Neuron 51, 811–822, September 21, 2006 ª2006 Elsevier Inc.DOI 10.1016/j.neuron.2006.09.006
Leptin Regulation of the Mesoaccumbens
Stephanie Fulton,1,3Pavlos Pissios,1
Ramon Pinol Manchon,1Linsey Stiles,2
Lauren Frank,2Emmanuel N. Pothos,2
Eleftheria Maratos-Flier,1,* and Jeffrey S. Flier1,*
1Department of Medicine and Division of Endocrinology
Beth Israel Deaconess Medical Center and
Harvard Medical School
Boston, Massachusetts 02115
2Department of Pharmacology and Experimental
Program in Neuroscience
Tufts University School of Medicine
Boston, Massachusetts 02111
Leptin is an adipose-derived hormone that acts on
hypothalamic leptin receptors to regulate energy bal-
ance. Leptin receptors are also expressed in extra-
hypothalamic sites including the ventral tegmental
area (VTA), critical to brain reward circuitry. We report
that leptin targets DA and GABA neurons of the VTA,
inducing phosphorylation of signal-transducer-and-
activator-of-transcription-3 (STAT3). Retrograde trac-
ing combined with pSTAT3 immunohistochemistry
show leptin-responsive VTA neurons projecting to nu-
cleus accumbens (NAc). Assessing leptin function in
the VTA, we showed that ob/ob mice had diminished
locomotor response to amphetamine and lacked loco-
motor sensitization to repeated amphetamine injec-
tions, both defects reversed by leptin infusion. Electri-
cally stimulated DA release from NAc shell terminals
was markedly reduced in ob/ob slice preparations,
and NAc DA levels and TH expression were lower.
These data define a role for leptin in mesoaccumbens
DA signaling and indicate that the mesoaccumbens
DApathway,critical tointegrating motivated behavior,
responds to this adipose-derived signal.
The brain plays a critical role in the integration and
regulation of physiologic systems controlling energy
balance. Among the most important signals to the brain
reporting the status of energy stores is the adipocyte-
derived hormone leptin. Leptin potently regulates neural
circuits controlling feeding, neuroendocrine function,
metabolism, and body weight, and the absence of leptin
(ob/ob mice) or its receptor (db/db mice) produces
hyperphagia, endocrine/metabolic dysfunction, and
profound obesity (Campfield et al., 1995; Halaas et al.,
1995; Pelleymounter et al., 1995; Ahima et al., 1996).
Leptin exerts its actions on energy balance through
the long-form of the leptin receptor (ObRb), via signals
including the JAK-STAT3 and PI-3 kinase pathways
(Tartaglia et al., 1995; Baumann et al., 1996; Vaisse
et al., 1996; Niswender et al., 2001; Zhao et al., 2002).
The ability of leptin to activate STAT3 via ObRb has
been most extensively studied in hypothalamus, and
critical circuits involving leptin signals within POMC
and NPY/AgRP neurons in the arcuate nucleus have
been defined through functional neuroanatomy and ge-
netic gain- and loss-of-function approaches (Erickson
et al., 1996; Elias et al., 1999; Balthasar et al., 2004; Elm-
quist et al., 2005; Luquet et al., 2005).
ObRb mRNA expression in the CNS is not limited to
the hypothalamus but includes expression within thala-
mus, cerebellum, and substantia nigra pars compacta
among other areas (Elmquist et al., 1998). The presence
of leptin receptor protein on midbrain dopamine (DA)
neurons has also been reported (Hay-Schmidt et al.,
2001; Figlewicz et al., 2003). DA neurons that originate
in the VTA and project to the prefrontal cortex, amyg-
dala, and ventral striatum are critical for the regulation
of behavior, including feeding behavior. DA release in
damental role in learning about rewarding stimuli and
behaviors and in the motor activity required to obtain re-
wards(Schultz, 2002;Kelley etal., 2005;Salamone etal.,
2005). Moreover, the VTA-NAc dopamine pathway
(‘‘mesoaccumbens’’) has been extensively implicated
in mechanisms of drug addiction, not only as the basis
of the locomotor activating and reinforcing effects of
certain drugs but also as a substrate for neuroadapta-
tions that underlie the development and progression of
addictive behaviors (Robinson and Berridge, 2003;
Vezina, 2004; Hyman et al., 2006).
Leptin has been shown to modulate brain reward
circuitry by altering performance for rewarding brain
stimulation (Fulton et al., 2000, 2004). Leptin reverses
the effect of food deprivation to reinstate drug-seeking
behavior (Shalev et al., 2001), increases amphetamine-
induced locomotion (Haoetal.,2004), andblocks condi-
2004). However, the role of leptin in midbrain DA func-
tion is poorly characterized, and actions of leptin on
NAc DA signaling have not been demonstrated. We
action on the mesoaccumbens DA system through a
combination of neuroanatomical, biochemical, behav-
ioral, and electrochemical approaches in wild-type and
leptin-deficient ob/ob mice. We found that leptin rapidly
induces tyrosine phosphorylation of signal-transducer-
and-activator-of-transcription-3 (STAT3), a key down-
stream mediator of leptin receptor signaling, in the
VTA of ob/ob and wild-type mice. To characterize these
leptin responsive VTA cells, we showed that pSTAT3
immunoreactive neurons colabeled with markers of DA
and GABA. We then combined pSTAT3 immunohisto-
chemistry with retrograde tract tracing and determined
that a subset of leptin-responsive cells in the VTA
*Correspondence: email@example.com (E.M-F.), jflier@
3Present address: Department of Pharmacology, Center for Re-
search in Neurological Sciences, Universite ´ de Montre ´al, Roger-
Gaudry Building, Succursale Centre-ville, Montre ´al, Que ´bec H3C
for the locomotor-activating and sensitizing effects of
drugs of abuse, we assessed the effects of leptin on
d-amphetamine (AMPH)-induced locomotion andsensi-
tization in both wild-type mice and ob/ob mice geneti-
motor response to AMPH and showed no sensitization
to repeated injection. Finally, to assess the impact of
leptin deficiency on DA neurotransmission within the
mesoaccumbens pathway, we analyzed electrically
hydroxylase (TH), the rate-limiting enzyme for DA bio-
synthesis, in the VTA and NAc, and levels of phosphory-
lated TH (ser40) and DA in the NAc of wild-type and ob/
ob mice. A substantial impairment of DA release and
biosynthetic capacity in the NAc was observed. Taken
together, the findings define an important role for leptin
action on the function of the mesoaccumbens DA path-
standing the neural circuitry by which homeostatic and
reward pathways may be integrated in the regulation
of appetite, locomotor control, and body weight.
Leptin Activates pSTAT3 in Dopaminergic
Neurons in the VTA
We tested the ability of leptin to functionally activate
leptin receptors in the VTA by examining the immuno-
histochemical expression of pSTAT3 in wild-type and
ob/ob mice 2 hr after peripheral injection of leptin.
Nuclear pSTAT3 labeling was found throughout the
A10 dopamine region of the midbrain in both leptin-
treated ob/ob and wild-type mice (Figures 1A and
1C). The most dense pSTAT3 labeling was visualized
in the paranigral (PN) and interfascicular subnuclei
(IF) of the VTA followed by more dorsal labeling in the
rostral linear nucleus (RLi) of the raphe. To a lesser ex-
tent, nuclear staining was also observed in the para-
brachial pigmented nucleus (PBN) of the VTA and the
caudal linear nucleus (CLi) of the raphe. Nuclear
pSTAT3 labeling in the VTA, RLi, and CLi was counted
in half of all brain slices collected (1-in-2 series).There
was no significant difference between estimates of
pSTAT3-positive cells in the A10 region between wild-
type and ob/ob mice following leptin administration
(Figure 1D). Labeling was absent in leptin-receptor-de-
ficient db/db mice after leptin administration, which
demonstrates the obligatory role of ObRb (Figure 1B).
To quantitate pSTAT3 protein, we carried out western
immunoblotting on VTA microdissections (Figure 1E).
As a major target of leptin action is the mediobasal
hypothalamus (MBH) which includes the arcuate, ven-
tromedial, and dorsomedial nuclei, we also measured
induction of pSTAT3 in MBH tissue samples for com-
parative analyses. As shown in Figure 1F, the mag-
nitude of leptin-induced pSTAT3 in the VTA at 90 min
after leptin injection was an average of 63% of that
Figure 1. Peripheral Leptin Injection Increased pSTAT3 Expression in the VTA and Linear Raphe
(A) Immunohistochemical expression of pSTAT3 in ob/ob mice injected with leptin (5 mg/kg, i.p.) or vehicle (PBS; n = 4 per group).
