ARCHIVAL REPORT

Presynaptic Leptin Action Suppresses Excitatory

Synaptic Transmission onto Ventral Tegmental Area
Dopamine Neurons
Jennifer L. Thompson and Stephanie L. Borgland
Background: Leptin is an adipocyte-derived cytokine that can act in the brain to suppress feeding and maintain energy homeostasis.
Additionally, leptin activates its receptors in the ventral tegmental area (VTA), a critical site for neuroadaptations to rewarding stimuli, to
modulate reward-seeking behaviors. Although leptin can decrease intrinsic excitability of dopamine neurons in the VTA, it is unknown
whether leptin can modulate excitatory synaptic transmission in this brain region. Because plasticity of glutamatergic synapses onto VTA
neurons can encode predictive information about reward, we hypothesized that leptin can decrease excitatory synaptic transmission
onto dopamine neurons.

Methods: Using whole-cell patch clamp electrophysiology in mouse midbrain slices, we tested the effects of leptin on evoked a-amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) or N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory postsynaptic
currents (EPSCs) onto VTA dopamine neurons.
Results: Leptin depressed both AMPAR and NMDAR EPSCs in VTA dopamine neurons and reduced frequency but not amplitude of
mini EPSCs. Bath application of the MEK1/2 inhibitor U0126 did not alter leptin-induced suppression of AMPAR EPSCs. However,
external, but not internal, application of the phosphoinositol 3-kinase (PI3K) or Janus kinase 2 (Jak2) tyrosine kinase inhibitors abolished
leptin-induced synaptic depression.

Conclusions: This study demonstrates that leptin causes a presynaptic inhibition of the probability of glutamate release onto VTA
dopamine neurons. This synaptic inhibition requires Jak2 and PI3K activation. Leptin-induced weakening of synaptic strength onto
dopamine cells may underlie its inhibitory effects on appetitive behavior for rewarding stimuli.

So far, there is little understanding of the mechanism behind

Key Words: AMPA, dopamine, leptin, presynaptic, synaptic leptin modulation of these effects. Supraphysiologic doses of
transmission, VTA systemic leptin decrease the firing rate of putative VTA dopamine
neurons in anesthetized rats (5). Furthermore, bath application
of leptin to midbrain slices decreases firing rate of putative

L
eptin is a cytokine released from adipocytes that circulates
throughout the central nervous system (CNS) to convey the
status of body energy stores and to control feeding and
energy expenditure. Leptin readily crosses the blood–brain
barrier through a saturable transport system (1–3) and binds to
its long-form signaling receptor, LepRb, in many brain regions
(2,4) including the ventral tegmental area (VTA) (5–8). VTA
dopamine neurons and their widespread neural targets in the
striatum, amygdala, prefrontal cortex, and elsewhere mediate the
incentive salience of food and other rewards (9). Leptin signaling
in the VTA may be one mechanism used to alter the motivation
to consume food or drugs of abuse. Indeed, intra-VTA leptin
decreases food consumption (5) and LepRb knockdown in the
VTA increases effort to obtain palatable food reinforcers (10).
Systemic leptin reduces the rewarding value of sucrose that is
dependent on dopamine neuron activation (11). Taken together,
a wealth of evidence suggests that leptin signaling in the
mesolimbic dopamine circuit can modulate natural and drug
reward-seeking behaviors (12).
From the Department of Anesthesiology, Pharmacology and Therapeutics,
University of British Columbia, Vancouver, Canada.
Address correspondence to Stephanie L. Borgland, Ph.D., Department of
Anesthesiology, Pharmacology and Therapeutics, University of British
Columbia, 212-2176 Health Sciences Mall, Vancouver BC V6T 1Z3,
Canada; E-mail: Borgland@mail.ubc.ca.
Received Jul 14, 2012; revised and accepted Oct 31, 2012.
dopamine neurons by approximately 20% (5) in an extracellular
signal-regulated kinase (ERK)-dependent manner (13). Leptin-
induced decrease in neuronal firing may be due to a change in
intrinsic neuronal properties of dopamine neurons or a result of
altered pre- or postsynaptic effects of glutamate or gamma-
aminobutyric acid (GABA) onto dopamine neurons. However, it
is unknown how leptin modulates excitatory or inhibitory
synaptic transmission onto VTA dopamine neurons. Here, we
test the hypothesis that leptin modulates excitatory synaptic
transmission onto dopamine neurons.
Glutamatergic synapses onto VTA dopamine neurons can
undergo both long-term potentiation (LTP) and long-term
depression (LTD) to regulate their synaptic strength and conse-
quent dopaminergic output (14–18). Enhanced synaptic efficacy
onto dopamine neurons has been linked not only to exposure
to drugs of abuse but to feeding-related peptides, such as
hypocretin/orexin (19,20) and ghrelin (21). Potentiation of synap-
tic strength onto dopamine neurons by feeding-promoting
peptides may underlie motivation to obtain food (20,22,23).
Therefore, modulation of excitatory inputs by anorectic feeding
peptides such as leptin could be particularly important in
decreasing dopamine neuron firing activity and ultimately dopa-
mine release.
Leptin modulation of glutamatergic synaptic transmission in
other brain regions has been demonstrated in the hippocampus
(24–26), hypothalamus (27), and cerebellum (28). Thus, we
hypothesized that leptin action in the VTA can modulate
glutamatergic synaptic transmission onto VTA dopamine neurons.

