateral side. Data are plotted
as the average fold expression ± SEM.
Cannulation of male Lepob/obmice in the VTA was performed essentially the
same as described for the LHA. VTA coordinates relative to Bregma were A/P,
?3.2; M/L, ?0.5; D/V, ?4.3; in accordance with Franklin and Paxinos, 1997.
VTA-cannulated mice were treated similar to LHA-cannulated mice over
24 hr, and VTA RNA was isolated and analyzed for Th expression (PBS, n =
7 mice; leptin, n = 9 mice).
Student’st testwasusedtocompare twotestgroupsascomputed withExcel.
One-way ANOVA with posttesting was used for multiple comparisons using
InStat software for Macintosh.
Supplemental Data include Supplemental Experimental Procedures, Supple-
mental References, one table, and four figures and can be found online at
We thank Amylin Pharmaceuticals for the generous gift of leptin and thank
Gary Schwartz, Bob Kennedy, Maura Perry, and members of the Myers lab
for helpful discussions. This work was supported by the Michigan Diabetes
Research and Training Center; Michigan Comprehensive Diabetes Center;
and grantsfrom theAmerican Diabetes Associationand AmericanHeart Asso-
ciation (M.G.M.), the NIH (M.G.M., C.J.R., and J.B.B.), and the Obesity Society
Received: December 5, 2008
Revised: May 27, 2009
Accepted: June 25, 2009
Published: August 5, 2009
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