(B) pSTAT3 expression was absent in leptin receptor-deficient db/db mice treated with leptin (n = 4).
(C) pSTAT3 immunoreactivity in wild-type mice administered leptin or vehicle (n = 4 mice per group).
(D) Estimates of pSTAT3-positive cells in the VTA and linear raphe (A10 region). There was no difference between ob/ob and lean wild-type mice
(F(1, 6) = 1.97, p > 0.05). Estimates are based upon counts from one of two series.
(E) Western immunoblotting detection of pSTAT3 and total STAT3 in the VTA and MBH 90 min following acute leptin (5 mg/kg, i.p.) or vehicle
injection in wild-type mice (n = 4–5 per group).
(F) Leptin induced a 2.63 6 0.4-fold increase in pSTAT3 levels in the VTA and a 4.15 6 0.8-fold increase in pSTAT3 in the mediobasal hypothal-
amus as compared to vehicle treated controls. Values are expressed as mean 6 SEM.
observed in the MBH and a lesser signal was seen at
60 min after injection (data not shown).
The VTA is populated by both DA and GABA neurons.
We therefore sought to determine whether either of
these cell types respond to leptin by colabeling pSTAT3
with antibodies to tyrosine hydroxylase (TH) and gluta-
mate decarboxylase (GAD). Estimates of the number of
TH-labeled neurons did not reveal any significant differ-
ences between saline-injected ob/ob and wild-type
mice, thus complete leptin-deficiency does not seem
to be associated with an altered number of DA neurons
in the A10 region. As shown in Figure 2A, pSTAT3
nuclear labeling was present in neurons expressing
TH. Neurons coexpressing TH and pSTAT3 account for
42% 6 4% of pSTAT3-positive cells. Double-labeled
neurons were found predominantly in the PN subpopu-
lation of the VTA and to a lesser extent the RLi and
CLi. Neighboring GABA neurons of the VTA are known
and thus we colabeled for the enzyme GAD which is re-
quired for GABA synthesis. Neurons coexpressing GAD
and pSTAT3 represented 23% 6 5% of total pSTAT3-
positive cells and were seen primarily in the VTA.
VTA Neurons Expressing pSTAT3 Project
to the Nucleus Accumbens
NAc (Beckstead et al., 1979; Swanson, 1982), and this
pathway is important for modulation of goal-oriented
behavior and the effect of systemically administered
drugs. There is evidence that separate populations of
VTA dopamine neurons innervate the NAc and the pre-
frontal cortex (Sesack and Carr, 2002). To determine
whether pSTAT3-positive VTA cells send projections to
the NAc, we combined in vivo retrograde tracing from
the NAc with immunohistochemistry. Of the 14 mice in-
jected with the tracer, the injection sites of eight mice
were confirmed to be in the NAc core and/or shell
(Figure 3A). Microbead tracers do not diffuse signifi-
cantlyfrom thesite ofinjection,and histological analysis
showed that the fluorescent beads were contained
around the tip of the micropipette in the eight mice. In
these eight mice, the tip of the micropipette was posi-
tioned in the NAc at the level of 1.18 to 1.40 (mm anterior
Bregma). In all eight mice, pSTAT3 immunoreactivity
was present in retrogradely labeled perikarya of the
VTA. The mean percentage of rhodamine labeled cells
that expressed pSTAT3 was 12% 6 2%.
Leptin Enhances Sensitization to Amphetamine
Amphetamine (AMPH) increases extracellular DA in the
terminal and cell-body regions of midbrain DA neurons
eitherthrough inhibitionofDAreuptake orreversetrans-
port of cytosolic DA through the DAT transporter. Ele-
vated extracellular DA in the NAc is associated with
AMPH-induced locomotor activity (Kalivas and Stewart,
1991; Heusner et al., 2003). Furthermore, the process
whereby animals become more sensitive to the locomo-
tor-activating effects of amphetamine upon repeated in-
jections (‘‘sensitization’’) is secondary to neuroadapta-
tions of midbrain DA neurons (Vezina, 2004). Thus, to
determine whether leptin has effects on behavioral
outcomes directly involving the mesoaccumbens DA
pathway, we examined the influence of peripheral leptin
infusion on AMPH-induced locomotion. Figure 4A out-
lines the experimental procedure. Mice were initially ac-
climatized in dummy activity-monitoring chambers and
then were implanted with 14 day osmotic minipumps
that infused leptin (500 ng/hour; 12 mg/day) or PBS sub-
cutaneously. This dose of leptin reverses the metabolic
phenotype of ob/ob mice (Halaas et al., 1997) and
slightly exceeds the lowest dose of leptin (10 mg/day)
that decreases food intake and body weights in wild-
type C57BL/6J mice (Harris et al., 1998). Mean body
weights immediately after pump implantation were
24 6 1.4 g (ob/ob—leptin), 24.8 6 1 g (ob/ob—vehicle),
Figure 2. Leptin-Induced pSTAT3 Immunoreactivity Colocalizes with Dopamine and GABA
(A) Representative photomicrographs of the VTA showing TH and pSTAT3 immunofluorescence in an ob/ob mouse. There was no difference in
the estimates of TH-labeled cells between ob/ob (800.3 6 51.7) and wild-type mice (836 6 93.3) as measured from a subset of coronal slices
(F(1, 6) = .263, p > 0.05) (n = 4 mice/group). Note by the merged images that pSTAT3 nuclear immunoreactivity (green) colocalizes with cytoplas-
mic TH expression (red). TH-labeled neurons represent 42% 6 4.7% of pSTAT3-positive cells.
(B)Photomicropgraphs oftheVTAillustrating GADcytoplasmicimmunofluorescence(green)andpSTAT3nuclearstaining(DAB).Notethatthere
is colocalization of pSTAT3 and GAD IHC. Cells labeled with GAD accounted for 23% 6 5.1% of pSTAT3 staining in this region. Values are
expressed as mean 6 SEM.
Leptin and the Mesoaccumbens Dopamine Pathway
18.9 6 0.4 g (wild-type—leptin), and 19.6 6 0.4 g (wild-
type—vehicle). Following 10 days of continuous leptin
infusion, average body weights were 26.8 6 0.8 g
(ob/ob—leptin), 32.6 6 0.9 g (ob/ob—vehicle), 20.6 6
0.6 (wild-type—leptin), and 23.2 6 0.7 g (wild-type—vehi-
cle). Locomotor activity was measured for 4 hr following
IP injections of (1) saline, (2) 1 mg/kg AMPH, (3) 4 mg/kg
AMPH and, (4) 1 mg/kg AMPH dose, each separated by
3 days. Locomotor activity counts collected during the
first hour after injection were used for all comparisons.
Following injection, locomotor activity was assessed
during the light phase of the circadian cycle since all
our biochemical assays were carried out in mice during
the light cycle phase. After initial saline injection, activity
was low in all groups (Figure 4B). Three days later, low-
dose (1mg/kg) AMPH injection elicited no increase inlo-
comotor activity compared to saline treatment for any
days after low-dose AMPH, high-dose AMPH resulted in
a robust induction of locomotor activity, the degree of
which was genotype and treatment specific (Figures
4D and 4F). First, the locomotor response to high-dose
AMPH (4 mg/kg) was lower in ob/ob mice: activity of ve-
hicle-infused ob/ob mice was on average 36% lower as
compared to vehicle-infused wild-type mice. Further,
leptin infusion markedly enhanced the locomotor re-
sponse to high-dose amphetamine in both groups,
with the incremental response after leptin infusion being
ing high-dose AMPH, animals were again treated with
low-dose AMPH to examine sensitization. The degree
of AMPH sensitization was determined by comparing
activity counts from the first hour after the initial low-
dose AMPH to those collected after the second low
dose of AMPH. Wild-type mice showed a 3- to 4-fold in-
creased response to the second low dose of AMPH,
confirming AMPH sensitization, both in the presence
and absence of leptin infusion (Figures 4E and 4G). In
ob/ob mice infused with vehicle, there were no signifi-
cant differences between the two low-dose AMPH con-
ditions, confirming absence of sensitization to AMPH
and suggesting dysregulation of the mesoaccumbens
pathway in these mice. Remarkably, leptin infusion fully
restored the behavioral sensitization to AMPH in ob/ob
mice, producing a 3.1-fold increase in activity in re-
sponse to the second dose of AMPH relative to the first
low dose (Figure 4G).