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http://dx.doi.org/10.1016/j.biopsych.2012.10.026 & 2013 Society of Biological Psychiatry
J.L. Thompson and S.L. Borgland
Here, we used whole-cell patch clamp electrophysiology in VTA
brain slices to determine the effect of leptin on excitatory synaptic
transmission.
Methods and Materials
Animals
All protocols were in accordance with the ethical guidelines
established by the Canadian Council for Animal care and were
approved by the University of British Columbia Animal Care
Committee. Male C57BL/6 mice (p21–25; University of British
Columbia) were housed in groups of 3 to 5. For some experi-
ments, mice expressing pituitary homeobox 3 tagged with green
fluorescent protein (Pitx3-GFP) knock-in mice (p21–25; bred
inhouse) were used to easily identify VTA dopaminergic neurons
because Pitx3 is a transcription factor necessary for the develop-
ment of midbrain dopamine neurons (29). Data from C57BL/6
mice and Pitx3-GFP mice were not significantly different and
therefore were grouped together. Mice were maintained on a 12-
hour light:dark schedule (lights on at 7:00 AM) and given food and
water ad libitum. All experiments were performed during the
animals light cycle.
Slice Preparation
Briefly, animals were deeply anaesthetized with isoflurane and
decapitated, and brains were rapidly extracted into ice-cold
sucrose solution containing (in mmol/L: 75 sucrose, 87 NaCl, 2.5
KCl, 1.25 NaH2PO4, 25 NaHCO3, 7 MgCl2, .95 CaCl2, 1 to 1.5
ascorbic acid. Horizontal brain sections containing the VTA (30)
were cut at 250 mm on a vibratome (Leica, Nussloch, Germany).
Slices were transferred to 250-mL bicarbonate-buffered solution
(artificial cerebrospinal fluid [aCSF]) containing (in mmol/L) 126
NaCl, 1.6 KCl, 1.1 NaH2PO4, 1.4 MgCl2, 26 NaHCO3, 11 glucose, 2.4
CaCl2 and incubated for a minimum of 45 min at 31.41 to 331C
before recording. All solutions were continuously saturated with
95% O2/5% CO2.
Electrophysiology
Slices were placed in the recording chamber and perfused
with aCSF with the addition of picrotoxin (100 mmol/L) to block
GABAA receptor-mediated inhibitory postsynaptic currents. Cells
were visualized on an upright microscope using “Dodt-type”
gradient contrast infrared optics (31). Whole-cell voltage clamp
recordings were made using a Multiclamp 700B amplifier (Molecular
Devices, Union City, California). Recording electrodes (3–5 MO)
were filled with (in mmol/L): 120 cesium methanesulfonate, 20 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid, .4 ethylene glycol
tetraacetic acid, 5 tetraethylammonium chloride, 2 MgCl2, 2.5
MgATP, .3 NaGTP, with a pH of 7.2 to 7.3 and 273 to 285 mOsm.
Putative dopaminergic VTA neurons were identified by the presence
of a large hyperpolarization-activated, cyclic nucleotide-regulated
cation (Ih) current (32–36). A subset of C57BL/6 neurons was
identified by biocytin (.2%) filling and post hoc staining for tyrosine
hydroxylase (TH; discussed below). Of the 11 biocytin-filled neurons
expressing Ih, only 2 did not express TH and were excluded from the
analysis. Dopamine neurons expressing Ih from Pitx3-GFP mice were
identified by GFP fluorescence. Of 15 VTA dopamine neurons
recorded from Pitx3-GFP mice, 3 cells were used for the experiments
with leptin on a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor (AMPAR) EPSCs, 2 cells with wortmannin in the internal
solution, 2 cells were used with wortmannin in the external solution,
1 cell for the paired pulse ratio, 3 cells for the miniature EPSCs
(mEPSCs) with leptin, and 3 cells for the mEPSCs with vehicle. To
BIOL PSYCHIATRY 2013;73:860–868 861
stimulate local presynaptic terminals, a bipolar stimulating electrode
was placed 100 to 300 mm rostral to the recording electrode and was
used to stimulate excitatory afferents at .1 Hz. Neurons were voltage-
clamped at –70 mV and ⫹40 mV to record AMPAR- and N-methyl-D-
aspartate receptor (NMDAR)-mediated evoked excitatory-
postsynaptic currents (EPSCs), respectively. EPSCs were filtered at 2
kHz, digitized at 5 to 10 kHz, and collected online using pClamp10
software (Molecular Devices). Series resistance (6–30 MO) and input
resistance were monitored online with a 5-mV depolarizing step (50
msec) given just after every afferent stimulus. Paired-pulse ratio was
elicited by evoking 2 EPSCs with an interval of 50 msec. mEPSCs
were recorded in the presence of tetrodotoxin (TTX; 500 nmol/L) to
block action potentials driven by spontaneous events as well as
picrotoxin (100 mmol/L) and (2R)-amino-5-phosphonovaleric acid AP-
5 (50 mmol/L). For these experiments, mEPSCs were recorded before
and 15 min after a 15-min application of leptin (100 nmol/L). mEPSCs
were selected on the basis of their amplitude (⬎10 pA), decay time
(⬍3 msec), and rise time (⬍3 msec) using the MiniAnalysis60
program (Synaptosoft, Decateur, Georgia). Average noise levels were
4 ⫾ .2 pA. Example traces for evoked EPSCs were constructed from
the averaged trace of 12 sweeps (2 min).
Drugs
Recombinant mouse leptin (R&D Systems, Minneapolis,
Minnesota), was reconstituted in phosphate-buffered saline
(pH 8.0–8.2) and stored at –801C. Leptin was bath applied at
100-nmol/L concentrations for 15 min in all experiments. Picrotoxin
(Sigma, St. Louis, Missouri) was dissolved in H20 and was present in
the external solution throughout all recordings. U0126 (Promega,
Madison, Wisconsin), wortmannin (Tocris, Ellisville, Missouri), or
AG490 (Calbiochem, Billerica, Massachusetts) were dissolved in
dimethyl sulfoxide and used at 1/1000 working concentrations.
AG490 was protected from light throughout experiments. Signal
transduction inhibitors were preincubated with slices for a mini-
mum of 20 min before recording and were also present throughout
the experiment. In some experiments, wortmannin (100 nmol/L) or
AG490 (50 mmol/L) was included in the patch pipette.
Statistical Analysis
All data are expressed as mean percent change in baseline
levels ⫾ SEM. To determine statistical significance, a section of
the baseline (average of 12 sweeps at minutes 6 through 7) was
compared with the maximal effect of drug treatment (average of
12 sweeps) using a paired t test. N refers to the number of cells
recorded from a minimum of 3 rats.
TH Immunocytochemistry
Brain slices from patch-clamp recording were fixed overnight
in cold 4% paraformaldehyde, rinsed in PBS, blocked in 10%
normal donkey serum, incubated with monoclonal mouse anti-
TH antibody (Sigma, 1:1000) for 48 hours at 41C. DyLight 594
streptavidin (Jackson ImmunoResearch Laboratories, West Grove,
Pennsylvania; 1:200) was applied overnight at 41C. Secondary
donkey anti-mouse fluorescein isothiocyanate antibody (1:50)
was applied for 2 hours at 41C. Slices were mounted using
Fluoromount (Sigma).
Results
Leptin Suppresses Excitatory Synaptic Transmission onto
VTA Dopamine Neurons
AMPAR EPSCs were recorded from VTA dopamine neurons of
mouse midbrain slices voltage-clamped at –70 mV. Bath
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862 BIOL PSYCHIATRY 2013;73:860–868 J.L. Thompson and S.L. Borgland