Leptin Replacement Elevates TH Levels
in the VTA and NAc of ob/ob Mice
To evaluate potential mechanisms underlying the ob-
served difference in amphetamine-induced locomotion
and behavioral sensitization between leptin-deficient
ob/ob andwild-type mice, wenext investigated molecu-
iting enzyme in DA synthesis. We measured protein
levels of TH in the VTA and NAc after 3 day minipump
infusion of leptin (500 ng/hr) or vehicle (PBS) via western
immunoblotting. In ob/ob mice, 3 day leptin infusion
increased TH protein concentrations in the VTA by
2.5-fold as compared to vehicle-treated controls (Fig-
ures 5A and 5C). In contrast, leptin administration failed
to alter TH levels in the MBH, another region where TH
is expressed (Figure 5B). In the NAc terminal region, TH
was reduced in ob/ob controls as compared to wild-
type controls, whereas leptin replacement reversed
this effect by increasing TH levels 2.2-fold relative to sa-
ulation of DA synthesis. In particular, increases in phos-
phorylation of Ser40 is associated with increased enzy-
matic activity of TH (Harada et al., 1996; Jedynak et al.,
2002). As shown in Figure 5D, leptin treatment of ob/ob
mice increased phosphoTH (ser40) levels by an average
of 28.2% and 46.2% in the NAc as compared to leptin-
and vehicle-treated wild-type mice, respectively.
As there was no difference in the number of TH immu-
noreactive neurons between ob/ob and wild-type mice
Figure 3. Leptin-Induced pSTAT3 Immunoreactivity in the VTA
Colocalizes with a Tract Tracer Retrogradely Transported from the
(A) Illustration and photomicrograph showing a representative injec-
tion site of the rhodamine microbeads. Injection sites for 8 of the 14
mice were in the NAc.
(B) Photomicrograph depicting the profile of retrogradely labeled
cells in the VTA.
(C) Confocal image of tracer labeling in the VTA.
(D) Confocal image of nuclear pSTAT3 immunofluorescence.
(E) Confocal image depicting colocalization of pSTAT3 (green) and
rhodamine microbead tracer (red). pSTAT3-positive cells accounted
for an average of 12% 6 2% of rhodamine-labeled cells.
(Figure 2), this suggests that the differences in VTA and
NAc TH and DA levels reflect changes in protein levels
Mice Lacking Leptin Have Diminished Evoked
To further evaluate DA dysregulation, we directly as-
sessed DA release using electrochemical methods to
measure evoked DA release in acute coronal slices of
ob/ob and wild-type mice. A carbon fiber electrode in-
serted into the NAc shell was used to collect ampero-
metric recordings from dopaminergic axons arising
from the VTA. An adjacent bipolar electrode delivered
single pulses (0.5 mA, 2 ms) to trigger the release of syn-
aptic DA. The current caused by the electrochemical ox-
idation oftheextracellular DAservesas ameasureofDA
release kinetics in real time. As shown in Figures 6A and
6B, the evoked DA signal in ob/ob mice was reduced by
on average 90% as compared to the signal measured in
wild-type mice. Real-time DA kinetics are primarily de-
termined primarily by exocytosis and rate of reuptake,
so to determine which of these factors contribute to re-
duced DA signal in ob/ob mice, we examined both the
width of the recorded current spike and the effect of
the DA reuptake blocker nomifensin, a DA transporter
(DAT)-specific inhibitor, on current amplitude and width.
The spike width of the recorded current was similar be-
tween ob/ob and wild-type controls in the absence of
nomifensine. In addition, 30 min bath application of no-
mifensine did not eliminate the reduction of DA signal
amplitude in ob mice. These data suggest that the rate
of DA reuptake is similar between the two genotypes
and that reduced DA signal in ob/ob mice is due to di-
minished synaptic DA release. As an additional assess-
ment, we measured DA release following a pattern of
stimulation pulses that approximates real-time firing
conditions (Figure 6E). As shown in Figure 6F, there
was a progressive decrease in the mean amplitude of
DA release in wild-type mice. In contrast, there was a
much lower mean amplitude of DA release in ob/ob
ulation pulses (Figure 6G).
To assess whether presynaptic DA levels were indeed
lower in the NAc of ob/ob mice relative to wild-type
mice, we measured DA levels via radioimmunoassay of
NAc tissue dissections. As illustrated in Figure 6E, NAc
DA levels were on average 47% lower in ob/ob mice as
compared to wild-type mice. These data are consistent
with our observations of reduced TH expression in
The adipocyte hormone leptin acts directly on CNS cir-
cuits to influence appetite, energy expenditure, neuro-
endocrine function, and other processes. The best
Figure 4. Chronic Leptin Infusion Enhances
Amphetamine-Induced Locomotor Activity
(A) Amphetamine sensitization procedure for
ob/ob and wild-type mice receiving continu-
ous infusion of leptin (500 ng/hr, SC) or PBS
for 13 days (n = 5–8 per group).
(B) Locomotor activity following saline injec-
(C) A subthreshold dose of amphetamine did
not increase locomotor activity.
(D and F) The high dose of amphetamine
augmented locomotor activity in all groups.
Locomotor activity of vehicle-infused ob/ob
mice was reduced as compared to vehicle-
treated wild-type mice (F(1, 82) = 18.38, p =
0.001). Leptin treatment more than doubled
activity levels in ob/ob (F(1, 58) = 18.38, p <
0.0001) and produced a 30% increase in
wild-type mice (F(1, 94) = 18.38, p = 0.003)
as compared to respective vehicle controls.
cle-infused ob/ob mice. Relative to the first
low dose of AMPH, activity levels were not el-
evated after the second low dose of AMPH in
vehicle-infused ob/ob mice (F(1, 58) = 0.78,
p = 0.38) whereas there was a 4-fold increase
in vehicle-infused wild-type mice (F(1, 64) =
16.51, p < 0.0001), a 3.7-fold increase in lep-
tin-infused wild-type mice (F(1, 70)= 18.04,
p < 0.0001) and a 3.1-fold increase in leptin-
treated ob/ob mice (F(1, 39)= 4.03, p = 0.05).
Values are expressed as mean 6 SEM. All
statistics were computed by ANOVA with
Fisher’s post-hoc comparison. *p < 0.05.
Leptin and the Mesoaccumbens Dopamine Pathway
characterized direct targets of leptin action in the CNS
are within the hypothalamus, most specifically the arcu-
ate nucleus and ventromedial hypothalamus. Although
the signaling form of the leptin receptor, ObRb, is ex-
tin on extrahypothalamic circuits have not as yet been
clearly linked to function. The mesoaccumbens DA
pathway plays a role in incentive motivation (Berridge
and Robinson, 1998; Kelley et al., 2005; Robinson
et al., 2005; Salamone et al., 2005) and reward-related
derlying the locomotor-activating, rewarding and addic-
tive effects of drugs of abuse (Wise, 2002; Kelley, 2004;
Vezina, 2004). In the present study, we demonstrate that
leptin directly targets TH and GABA neurons within the
mesoaccumbens pathway and has actions consistent
with an ability to modulate DA tone in this circuit. In ag-
gregate, these observations reveal a mechanism by
and suggests that modulation of midbrain DA as a con-
sequence of leptin action is one way in which leptin may
influence behaviors relevant to energy homeostasis.
After peripheral injection of leptin, tyrosine phosphor-
ylation of STAT3 was identified in the VTA both by west-
ern blotting and by immunohistochemical analysis, with
the latter demonstrating colocalization of pSTAT3 in
a subset of DA neurons of the VTA . In all previous stud-
ies, leptin-induced pSTAT3 in neurons is taken to indi-
cate a direct action of leptin, mediated by the long form
of the leptin receptor (ObRb) on the responsive cell. In
fact, ObRb protein has been colocalized with TH in VTA
neurons (Figlewicz et al., 2003), and ObRb coexpression
in VTA DA neurons has been demonstrated using a ge-
to Rosa26-EYFP reporter mice to generate ObRb-EYFP
reporter mice (J. Lachey, J. Friedman, and J. Elmquist,
personal communication). Localization of pSTAT3 to
DA neurons was most prominent in mid A10 regions in-
known to richly innervate the NAc (Phillipson and Grif-
fiths, 1985). Indeed, we found that a retrograde tracer
injected into the Nac-labeled VTA perikarya that
expressed pSTAT3 demonstrating that at least a subset
of the VTA leptin-regulated neurons innervate the NAc.
We also identified a smaller subset of pSTAT3-immuno-
reactive cells that were colabeled with a marker for
GABA, the second major neurotransmitter found in the
VTA. As several lines of evidence suggest that VTA
GABAergic neurons have an inhibitory influence on DA
neurotransmission, regulation of GABA is an additional
mechanism by which leptin might alter DA function.
To link the ability of leptin to activate STAT3 in VTA DA
neurons to a behavioral phenomenon contingent on
mesoaccumbens DA neurotransmission, we examined
the ability of leptin deficiency and treatment to alter am-
phetamine-induced locomotion. One of the principle ac-
tions of amphetamine is to increase extracellular levels
of DA through blocking reuptake and reversing DA
transport via the dopamine transporter. These actions
ner et al., 2003), whereas its actions in the VTA are asso-
ciated with the induction of behavioral sensitization
Figure 5. Leptin Infusion Increases TH Levels
(A) Three day leptin infusion (500 ng/hr) in
ob/ob mice increased TH levels in the VTA
by 2.5-fold relative to vehicle-treated ob/ob
mice (n = 8–12 per group) (F(3, 40) = 3.98,
p = 0.014).
(B) Leptin infusion failed to alter TH levels in
MBH samples (F(3, 18) = 1.50, p = 0.24).
(C) TH protein levels in the NAc of ob/ob mice
were elevated 2.2-fold by leptin relative to
controls (F(1, 14) = 3.98, p = 0.007). Vehicle-
infused ob/ob mice had 54% less NAc TH
than wild-type controls (n = 7–8 per group)
(F(3, 27) = 3.98, p = 0.008).
(D) Leptin increased phosphoTH (ser40)
levels in the NAc of ob/ob mice by 28.2%
relative to leptin-treated wild-type mice and
by 46.2% as compared to saline-treated
wild-type mice (n= 6–8 per group) (F(3, 25) =
3.36, p = 0.035). Values for immunoblotting
are arbitrary units. All values are expressed
as mean 6 SEM. Statistics were computed
by ANOVA with Fisher’s post hoc compari-
sons. *p < 0.05.
tween any of the genotypes or treatment groups after
saline or the first low-dose amphetamine injection.
This reflects the low basal locomotor activity during
the light phase of the circadian cycle and indicates
that the different responses between genotypes and
treatment groups (see below) were not due to differ-
ences in basal activity. The locomotor response to
high-dose amphetamine was substantially diminished
in ob/ob mice relative to the wild-type mice. Peripheral
leptin infusion at doses in the physiological range re-
versed this defect and elevated amphetamine-induced
locomotor activity in ob/ob to levels above that
observed in any of the other treatment groups. Leptin
infusion increased locomotion in wild-type mice as
well, although to a smaller extent.
Remarkably, in addition to an attenuated locomotor
response to high-dose amphetamine, ob/ob mice
showed no behavioral sensitization to repeat low-dose
amphetamine injection. Unlike wild-type mice, which
displayed the expected enhanced locomotor response
to a second low dose of amphetamine, saline-treated
ob/ob mice had no increased locomotor activity follow-
ing a repeat low-dose amphetamine injection as com-
pared to that after the first low-dose injection. Strikingly,
leptin treatment of ob/ob mice completely reversed this
deficiency, asleptin treatedob/obmicehad asensitized
locomotor response similar to that in wild-type mice.
These data highlight a critical role for leptin in the devel-
opment of AMPH sensitization and thus the reorganiza-
tion of neural circuits that underlies the sensitization
process. Linked to the sensitizing effects of drugs of
abuse such as cocaine and AMPH are alterations in
gene transcription and increased dendritic branching
of medium spiny neurons of the NAc (Robinson and
Berridge, 2003; Hyman et al., 2006).The requirement of
leptin for AMPH sensitization suggests a role for leptin
in such molecular and morphological adaptations.
To explain the diminished amphetamine-induced
locomotor response of ob/ob mice, we sought to deter-
DA release in the mesoaccumbens pathway. There is
prior evidence in rats that AMPH-induced changes in
NAc DA release correlate with the presynaptic DA levels
(Pothos et al., 1998). TH protein levels, both in VTA cell
body and NAc terminal regions, were reduced in ob/ob
mice as compared to leptin-infused ob/ob mice. In addi-
TH and phosphorylated TH ser40 in the NAc.
To directly probe DA release in the NAc, we measured
the amount of evoked-DA release from NAc terminals in
acute slice preparations of ob/ob and wild-type mice.
Figure 6. Reduced Evoked DA Release in the
NAc of ob/ob Mice
(A) Representative amperometric traces of
electrical stimulation-evoked dopamine re-
lease in acute coronal accumbens slices
from ob/ob and wild-type mice. Stimulation
electrodes and carbon fiber recording micro-
electrodes were positioned in the shell region
of the posterior nucleus accumbens, which
receives the majority of the dopaminergic
projections fromthe VTA. Datawereacquired
at 50 kHz and digitally postfiltered at 1 kHz.
Arrows point to onset of electrical single
pulse (2 ms of 0.5 mA).
(B) The mean evoked dopamine signal ampli-
tude was significantly reduced from 50 6 12
pA (mean 6 SEM) in wild-type slices to 5 6
1 pA in ob/ob slices (n = 25 stimulations in
seven slices/mice per genotype, (F(1, 46) =
11.97, *p < 0.01 by one-way ANOVA).
(C) Mean signal width was not different, sug-
gesting that dopamine reuptake rate did not
change in ob/ob slices.
(D) In the presence of the DAT-specific reup-
take blocker nomifensin, the reduction in
evoked dopamine signal wasmaintained,fur-
therindicatingthat thesignalreduction isdue
increase in DA reuptake. *p < 0.01.
(E) Representative amperometric traces from
300 mm nucleus accumbens slices following
a train of five electrical pulses of 500 mA
each with an interpulse interval of 500 ms.
These traces were used to determine the
mean amplitude for each pulse in ob/ob
mice and wild-type controls.
(F and G) There was a significant and pro-
gressive decrease in the mean amplitude
for wild-type mice (F(33, 4) = 130.1541,
*p < 0.01) while the ob/ob mice had an overall lower mean amplitude, which, however, remained stable throughout the five train pulses.
(H) As measured by radioimmunoassay of NAc microdissections, cytosolic DA levels were on average 47% lower in ob/ob mice relative to wild-
type mice (F(1, 22) = 7.8, p = 0.01). All values are expressed as mean 6 SEM.
Leptin and the Mesoaccumbens Dopamine Pathway
Amperometric recordings detected dramatically lower
DA signal in ob/ob mice relative to wild-type mice in re-
sponse to both a single electrical pulse or a train of
pulses. Moreover, during the train of electrical pulses
that resembles real-time firing of DA neurons, the ampli-
tude of DA release did not progressively decrease in
ob/ob mice as it did in wild-type mice. Under normal
conditions DA release is rapidly reduced via the actions
of the D2 autoreceptor (Farnebo and Hamberger, 1971).
That the amplitude of DA release remained relatively
constant over stimulation pulses in ob/ob mice sug-
gests that mechanisms underlying D2 autoreceptor
function may be disabled in ob/ob mice. Despite impair-
ments in DA release in ob/ob mice, the rate of DA clear-
ance did not differ between genotypes, suggesting that
mechanisms controlling DA reuptake are not responsi-
ble for the observed defects in DA release in ob/ob
mice. Therefore, diminished evoked DA release in ob/
able for release, potentially due to diminished synthesis.
Consistent with this possibility, DA levels measured in
NAc punches were reduced by 47%. It remains to be
elucidated however whether additional mechanisms
influencing DA release intheNAc couldalso beimpaired
in ob/ob mice.
It is well known that ob/ob mice lacking leptin have re-
ducedlocomotor activity, andthiscanbecorrected with
leptin therapy (Pelleymounter et al., 1995), but the neural
circuitry underlying this effect is not well described.
Might this be related to our finding that ob/ob mice
have reduced locomotor response to amphetamine
which is reversed by leptin therapy? Amphetamine is
well known to induce hyperlocomotion in mice, and
this is associated with the action of amphetamine on
DA within the NAc. Recent studies using genetic ap-
specific brain regions strongly support the notion that
the locomotor effect of amphetamine requires DA syn-
this response (Heusner et al., 2003). Thus, our finding
that ob/ob mice have reduced locomotor responses to
amphetamine and reduced DA release in the NAc are
fully consistent with these conclusions. It remains to
be determined to what extent this neural circuit is re-
ity consequent to leptin deficiency in ob/ob mice.