Figure 1. Leptin depresses a-amino-

3-hydroxy-5-methyl-4-isoxazolepropionic
acid receptor (AMPAR)- and N-methyl-
D-aspartate receptor (NMDAR)-mediated
150 -200
synaptic transmission onto ventral teg-
AM PAR EPSCs (% Baseline)

mental area (VTA) dopamine neurons.

AM PAR EPSCs (pA)

125 -150 Evoked AMPAR (–70 mV) or NMDAR
(⫹40 mV) excitatory postsynaptic currents
(EPSCs) were recorded before, during,
100 -100 and after bath application of leptin
(100 nmol/mL). (A) Fifteen-minute bath
application of leptin caused a long-lasting
75 -50
decrease of AMPAR-mediated EPSCs.
100 nM leptin
(B) Example recording of leptin on evoked
50 0 AMPAR EPSCs from a VTA dopamine
0 10 20 30 40 0 10 20 30 40 neuron. Filled bar indicates presence of
Time (min) Time (min) leptin in the bath. Inset, representative
traces of AMPAR EPSCs before (black) and
after leptin (gray). Scale bars, 10 msec,
100 pA. (C) Fifteen minute bath applica-
150 200 tion of leptin caused a lasting decrease in
NMDAR EPSCs (% baseline)

NMDAR-mediated EPSCs of VTA dopamine

neurons. (D) Example recording of leptin
NM DAR EPSCs (pA)