Therole ofneurotransmitter systems within theNAcin
reward and addiction has been the subject of much in-
vestigation (Di Chiara et al., 2004; Kelley et al., 2005; Sal-
amoneetal., 2005). Functional inhibition oftheNAcshell
via GABA agonism produces intense hyperphagia in ad
libitum fed animals (Kelley et al., 2005), while dietary
obese rats have low basal extracellular DA levels in the
NAc (Pothos et al., 1998). These observations are con-
sistent with our findings that hyperphagic ob/ob mice
have reduced DA signaling in the NAc. How does this
finding relate to existing concepts of the role of DA in
the regulation of feeding? Much of the literature on the
subject originates in studies with drugs of abuse, not
feeding and natural reinforcers. Those studies that
have focused on the link between mesoaccumbens do-
extracellular dopamine increases during the appetitive
and/or consummatory phase of feeding, and in some
cases it outlasts the consummatory response (Hernan-
dez and Hoebel, 1988; Pothos et al., 1995; Martel and
Fantino, 1996; Bassareo and Di Chiara, 1999; Ahn and
Phillips, 2002; Hajnal et al., 2004). One interpretation of
is a signal associated with food-related learning espe-
cially as it pertains to satiation. Evidence directly linking
reduced mesoaccumbens DA signaling with obesity is
limited. Basal extracellular dopamine in the NAc is low
in rats with dietary obesity, and this is consistent with
the hypothesis that these animals compensate for a def-
icit in NAc DA by increasing food intake (Pothos et al.,
1998). The hypothesis that decreased NAc dopamine
signaling leads to increased feeding is compatible with
the finding that obese humans have reduced central
D2 receptor levels (Wang et al., 2001) and that adminis-
tration of DA agonists to ob/ob mice normalizes hyper-
phagia (Scislowski et al., 1999; Bina and Cincotta,
2000). Therefore, it is possible to hypothesize that mes-
oaccumbens dopamine release and signaling in the
leptin deficient ob/ob mouse are too low to serve as
an adequate reward-relevant learning and satiety signal.
Based on the studies presented here, a defect in NAc
dopamine release may now be attributed at least partly
to deficient leptin action upon this circuit. Since chronic
leptin administration reduces food intake, increases en-
ergy expenditure, and promotes weight loss, and these
in turn can affect other metabolic and neural signals, it is
possible that the actions of leptin on the mesoaccum-
bens pathway responsible for the enhancement of
amphetamine-induced locomotion are both direct, via
actions on the VTA-NAc circuit described here, and indi-
rect. The extent to which this results from direct leptin
action on neurons within the mesoaccumbens circuit,
as opposed to indirect effects via changes in other hor-
mones or neuropeptides, will require additional study.
eral leptin to induce signaling in neurons within the mes-
oaccumbens DA circuit. In leptin-deficient ob/ob mice,
deficient leptin action causes reduced NAc TH levels
and reduced evoked DA release from coronal slices of
NAc shell. Finally, these neuroanatomic and biochemi-
cal findings are accompanied by important behavioral
consequences previously linked to this circuit, since
leptin action regulates the ability of AMPH to stimulate
locomotor activity and leptin is required for sensitization
of the locomotor response to low-dose amphetamine.
Taken together, these data suggest that the actions of
leptin go beyond well-known targets in the hypothala-
mus and brainstem and that the modulation of mesoac-
cumbens DA circuitry by leptin may be important for the
regulation of behaviors that influence appetite, locomo-
tor control, and body weight.
Male B6.V-Lepob/J (ob/ob), wild-type controls (LepOb+/?), Leprdb
chased from Jackson Laboratory. Mice, between 4 and 10 weeks of
age, were housed in a temperature- and humidity-controlled room
that was maintained on a 14:10 light/dark cycle, with lights on at
0600 hr. All experiments were conducted in accordance with the
guidelines and approval of the Harvard Medical School and Beth
Israel Deaconess Medical Center and Tufts University-New England
Medical Center Institutional Animal Care and Use Committees.
Immunohistochemical Characterization of Leptin Signaling
Recombinant mouse leptin was obtained from Dr. A.F. Parlow (Na-
tional Hormone and Peptide Program, National Institute of Diabetes
and Digestive Kidney Diseases, Torrance, California). Leptin (5 mg/
kg body weight) or vehicle (PBS) was injected i.p. following an over-
night fast. Two hours after injection, mice were anesthetized with
ketamine/xylazine (45 mg/kg/5 mg/kg, i.p.) and perfused transcar-
dially with ice-cold saline followed by 10% neutral buffered formalin.
Brains were removed and postfixed for 4–8 hr and then cryopro-
tected by overnight immersion in a 20% sucrose solution. Frozen
brains were sliced in 25 mm coronal sections using a microtome.
For pSTAT3 immunohistochemistry, free-floating sections werepre-
treated with 1% NaOH, 1% H2O2, and then 0.6% glycine, blocked in
goat serum, and then incubated in anti-pSTAT3 (tyr705) antibody
(1:1000, Cell Signal Technology) for 48–54 hr at 4?C. Sections were
rinsed and incubated with a secondary biotinylated anti-rabbit anti-
body (1:1000; Vector Laboratories), labeled with avidin-biotin com-
plex, and then stained with nickel-enhanced diaminobenzidine.
For double-labeling experiments examining the cell types express-
ing pSTAT3 in the VTA, separate series of brain sections were addi-
tionallyprocessed with amouse monoclonalantibody to TH (1:1000;
Chemicon International) or a rabbit polyclonal antibody to GAD65/67
(1:1000; Chemicon). Fluorescent secondary detection Alexafluor
595 and 488 (1:200, Vector Laboratories) was used to visualize TH
and GAD65/67, respectively. Brain sections for light microscopy
were mounted on gelatin-coated slides, dehydrated, and coverslip-
ped with mounting solution. Fluorescently labeled sections were
mounted and coverslipped with aqueous mounting medium for fluo-
rescence. Staining was visualized using a Zeiss Axio Imager A1 fluo-
rescent microscope with an Axiocam HR cooled CCD digital camera
and Axiovision 4AC image acquisition software (Carl Zeiss Microi-
maging, Inc.). Estimates of pSTAT3-labeled cell numbers were
made by counting nickel-enhanced DAB nuclear staining in the mid-
brain area corresponding to the A10 dopamine cell group based
upon the description by Dahlstrom and Fuxe (1964) and its applica-
tion to C57BL/6J mice (Zaborszky and Vadasz, 2001). Accordingly,
the A10 cell group includes the VTA, RLi, and CLi. Nuclear pSTAT3
was manually counted in the VTA, RLi, and CLi in every mounted
section of a 1-in-2 series using Image J software (Rasband, WS;
Image J, US, NIH). For TH and pST T3 double labeling, the number
of TH-positive neurons was counted in coronal sections that corre-
spond to five rostrocaudal levels of the A10 DA region as based on
the mouse atlas of Paxinos and Franklin (2001) (distance caudal to
Bregma): 23.08 mm, 23.16 mm, 23.40 mm, 23.64 mm, 3.80 mm.
Percentages of pSTAT3-positive cells that colocalized with TH or
GAD immunoreactivity were derived from sections corresponding
to these same five coronal levels.
Subcutaneous Leptin Infusion
Leptininfusionfor either 3 days (westernblotting) or14 days (behav-
ioral testing) was delivered using Alzet micro-osmotic pumps (Alta
Corp.). Pumps were filled with either leptin (500 ng/hr; 12 mg/day)
or vehicle (PBS) and primed for 4–6 hr at 37?C before implantation.
The pump was inserted into the interscapular region under Isoflur-
ane anesthesia. The incision was closed with wound clips.
SDS-PAGE and Immunoblotting
Animals were anesthetized with CO2before decapitation. Brains
were rapidly removed and froze on powdered dry ice. Frozen brains
were placed in a cold aluminum brain slicer with 0.5 mm dividers.