125 150 on evoked NMDAR EPSCs from a dopa-

mine neuron in the VTA. Filled bar indi-
cates presence of leptin in the bath. Inset,
100 100
representative traces of NMDAR EPSCs
before (black) and after leptin (gray). Scale
75 50 bars: 20 msec, 100 pA. Stimulus artifacts
have been removed for clarity. Error bars
100 nM leptin indicate SEM.
50 0
0 10 20 30 40 0 10 20 30 40
Time (min) Time (min)

application of leptin for 15 min caused a long-lasting depression
of AMPAR EPSCs (Figure 1A,B; baseline: 98% ⫾ 1% vs. leptin:
77% ⫾ 6%; n ¼ 7, p ⬍ .05). Leptin depressed AMPAR EPSCs in all
four neurons that were post hoc labeled for TH. To measure
NMDAR EPSCs, neurons were voltage clamped at ⫹40 mV and
measurements were taken 20 ms after the stimulus artifact,
a time point at which the glutamatergic EPSC is mediated
primarily by NMDARs (14). Leptin caused a long-lasting depres-
sion of NMDAR EPSCs (Figure 1C,D; baseline: 100% ⫾ 2% vs.
leptin: 77% ⫾ 7%; n ¼ 6, p ⬍ .05). The maximum effect of leptin
on AMPAR or NMDAR was not significantly different (p ⬎ .05).
Furthermore, of the 9 biocytin-labeled neurons included in the
analysis, leptin depressed AMPAR EPSCs in five neurons and
NMDAR in three neurons expressing TH, suggesting that leptin
inhibition of glutamatergic synaptic transmission can occur onto
VTA dopamine neurons.
Because leptin suppressed AMPAR or NMDARs with equal
magnitude, leptin may be decreasing glutamate release. Therefore,
to investigate whether leptin altered the number or function of
postsynaptic AMPARs or caused a presynaptic inhibition of gluta-
mate release, we measured AMPAR-mediated miniature EPSCs
(mEPSCs), a standard method for determining the locus of synaptic
change (37). Fifteen to 18 minutes after bath application of leptin
(100 nmol/L, 15 min), mEPSCs frequency was significantly reduced
(1.2 ⫾ .3 Hz) compared with baseline mEPSCs (Figure 2A–D;
1.9 ⫾ .4 Hz; n ¼ 12, p ⬍ .05). This effect was not due to rundown
over time because there was no significant difference between the
baseline and vehicle treatment (Figure 2A–2D; baseline: 1.7 ⫾ .3 Hz
vs. vehicle: 1.7 ⫾ .3 Hz, n ¼ 12, p ⬎ .05). Leptin did not alter mEPSC
amplitude (Figure 2B,C,E; baseline: 16.6 ⫾ .9 pA vs. leptin: 16.0 ⫾ .9
pA, n ¼ 12, p ⬎ .05) nor were there changes in mEPSC amplitude
after vehicle application (baseline: 18.6 ⫾ 1.1 pA vs. vehicle:
17.9 ⫾ .9 pA, n ¼ 12, p ⬎ .05).
A reduction of mEPSCs frequency concomitant with unchanged
amplitude likely reflects a decrease in the probability of presynaptic
neurotransmitter release (38). To further verify whether leptin-
mediated depression of AMPARs was mediated pre- or postsynap-
tically, we examined the effects of leptin on the probability of
transmitter release, comparing the response to paired pulses, a
measure that changes in a highly predictable fashion with release
probability. We recorded AMPAR EPSCs at –70 mV using a paired-
pulse stimulation protocol with a 50 msec interval and observed
significant paired pulse facilitation after leptin bath application
(Figure 2F,G; n ¼ 6, p ⬍ .05). Taken together, these data suggest
that leptin-induced synaptic depression is consistent with a reduc-
tion in the probability of presynaptic glutamate release.
Leptin-Induced Synaptic Depression Occurs by Activation
of the Jak2–PI3K Pathway
LepRb activation triggers three main signal transduction
cascades upon association with Janus kinase 2 (Jak2) tyrosine
kinase. First, Tyr(985) of LepRb recruits SH2-containing tyrosine
phosphatase (SHP-2) resulting ERK activation. Second, Tyr(1138)
of LepRb recruits signal transducer and activator of transcription
3 (STAT3). Third, tyrosine phosphorylation sites on the receptor-
associated Jak2 leads to activation of phosphatidylinositol 3-
kinase (PI3K) (3).
To determine whether Jak2 signaling was required for leptin-
induced synaptic depression, we bath applied the Jak2 inhibitor,
AG-490 (50 mmol/L) (39) to midbrain slices. Bath application
of AG-490 significantly inhibited leptin-induced depression of
AMPAR EPSCs (Figure 3A,B; leptin: 100% ⫾ 6%, n ¼ 7, compared
with maximum leptin response from Figure 1A (77% ⫾ 6%),
p ⬍ .05).
Leptin-induced synaptic depression may be due to activation
of postsynaptic leptin receptors on dopamine neurons, leading to

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J.L. Thompson and S.L. Borgland BIOL PSYCHIATRY 2013;73:860–868 863

Figure 2. Leptin-induced synaptic inhibi-

3 30 tion is mediated presynaptically. a-amino-

mEPSC peak amplitude (pA)

* 3-hydroxy-5-methyl-4-isoxazolepropionic
mEPSC fre que ncy (Hz)

acid receptor (AMPAR) miniature excita-

2 20 tory postsynaptic currents (mEPSCs) were
recorded at –70 mV in the presence of
picrotoxin, TTX and AP-5 before and after
a 15 min bath application of leptin
1 10 (100 nmol/L). (A) Frequency of mEPSCs
events was significantly decreased after
leptin application (filled bars) compared
with baseline (open bars, n ¼ 8, p ⬍ .05).
0 0
Leptin Vehicle Leptin Vehicle
Vehicle treatment (filled bar) did not alter
mEPSC frequency compared to baseline
(p ⬎ .05). (B) AMPAR mEPSCs amplitude
Baseline Baseline was not significantly different before
(open bar) or after (filled bars) bath appli-
cation of leptin (right panel) or vehicle (left
Leptin Vehicle panel; p ⬎ .05). (C) Example recordings of
AMPAR mEPSCs before or after leptin (left
side) or vehicle (right side) application.
Scale bars: 100 msec, 20 pA. (D) Cumula-
1.0
tive probability plots for interevent interval
1.0
for before (black) and after (gray) leptin
Cumulative Proba bility

application. A significant right-shift in the

Cumulative Proba bility

cumulative probability of mEPSC fre-

quency was detected after leptin applica-
0.5
tion compared to the baseline (p ⬍ .001,
0.5
Baseline Kolmogorov–Smirnov test). (E) Cumulative
Leptin probability plots for amplitude for before
Baseline (VEH) (black) and after (gray) leptin application.
Vehicle No significant difference was detected in
cumulative probability plots of mEPSC
0.0 0.0
0 10 20 30 40 50 60
amplitude before or after leptin (p ⬎ .05,
0 1 2 3 4 5
Kolmogorov-Smirnov test). (F) Bar graph
inter-event interval (s) mEPSC peak amplitude (pA)
illustrating a significant increase in the
paired pulse ratio after leptin administra-
* tion (*p ⬍ .05). (G) Example recording of
1.0 paired (50 msec interval) AMPAR EPSCs
before (black) and after leptin (gray) appli-
Paired Pulse Ratio (P2/P1)

0.8 cation. Scale bars: 20 msec, 50 pA. Stimu-

lus artifact has been removed for clarity.
0.6 Bars represent mean ⫾ SEM. VEH, vehicle.

0.4

0.2

0.0
Baseline Leptin

presynaptic inhibition of glutamate release via retrograde med- (100 nmol/L) (5,13) abolished the leptin-induced synaptic depression
iators. Another possibility is that activation of presynaptic LepRbs (Figure 4C,D; baseline: 102% ⫾ 2% vs. leptin: 96% ⫾ 5%, n ¼ 7,
induces a suppression of glutamate release. To investigate if p ⬎ .05). Furthermore, with postsynaptic inhibition of PI3K, leptin
postsynaptic receptor activation was required for leptin-induced significantly depressed AMPAR EPSCs (Figure 4E,F; baseline:
synaptic depression, we applied AG-490 via the patch pipette. 102% ⫾ 2% vs. leptin: 82% ⫾ 6%; n ¼ 7, p ⬍ .05). These data
Leptin-induced a significant synaptic depression of AMPAR- suggest that leptin likely activates presynaptic LepRbs to inhibit
mediated synaptic transmission with intracellular inhibition of glutamate release and that leptin activation of Jak2-PI3K is necessary
Jak2 in dopamine neurons (Figure 3C,D, baseline: 99% ⫾ 4% vs. to induce a reduction in synaptic efficacy of VTA dopamine neuron
leptin: 78% ⫾ 7%; n ¼ 5, p ⬍ .05). inputs.
To test the involvement of the SHP-2/ERK pathway, we pre- Discussion
incubated slices with U0126 (10 mmol/L) (13) a MEK2 inhibitor
upstream of ERK activation. In the presence of U0126, leptin The data presented here establish a novel mechanism for
significantly depressed AMPAR-mediated EPSCs (Figure 4A,B, base- leptin action in the mesolimbic incentive motivation/reward
line: 100% ⫾ 2% vs. leptin 80% ⫾ 5%, n ¼ 5, p ⬍ .05). These data system. Leptin reduced both NMDAR- and AMPAR-mediated
indicate that unlike leptin suppression of firing rate (13), leptin- synaptic transmission onto dopamine neurons by reducing
induced synaptic depression is via a different signaling pathway. We glutamate release from presynaptic terminals. This effect was
next tested the involvement of the Jak2/PI3K pathway using the mediated by presynaptic Jak2 and PI3K signaling, suggesting that
PI3K inhibitor, wortmannin. Extracellular application of wortmannin leptin is not directly activating dopamine neurons to induce