A 1 mm thick frozen coronal section limiting the VTA and 2 mm cor-
onal section containing the arcuate, ventromedial, and dorsomedial
nuclei (‘‘mediobasal hypothalamus’’) was sliced and mounted on a
slide. Brain nuclei were rapidly microdissected using a brain punch
set (Stoelting Co). Two tissue punches, one for each hemisphere,
were collected for the VTA (0.75 mm diameter), and the MBH (1.0
mm diameter). Microdissected tissues were homogenized on ice
in 100 ml of cell lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl,
1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophos-
phate, 1mM b-glycerophosphate, 1mM Na3VO4, 1 mg/ml leupeptin)
with added protease (PMSF 100 mM) and phosphatase inhibitors
(Sigma phosphatase inhibitor cocktails I and II) in 1.5 ml tubes using
for 30 min at 4?C to allow for complete lysis and then centrifuged for
15 min at 14,000 3 g. Protein concentrations were measured using
BCA protein assay (Pierce Biotechnology). For the experiment ex-
amining pSTAT3 protein levels following IP leptin (5mg/kg body
weight) or vehicle (PBS), 70 mg of protein was loaded into 10% poly-
acrylamide gels. Protein of concentration representing 50%, 100%,
150% of sample protein concentrations from saline-treated wild-
type mice served as standards for each gel. Protein was transferred
overnight (30V) onto PVDF membranes using transfer buffer (50 mM
Tris, 20 mM glycine, 20% methanol). Membranes were blocked in
5% nonfat milk (20 mM Tris [pH 7.4], 0.9% NaCl, 0.1% Tween 20)
and incubated with anti-pSTAT3 antibody (1:1000; Cell Signal) over-
night at 4?C. Membranes were then washed and incubated with
secondary antibody (anti-rabbit horseradish peroxidase coupled;
1:1000; Cell Signal), washed and developed by enhanced chemilu-
minescence, and imaged on the Genegnome digital imaging system
(Syngene). Protein volume from the generated digital images was
measured using Genetools analysis software (Syngene). For total
STAT3 detection, membranes were stripped with Restore Western
stripping buffer (Pierce Biotechnologies) and reprobed with an
anti-STAT3 antibody (1:1000; Cell Signaling) and detected as de-
For experiments examining the influence of subcutaeuous leptin
following 3 days continuous minipump infusion (see above, Subcu-
taneous Leptin Infusion). Microdissections from the VTA, MBH, and
NAc (1.5 mm diameter punch) were processed as described above.
Protein samples (30 mg) were separated by SDS-PAGE, transferred
to membrane and then probed for TH (1:1000; Chemicon), phos-
phoTH (ser40) (1:1000; Chemicon). Equal loading and transfer was
verified by probing for the housekeeping protein, GADPH (1:1000;
Combined Retrograde Tracing and Immunohistochemistry
To determine whether cells targeted by leptin project to the Nac, we
combined retrograde tract tracing with pSTAT3 immunohistochem-
istry. Fourteen male C57BL/6J mice anesthetized with ketamine/
xylazine (45 mg/kg/5 mg/kg, i.p.) received unilateral injections of
rhodamine-filled latex microbeads (Retrobeads, Lumafluor Inc.)
into the NAc. With the aid of an Ultraprecise Small Animal Stereo-
taxic Instrument (Kopf), injections were made using a 1 ml Hamilton
digital syringe attached to a pulled glass micropipette with a 60–80
mm external tip diameter. The coordinates for NAc injections were
0.4 mm rostral to Bregma, 1.40 mm lateral to the midline, and
4.25 mm ventral to the skull surface, based upon the mouse brain
atlas of Paxinos and Franklin (2001). The micropipette remained in
place for 15 min following the delivery of 50–100 nl of tracer. The
scalp incision was closed with wound clips and treated with antibi-
otic ointment. After a 4 to 7 day survival time, mice were fasted over-
night and then injected with leptin (5 mg/kg bodyweight, i.p.). Two
hours later, animals were anesthetized with ketamine/xylazine
(45 mg/kg/5 mg/kg, i.p.) and underwent cardiac perfusion with ice-
cold saline followed by 10% formalin. IHC procedures for pSTAT3
wereasdescribed above. Sections weremounted on gelatin-coated
slides, coated with Clearmount mounting solution (Zymed), and
heat-dried in an oven to form a liquid coverslip. A Zeiss A1 fluores-
cent microscope, Axiocam HR CCD camera, and Axiovision 4AC
software (Carl Zeiss Microimaging, Inc.) was used to visualize the in-
jection sites and the retrogradely labeled perikarya profiles in the
VTA. Injection sites in which the tracer did not target any part of
the NAc served as negative controls. Colocalization of tracer with
pSTAT3 in the VTA was visualized and photographed with a Zeiss
LSM META 510 confocal microscope and LSM META software
(Carl Zeiss Microimaging, Inc.). Counts of rhodamine-labeled cells
and pSTAT3 double-labeled cells in the VTA were made in sections
corresponding to five different coronal levels of the A10 DA region
according to the mouse atlas of Paxinos and Franklin (2001)
(described here as distance caudal to Bregma): 23.08 mm, 23.16
mm, 23.40 mm, 23.64 mm, 3.80 mm. The percentage of rhoda-
mine-labeled perikarya in the A10 region that expressed pSTAT3
Leptin and the Mesoaccumbens Dopamine Pathway
Amphetamine-Induced Locomotor Activity and Sensitization
The experiment described here used a Comprehensive Lab Animal
Monitoring System (CLAMS; Columbus Instruments) that consists
of individual live-in cages for mice that allow automated, noninva-
sive data collection. The system allows measurement of locomotor
activity by quantifying beam breaks in x-y-z planes. A total of 32
drug naı ¨ve mice (16 ob/ob and 16 wild-type controls) were tested.
Mice were initially placed in dummy acclimation chambers for
2 days. On the third day, a 14 day Alzet micro-osmotic pump (model
1014D, Alza Corp.) was implanted SQ in the intrascapular region
under Isoflurane anesthesia. The pumps delivered either leptin
(500 ng/hr; 12 mg/day) or PBS for the duration of the experiment
(11 days).Over the subsequent 10days,all micereceived fouri.p. in-
jections, each separated by 3 days, in the following order: (1) saline,
(2) 1 mg/kg amphetamine, (3) 4 mg/kg amphetamine, (4) 1 mg/kg
amphetamine. Mice were acclimatized O/N to the CLAMS chambers
before the injections and returned to their home cages after the ac-
tivity recording. All injections were given at the same time of day
which corresponded to the middle of the light phase of the cycle.
Beam breaks were measured in 10 min bins for 4 hr after the injec-
tion. Body-weight values were collected daily. One-way ANOVAs
with post hoc pairwise comparisons (Fisher PLSD) were used to
compare activity counts from the first hour following the injection
for each of the four injections. The ability of amphetamine to sensi-
tize the locomotor response to the second low dose (1mg/kg) of am-
phetamine was assessed using a one-way ANOVA that compared
activitycountsbetween the first andsecond lowdose foreachtreat-
Mice were anesthetized with ketamine (200 mg/kg IP)/xylazine
(20 mg/kg IP). Upon removal, the brain was immediately placed
into ice-cold and carbogenated (95% O2, 5% CO2) dissection solu-
tion (Pelletier/Carlen) containing 210 mM sucrose, 3.5 mM KCl,
1 mM CaCl2dihydrate, 4 mM MgCl2hexahydrate, 1.25 mM NaH2PO4
hydrate, 10 mM glucose, and 26 mM NaHCO3. Coronal slices con-
taining the posterior nucleus accumbens shell region, 300 mm thick,
were cut using a vibratome. Following a 1 hr recovery period in car-
bogenated ACSF (124 NaCl mM, 2.0 KCl mM, 1.25 KH2PO4mM,
2.0 MgSO4mM, 25 NaHCO3mM, 1.0 CaCl2mM, 11 glucose mM
[pH 7.3]), each slice was transferred into a recording chamber main-
tained at 37?C. Slices were continually perfused with ACSF at a rate
of 1 ml/min. A disk carbon fiber electrode, 5 mm in diameter, was
placed in the posterior nucleus accumbens shell, posterior dorso-
medial striatum, or medial prefrontal cortex at a depth of w50 mm.
The reference electrode (Ag/AgCl wire) was inserted into the ACSF
bath. Voltage was set to +700 mV (Axopatch 200B, Axon Instr.).