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864 BIOL PSYCHIATRY 2013;73:860–868
synaptic inhibition. A model of leptin inhibition of excitatory
synaptic transmission onto VTA dopamine neurons is illustrated
in Figure 5.
Previous studies have implicated leptin action in the VTA in
reducing food intake (5,40,41). Interestingly, Morton et al. (40)
demonstrated that leptin’s effect on food intake was not sensitive
to PI3K inhibition, suggesting that our results may dissociate
from the effects of leptin on food intake. To inhibit PI3K, this
study used a concentration of LY-294002 (2 mmol/L) intra-VTA,
which may have several off-target effects including action at
several ion channels (42–44), phosphodiesterases (45), and the
estrogen receptor (46), potentially confounding the feeding
results. Furthermore, it is important to consider that leptin acting
on GABAergic inputs to VTA neurons (excluded in our study) may
play a role in feeding. Indeed, leptin potentiates inhibitory
synaptic transmission in the hippocampus (47). Future studies
will be aimed at directly testing leptin modulation of GABAergic
inputs to VTA dopamine neurons. Interestingly, knocking down
LepRb expression within the VTA (using short hairpin RNA
[shRNA]) or knocking out LepRb on VTA dopamine neurons
yielded differential effects with regards to feeding. Although
neither method alters body weight, shRNA to LepRb in VTA
increased consumption of high fat food and increased the work
required to obtain sucrose (5,10). In contrast, there was no
difference in regular or high fat food intake in mice with a
conditional LepRb knockout in dopamine transporter expressing
neurons compared with wildtype mice (48). Possible discrepan-
cies between these studies may be because LepRb is removed
only from dopamine neurons of the substantia nigra and VTA in
the conditional knockout mice, whereas the LepRb shRNA would
target dopamine as well as GABAergic neurons only in the VTA.
Furthermore, conditional LepRb mice are missing LepRbs
throughout development, possibly leading to compensatory
changes.
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J.L. Thompson and S.L. Borgland
Figure 3. Leptin-induced synaptic inhibi-
tion requires PI3K activation. Evoked
a-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid receptor (AMPAR) excitatory
postsynaptic currents (EPSCs) were recorded
before, during, and after bath application of
leptin (100 nmol/L) for 15 min. Slices were
preincubated in inhibitors for a minimum
20 min before and throughout the experi-
ment. Filled bars indicate the presence of
leptin in the bath. (A) Bath application of
AG490 (50 mmol/L) abolished leptin-induced
synaptic depression. (B) Example recording
of AMPAR EPSCs from a representative
dopamine neuron in the presence of
AG490 and leptin. Inset, example traces
before (black) and after (gray) leptin applica-
tion. (C) AG490 (50 mmol/L) applied intracel-
lularly did not inhibit leptin-induced synaptic
depression. (D) Example recording of
AMPAR EPSCs before and after leptin appli-
cation in the presence of intracellular AG-
490. Inset, example traces before (black) and
after (gray) leptin application. Scale bars: 20
msec, 100 pA. Stimulus artifacts have been
removed for clarity. Error bars indicate SEM.
Several lines of evidence suggest that leptin modulates reward-
seeking behavior. Intra-VTA leptin reduced the reward value of
sucrose via a reduction in dopamine neuronal activity (11), intra-VTA
or intracerebroventricular leptin suppressed the rewarding effects of
intracranial self-stimulation (41), intracerebroventricular leptin
attenuates acute food deprivation-induced relapse to drug seeking
(40) and reverses conditioned place preference for sucrose (49) or
high fat foods (41). Furthermore, firing rate of putative dopamine
neurons is decreased after intravenous leptin administration or bath
application of leptin to midbrain slices (5,13). Studies using leptin
deficient ob/ob mice have provided conflicting results. Fulton et al.
(6) reported that the baseline and electrically evoked release of
dopamine in the nucleus accumbens was significantly reduced in
ob/ob mice. In contrast, Rosenberry et al. (50) demonstrated that
basal or cocaine-induced dopamine levels in the nucleus accum-
bens was increased in ob/ob mice when measured via microdialysis.
This discrepancy is likely due to technical differences such as the use
of in vivo microdialysis (50) compared with amperometry in
forebrain slices (6). Amphetamine-induced locomotion and sensiti-
zation was reduced in ob/ob mice compared with wildtype mice (6).
However, basal locomotor activity is lower in ob/ob mice compared
with wildtype mice, potentially confounding these results (50–54).
Taken together, these studies suggest that leptin in the VTA reduces
activity of dopamine neurons and modulates reward seeking
behavior. Our findings that leptin reduces excitatory synaptic
transmission onto dopamine neurons are consistent with these
studies and suggest that in the constitutive presence of leptin,
selective glutamatergic afferents to the VTA are tonically dampened.
Leptin can modulate excitatory synaptic transmission in other
brain regions. For example, mice with mutations in LepRb (db/db
mice or fa/fa rats) have decreased LTP or LTD of hippocampal
neurons (55). Leptin bath applied to hippocampal slices facilitates
LTP (56), likely because of leptin-induced forward trafficking on
NMDARs to the synapse (26). Additionally, leptin is reported
J.L. Thompson and S.L. Borgland
to preferentially enhance NR2B-mediated NMDAR responses in
cerebellar granule cells (28). Thus, unlike the postsynaptically
mediated alteration of glutamatergic synapses in the hippocam-
pus or cerebellum described earlier, leptin action in the VTA
caused a presynaptic suppression of glutamate release.
Interestingly, leptin-induced synaptic depression was not due
to a direct effect of leptin on dopamine neurons. Several lines of
evidence support our conclusion that depression of AMPAR-
mediated EPSCs by leptin was mediated presynaptically. First,
leptin depressed NMDAR-mediated EPSCs with a similar time
course and maximum to that of AMPAR EPSCs, suggesting
reduced availability of synaptic glutamate to activate either
glutamate receptor subtype as opposed to a postsynaptic
decrease in number or function of both receptors. Second,
decreased mEPSC frequency concomitant with unchanged mEPSC
amplitude suggests that leptin reduced synaptic glutamate with-
out affecting the number or function of AMPARs on dopaminergic
neurons. Third, leptin induced paired-pulse facilitation suggesting
a decreased release probability. Finally, while postsynaptic inhibi-
tion of Jak2 or PI3K did not alter leptin-induced synaptic
depression, bath application of AG490 or wortmannin abolished
BIOL PSYCHIATRY 2013;73:860–868 865
Figure 4. Leptin-induced synaptic inhibi-
tion requires PI3K activation. Evoked
a-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid receptor (AMPAR) excita-
tory postsynaptic currents (EPSCs) were
recorded before, during, and after bath
application of leptin (100 nmol/L) for 15
min. Slices were preincubated in inhibitors
for a minimum 20 min before and
throughout the experiment. Filled bars
indicate the presence of leptin in the bath.
(A) U0126 (10 mmol/L) does not alter
leptin-induced depression of AMPAR
EPSCs of ventral tegmental area dopamine
neurons. (B) Example recording of AMPAR
EPSCs from a representative dopamine
neuron in the presence of U0126 and
leptin. (C) Bath application of wortmannin
(100 nmol/L) abolished leptin-induced
synaptic depression. (D) Example record-
ing of AMPAR EPSCs from a representative
dopamine neuron in the presence of
wortmannin and leptin. Inset, example
traces before (black) and after (gray) leptin
application. Scale bars: 20 msec, 100 pA.
(E) Wortmannin (100 nmol/L) applied
intracellularly did not inhibit leptin-
induced synaptic depression. (F) Example
recording of AMPAR EPSCs before and
after leptin application in the presence of
intracellular wortmannin. Inset, example
traces before (black) and after (gray) leptin
application. Scale bars: 20 msec, 100 pA.
Stimulus artifacts have been removed for
clarity. Error bars indicate SEM.
the effect of leptin, suggesting that LepRb activation does not
occur directly on dopamine neurons.
Several studies have demonstrated that there is high LepRb
expression in the VTA using immunohistochemistry to LepRb
protein in rats (57); fluorescent in situ hybridization to LepRb
messenger RNA colocalized with TH messenger RNA in rats (5)
and immunohistochemistry to leptin activated pSTAT3 colocaliza-
tion with TH in mice (6). However, in mice expressing enhanced
green fluorescent protein (EGFP) in LepRb-containing neurons, only
approximately 6% overlapped with TH expression in the VTA (8).
Low promoter activity driving EGFP expression in the LepRbEGFP
mice may account for some of this discrepancy; however, it is clear
that LepRb expression on dopamine neurons is less than originally
described. Our results are consistent with the idea that leptin
control of the mesolimbic dopamine system occurs indirectly.
Leptin acting at LepRb expressed on presynaptic glutamatergic
terminals in the VTA may be one mechanism through which leptin
inhibits excitatory synaptic transmission. Terminals expressing LepR-
bEGFP in the VTA include those from the lateral hypothalamus (LH),
periaqueductal gray area, and hypothalamic preoptic area (8).
Whereas POA neurons are mainly inhibitory (58,59), periaqueductal
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866 BIOL PSYCHIATRY 2013;73:860–868
STAT3
STAT3
U0126
MEK1/2
JA
wortmannin
PI3-K
? ?
glutamate
EAAT4
VTA dopamine neuron
gray area neurons provide glutamatergic input to the VTA (60).
Interestingly, activation of LepRb-expressing neurons in the LH
modulates dopamine production and content in the VTA, con-
tributing to a decrease in incentive value of food (61). However,
LepRb expression was mainly on LH GABAergic neurons.
Although it is possible that LepRb-containing LH GABAergic
terminals within the VTA synapse onto glutamatergic afferents
to control glutamate release, this effect is unlikely to occur in our
slices because of the presence of picrotoxin in all extracellular
solutions. Another possibility is that leptin activates LepRb on
somata outside the VTA in our horizontal midbrain slices. These
slices contain a significant portion of the posterior hypothalamic