The bipolar stimulating electrode (diameter 0.005 inch—MS 303/3,
Plastics One, Inc.) was placed within a distance of 200 mm from
the carbon fiber electrode. A constant monophasic current stimulus
(delivered by Isoflex stimulus isolator, AMPI Inc., controlled by a
Grass Instruments S88 Stimulator) was applied through the bipolar
stimulating electrode with the following stimulation parameters: 4
ms single rectangular pulse, 300 mA current; a train of five single
pulses is delivered with an interstimulus interval of 5 min to allow
for full recovery. Local bath application of the dopamine reuptake
blocker nomifensin (3 mM) for at least 30 min was used to assess
the contribution of reuptake in the evoked dopamine signal. To
imates real-time firing rate, an additional set of recording was col-
lected following a train of five electrical pulses of 500 mA each with
an interpulse interval of 500 ms. Amperometric electrode recordings
were monitored and quantified by a locally written routine on the
Superscope II platform (GW Instruments). Data acquisition occurred
at 50 kHz and was digitally postfiltered at 1 kHz background-
subtracted cyclic voltammograms serve to calibrate the electrodes
and to identify the released substance as DA.
Data Analysis and Statistics
Data analysis included spike amplitude, spike width, and number of
molecules as derived by the charge of each spike. Specifically, the
total charge of the event between the baseline intercepts was deter-
mined and the number of molecules estimated by the relation N =
Q/nF, where Q is the charge, n the number of electrons donated
per molecule, and F is Faraday’s constant (96,485 coulombs per
equivalent). Estimates were based on an assumption of two elec-
trons donated per oxidized molecule of dopamine (Ciolkowski
et al., 1994). Amperometric spikes were identified as events with
greater than 4.53 the Rms noise of the baseline following the stim-
ulus artifact. This cutoff excludes all transients observed during
background recordings in the bath. The event width is the duration
between (a) the baseline intercept of the maximal incline from the
baseline to first point that exceeded the cut-off and (b) the first
data point following the maximal amplitude that registered a value
of %0 pA. The maximum amplitude (imax) of the event is the highest
value within the event. The width at half height (t1/2) was determined
by the duration of the spike trace at one-half the maximal amplitude.
The five single pulses per slice were averaged into a grand mean
and the two groups (ob/ob versus wild-type) were compared with
a one-way ANOVA.
as described above (SDS-PAGE and Immunoblotting) and homoge-
nized in 100 ml of 0.1 N HCl. Aliquots containing the homogenates
were shaken for 30 min at 4?C to allow for complete lysis and then
centrifuged for 15 min at 14,000 3 g. Protein concentrations were
were processed according to dopamine RIA kit instructions
(RE29345, IBL Hamburg) while ensuring proper standards and
We are grateful to Clifford Saper for helpful comments on this paper.
We would also like to thank Maia Kokoeva and FrankMarino for their
help. Thanksto the Canadian Institute of Health Research for apost-
doctoral fellowship supporting S.F. and grants from NIH to J.S.F.
(DKR37 28082), to J.S.F., E.M.-F. and E.N.P. (DK069983), and to
E.N.P. (1RO1DK065872 and Smith Family Investigator Award-
Medical Foundation). J.S.F. is on the scientific advisory board of
Elixir Pharmaceuticals and receives sponsored research from
Received: June 13, 2006
Revised: August 17, 2006
Accepted: September 5, 2006
Published: September 20, 2006
Ahima, R.S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Mar-
atos-Flier, E., and Flier, J.S. (1996). Role of leptin in the neuroendo-
crine response to fasting. Nature 382, 250–252.
Ahn, S., and Phillips, A.G. (2002). Modulation by central and basolat-
eral amygdalar nuclei of dopaminergic correlates of feeding to sati-
ety in the rat nucleus accumbens and medial prefrontal cortex.
J. Neurosci. 22, 10958–10965.
Balthasar, N., Coppari, R., McMinn, J., Liu, S.M., Lee, C.E., Tang, V.,
Kenny, C.D., McGovern, R.A., Chua, S.C., Jr., Elmquist, J.K., and
Lowell, B.B. (2004). Leptin receptor signaling in POMC neurons
is required for normal body weight homeostasis. Neuron 42, 983–
Bassareo, V., and Di Chiara, G. (1999). Modulation of feeding-
induced activation of mesolimbic dopamine transmission by appeti-
tive stimuli and its relation to motivational state. Eur. J. Neurosci. 11,
Baumann, H., Morella, K.K., White, D.W., Dembski, M., Bailon, P.S.,
Kim, H., Lai, C.F., and Tartaglia, L.A. (1996). The full-length leptin re-
ceptor has signaling capabilities of interleukin 6-type cytokine re-
ceptors. Proc. Natl. Acad. Sci. USA 93, 8374–8378.
Beckstead, R.M., Domesick, V.B., and Nauta, W.J. (1979). Efferent
connections of the substantia nigra and ventral tegmental area in
the rat. Brain Res. 175, 191–217.
Berridge, K.C., and Robinson, T.E. (1998). What is the role of dopa-
mine in reward: hedonic impact, reward learning, or incentive sa-
lience? Brain Res. Brain Res. Rev. 28, 309–369.
Bina, K.G., and Cincotta, A.H. (2000). Dopaminergic agonists nor-
malize elevated hypothalamic neuropeptide Y and corticotropin-
releasing hormone, body weight gain, and hyperglycemia in ob/ob
mice. Neuroendocrinology 71, 68–78.
Campfield, L.A., Smith, F.J., Guisez, Y., Devos, R., and Burn, P.
(1995). Recombinant mouse OB protein: evidence for a peripheral
signal linking adiposity and central neural networks. Science 269,
Ciolkowski, E.L., Maness, K.M., Cahill, P.S., Wightman, R.M., Evans,
D.H., Fosset, B., and Amatore, C. (1994). Disproportionation during
electrooxidation of catecholamines at carbon-fiber microelec-
trodes. Anal. Chem. 66, 3611–3617.
Dahlstrom, A., and Fuxe, K. (1964). Localization of monoamines in
the lower brain stem. Experientia 20, 398–399.
Di Chiara, G., Bassareo, V., Fenu, S., De Luca, M.A., Spina, L.,
Cadoni, C., Acquas, E., Carboni, E., Valentini, V., and Lecca, D.
(2004). Dopamine and drug addiction: the nucleus accumbens shell
connection. Neuropharmacology 47 (Suppl 1), 227–241.
Elias, C.F., Aschkenasi, C., Lee, C., Kelly, J., Ahima, R.S., Bjorbaek,
C., Flier, J.S., Saper, C.B., and Elmquist, J.K. (1999). Leptin differen-
tially regulates NPY and POMC neurons projecting to the lateral hy-
pothalamic area. Neuron 23, 775–786.
Elmquist, J.K., Bjorbaek, C., Ahima, R.S., Flier, J.S., and Saper, C.B.
(1998). Distributions of leptin receptor mRNA isoforms in the rat
brain. J. Comp. Neurol. 395, 535–547.
Elmquist, J.K., Coppari, R., Balthasar, N., Ichinose, M., and Lowell,
B.B. (2005). Identifying hypothalamic pathways controlling food in-
take, body weight, and glucose homeostasis. J. Comp. Neurol.
Erickson, J.C., Hollopeter, G., and Palmiter, R.D. (1996). Attenuation
of the obesity syndrome of ob/ob mice by the loss of neuropeptide
Y. Science 274, 1704–1707.
Farnebo, L.O., and Hamberger, B. (1971). Drug-induced changes in
the release of 3 H-monoamines from field stimulated rat brain slices.
Acta Physiol. Scand. Suppl. 371, 35–44.
Figlewicz, D.P., Evans, S.B., Murphy, J., Hoen, M., and Baskin, D.G.
(2003). Expression of receptors for insulin and leptin in the ventral
tegmental area/substantia nigra (VTA/SN) of the rat. Brain Res.
Figlewicz,D.P., Bennett, J.,Evans,S.B.,Kaiyala,K.,Sipols, A.J., and
Benoit, S.C. (2004). Intraventricular insulin and leptin reverse place
preference conditioned with high-fat diet in rats. Behav. Neurosci.
Fulton, S., Woodside, B., and Shizgal, P. (2000). Modulation of brain
reward circuitry by leptin. Science 287, 125–128.
Fulton, S., Richard, D., Woodside, B., and Shizgal, P. (2004). Food
restriction and leptin impact brain reward circuitry in lean and obese
Zucker rats. Behav. Brain Res. 155, 319–329.
Hajnal, A., Smith, G.P., and Norgren, R. (2004). Oral sucrose stimula-
tion increases accumbens dopamine in the rat. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 286, R31–R37.
Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, S.L., Chait, B.T., Ra-
binowitz, D., Lallone, R.L., Burley, S.K., and Friedman, J.M. (1995).
Weight-reducing effects of the plasma protein encoded by the
obese gene. Science 269, 543–546.
Halaas, J.L., Boozer, C., Blair-West, J., Fidahusein, N., Denton, D.A.,
and Friedman, J.M. (1997). Physiological response to long-term
peripheral and central leptin infusion in lean and obese mice. Proc.
Natl. Acad. Sci. USA 94, 8878–8883.
Hao, J., Cabeza de Vaca, S., and Carr, K.D. (2004). Effects of chronic
ICV leptin infusion on motor-activating effects of D-amphetamine in
food-restricted and ad libitum fed rats. Physiol. Behav. 83, 377–381.
Harada, K., Wu, J., Haycock, J.W., and Goldstein, M. (1996). Regula-
tion of L-DOPA biosynthesis by site-specific phosphorylation of
tyrosine hydroxylase in AtT-20 cells expressing wild-type and serine
40-substituted enzyme. J. Neurochem. 67, 629–635.
Harris, R.B.,Zhou,J.,Redmann,S.M., Jr., Smagin, G.N., Smith, S.R.,
Rodgers, E., and Zachwieja, J.J. (1998). A leptin dose-response
study in obese (ob/ob) and lean(+/?)mice.Endocrinology 139,8–19.
Hay-Schmidt, A., Helboe, L., and Larsen, P.J. (2001). Leptin receptor
immunoreactivity is present in ascending serotonergic and cate-
cholaminergic neurons of the rat. Neuroendocrinology 73, 215–226.
Hernandez, L., and Hoebel, B.G. (1988). Food reward and cocaine
increase extracellular dopamine in the nucleus accumbens as mea-
sured by microdialysis. Life Sci. 42, 1705–1712.
Heusner, C.L., Hnasko, T.S., Szczypka, M.S., Liu, Y., During, M.J.,
and Palmiter, R.D. (2003). Viral restoration of dopamine to the nu-
cleus accumbens is sufficient to induce a locomotor response to
amphetamine. Brain Res. 980, 266–274.
Hoebel, B.G., Monaco, A.P., Hernandez, L., Aulisi, E.F., Stanley,
B.G., and Lenard, L. (1983). Self-injection of amphetamine directly
into the brain. Psychopharmacology (Berl.) 81, 158–163.
Hyman, S.E., Malenka, R.C., and Nestler, E.J. (2006). Neural mecha-
nisms of addiction: the role of reward-related learning and memory.
Annu. Rev. Neurosci. 29, 565–598.
Jedynak, J.P., Ali, S.F., Haycock, J.W., and Hope, B.T. (2002). Acute
administration of cocaine regulates the phosphorylation of serine-
19, -31 and -40 in tyrosine hydroxylase. J. Neurochem. 82, 382–388.
Kalivas, P.W., and Stewart, J. (1991). Dopamine transmission in the
initiation and expression of drug- and stress-induced sensitization
of motor activity. Brain Res. Brain Res. Rev. 16, 223–244.
Kelley, A.E. (2004). Memory and addiction: Shared neural circuitry
and molecular mechanisms. Neuron 44, 161–179.
Kelley, A.E., Baldo, B.A., Pratt, W.E., and Will, M.J. (2005). Cortico-
striatal-hypothalamic circuitry and food motivation: integration of
energy, action and reward. Physiol. Behav. 86, 773–795.
Luquet, S.,Perez,F.A.,Hnasko, T.S., and Palmiter, R.D.(2005).NPY/
AgRP neurons are essential for feeding in adult mice but can be ab-
lated in neonates. Science 310, 683–685.
Martel, P., and Fantino, M. (1996). Mesolimbic dopaminergic system
activity as a function of food reward: a microdialysis study. Pharma-
col. Biochem. Behav. 53, 221–226.
Niswender, K.D., Morton, G.J., Stearns, W.H., Rhodes, C.J., Myers,
M.G., Jr., and Schwartz,M.W. (2001). Intracellularsignalling. Key en-
zyme in leptin-induced anorexia. Nature 413, 794–795.
Paxinos, G., and Franklin, K.B.J. (2001). The Mouse Brain in Stereo-
taxic Coordinates (San Diego, CA: Academic Press).
Pelleymounter, M.A., Cullen, M.J., Baker, M.B., Hecht, R., Winters,
D., Boone, T., and Collins, F. (1995). Effects of the obese gene prod-
uct on body weight regulation in ob/ob mice. Science 269, 540–543.
Phillipson, O.T., and Griffiths, A.C. (1985). The topographic order of
inputs to nucleus accumbens in the rat. Neuroscience 16, 275–296.
Pothos, E.N., Creese, I., and Hoebel, B.G. (1995). Restricted eating
with weight loss selectively decreases extracellular dopamine in
the nucleus accumbens and alters dopamine response to amphet-
amine, morphine, and food intake. J. Neurosci. 15, 6640–6650.
Pothos, E.N., Sulzer, D., and Hoebel, B.G. (1998). Plasticity of quan-
tal size in ventral midbrain dopamine neurons: possible implications
for the neurochemistry of feeding and reward. Appetite 31, 405.
Robinson, T.E., and Berridge, K.C. (2003). Addiction. Annu. Rev.
Psychol. 54, 25–53.
Robinson, S., Sandstrom, S.M., Denenberg, V.H., and Palmiter, R.D.
(2005). Distinguishing whether dopamine regulates liking, wanting,
and/or learning about rewards. Behav. Neurosci. 119, 5–15.
Salamone, J.D., Correa, M., Mingote, S.M., and Weber, S.M. (2005).
Beyond the reward hypothesis: alternative functions of nucleus ac-
cumbens dopamine. Curr. Opin. Pharmacol. 5, 34–41.
Schultz, W. (2002). Getting formal with dopamine and reward. Neu-
ron 36, 241–263.
Scislowski, P.W., Tozzo, E., Zhang, Y., Phaneuf, S., Prevelige, R.,
and Cincotta, A.H. (1999). Biochemical mechanisms responsible
for the attenuation of diabetic and obese conditions in ob/ob mice
treated with dopaminergic agonists. Int. J. Obes. Relat. Metab. Dis-
ord. 23, 425–431.
Leptin and the Mesoaccumbens Dopamine Pathway
Sesack, S.R., and Carr, D.B. (2002). Selective prefrontal cortex Download full-text
inputs to dopamine cells: implications for schizophrenia. Physiol.
Behav. 77, 513–517.
Shalev, U., Yap, J., and Shaham, Y. (2001). Leptin attenuates acute
food deprivation-induced relapse to heroin seeking. J. Neurosci.
Swanson, L.W. (1982). The projections of the ventral tegmental area
and adjacent regions: a combined fluorescent retrograde tracer and
immunofluorescence study in the rat. Brain Res. Bull. 9, 321–353.
Tartaglia, L.A., Dembski, M., Weng, X., Deng, N., Culpepper, J., De-
vos, R., Richards, G.J., Campfield, L.A., Clark, F.T., Deeds, J., et al.
(1995). Identification and expression cloning of a leptin receptor,
OB-R. Cell 83, 1263–1271.
reward value by dopamine neurons. Science 307, 1642–1645.
Vaisse, C., Halaas, J.L., Horvath, C.M., Darnell, J.E., Jr., Stoffel, M.,
and Friedman, J.M. (1996). Leptin activation of Stat3 in the hypothal-
amus of wild-type and ob/ob mice but not db/db mice. Nat. Genet.
Vezina, P. (2004). Sensitization of midbrain dopamine neuron reac-
tivity and the self-administration of psychomotor stimulant drugs.
Neurosci. Biobehav. Rev. 27, 827–839.
Wang, G.J., Volkow, N.D., Logan, J., Pappas, N.R., Wong, C.T., Zhu,
W., Netusil, N., and Fowler, J.S. (2001). Brain dopamine and obesity.
Lancet 357, 354–357.
Wise, R.A. (2002). Brain reward circuitry: Insights from unsensed in-
centives. Neuron 36, 229–240.
Zaborszky, L., and Vadasz, C. (2001). The midbrain dopaminergic
system: anatomy and genetic variation in dopamine neuron number
of inbred mouse strains. Behav. Genet. 31, 47–59.
Zhao, A.Z., Huan, J.N., Gupta, S., Pal, R., and Sahu, A. (2002). A
phosphatidylinositol 3-kinase phosphodiesterase 3B-cyclic AMP
pathway in hypothalamic action of leptin on feeding. Nat. Neurosci.