area, a region with dense LepRb expression (2). Therefore, it is
feasible that leptin could activate receptors on cell bodies in
these regions that could project directly or indirectly to the VTA,
thus inhibiting glutamate release. It should be noted that
differences in leptin signaling and receptor expression levels
may differ between young mice used in this study and adult
animals used in other studies. However, location of receptor
expression is not expected to change with age. Furthermore,
others have reported consistent effects of leptin in the VTA of
adult and 3 week old mice (13). Taken together, leptin likely
activates LepRbs on glutamatergic inputs directly or indirectly
projecting to the VTA.
Interestingly, activation of PI3K, but not ERK, was required for
leptin-induced synaptic depression in the VTA. Leptin-induced
activation of Jak2 recruits insulin receptor substrate-1 (IRS-1),
leading to PI3K activation. Inhibition of PI3K by the potent
inhibitor wortmannin blocked leptin-induced synaptic depression
only when applied in the bath solution. In hippocampal cultures,
leptin caused a PI3K-dependent forward trafficking of postsynap-
tic GluA1 subunits (62). This discrepancy is likely because in VTA,
leptin inhibition of excitatory synaptic transmission is mediated
presynaptically. Leptin-induced inhibition of firing of VTA neu-
rons was mediated by ERK signaling as bath application of the
MEK inhibitor U0126 abolished leptin-induced suppression of
firing rate (13). Although these experiments did not test whether
www.sobp.org/journal
J.L. Thompson and S.L. Borgland
Figure 5. Putative mechanisms of leptin-
induced synaptic depression onto ventral
tegmental area (VTA) dopamine neurons.
Leptin may act via presynaptic LepRbs on
glutamatergic terminals. Inhibition of Jak2
and PI3K, but not MEK1/2, could block the
leptin-induced reduction in presynaptic
LepRb glutamate release. Leptin may also act
via PI3K-dependent upregulation of glu-
tamate transporters (i.e., EAAT4) to
K2 increase clearance of glutamate from the
K2 synapse.
JA
AG490
leptin-induced suppression of firing was mediated by direct or
indirect depolarization of dopamine neurons, it does suggest that
leptin-modulation of firing rate is mediated by an independent
mechanism to leptin-induced synaptic inhibition.
One interesting hypothesis for the mechanism underlying a
leptin-induced synaptic inhibition is that leptin may increase
glutamate clearance from the synapse by upregulating transpor-
ter function. Recent evidence demonstrated that leptin activation
of Jak2 led to upregulation of Na⫹/K⫹-dependent excitatory
amino acid transporters (EAATs), particularly EAAT2 and EAAT4
(63). Consistent with this, PI3K/Akt signaling has been implicated
in forward trafficking of both EAAT2 and EAAT4 (64,65). Interest-
ingly, in addition to astrocytic EAAT4 expression, EAAT4 is
densely expressed on neurons within the VTA (66). Notably,
leptin caused modest paired pulse facilitation in our experiments,
suggesting that while synaptic glutamate release was depressed,
the probability of release was less affected, consistent with an
increase in glutamate reuptake. Thus, leptin-induced synaptic
depression may be due to an upregulation of glutamate
clearance in the VTA.
Increased excitatory synaptic transmission onto dopamine
neurons is associated with learning of cues predicting food
reward (67). A reduction in excitatory synaptic transmission
may raise the threshold required for burst firing of dopamine
neurons (17). Because burst firing has been implicated in
promoting dopamine release and driving salience of food related
cues, a reduction of salience to food-related cues may be a
functional consequence of leptin-induced inhibition of glutamate
release. This idea is consistent with studies demonstrating a
leptin-induced reduction in hedonic food intake (11) and reduced
motivation to obtain food (10). Because leptin’s action was
selective to presynaptic glutamatergic inputs to the VTA, it is
possible that circulating leptin may tonically dampen select
inputs to the VTA, thereby reducing their influence to promote
burst firing of dopamine neurons. Fasting decreases circulating
leptin (68); therefore, one can speculate that tonic suppression
of glutamatergic inputs to the VTA would be relieved in
J.L. Thompson and S.L. Borgland
this situation, resulting in heightened salience to food-related
cues.
Here, we propose a novel mechanism for leptin signaling in the
VTA that is, to our knowledge, the first report of a presynaptic
inhibition of excitatory synaptic transmission induced by leptin.
Leptin-induced synaptic inhibition in the VTA is dependent on
activation of PI3K of nondopaminergic neurons. Taken together, this
study provides new insight for how leptin modulates the mesolimbic
dopamine system to suppress reward-seeking behaviors.
This work was supported by an Natural Sciences and Engineer-
ing Research Council discovery grant and a Canadian Institutes of
Health Research new investigator award to SLB. We thank Haiyan
Zou for breeding the Pitx3 mice.
The authors have no biomedical financial interests or potential
conflicts of interest.
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