Neuron

ArticleNeuropeptide Exocytosis Involving Synaptotagmin-4
and Oxytocin in Hypothalamic Programmingof Body Weight and Energy Balance
Guo Zhang,1,2 Hua Bai,2 Hai Zhang,1,2,3 Camin Dean,2 Qiang Wu,2 Juxue Li,1,2 Sara Guariglia,1 Qingyuan Meng,1and Dongsheng Cai1,2,*
1Department of Molecular Pharmacology and Diabetes Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2Department of Physiology
3Cellular and Molecular Biology Program
University of Wisconsin-Madison, Madison, WI 53706, USA*Correspondence: dongsheng.cai@einstein.yu.edu
DOI 10.1016/j.neuron.2010.12.036

SUMMARY

Hypothalamic neuropeptides play essential roles inregulating energy and body weight balance. Energy
imbalance and obesity have been linked to hypothalamic signaling defects in regulating neuropeptide
genes; however, it is unknown whether dysregulationof neuropeptide exocytosis could be criticallyinvolved. This study discovered that synaptotagmin-4, an atypical modulator of synaptic exocytosis,
is expressed most abundantly in oxytocin neurons of
the hypothalamus. Synaptotagmin-4 negatively
regulates oxytocin exocytosis, and dietary obesity
is associated with increased vesicle binding of synaptotagmin-4 and thus enhanced negative regulationof oxytocin release. Overexpressing synaptotagmin4 in hypothalamic oxytocin neurons and centrally
antagonizing oxytocin in mice are similarly obesogenic. Synaptotagmin-4 inhibition prevents against
dietary obesity by normalizing oxytocin release andenergy balance under chronic nutritional excess. In
conclusion, the negative regulation of synaptotagmin-4 on oxytocin release represents a hypothalamicbasis of neuropeptide exocytosis in controllingobesity and related diseases.
INTRODUCTION
The hypothalamus in the central nervous system (CNS) is known
as the central regulator of feeding, energy, and body weighthomeostasis (Coll et al., 2007; Flier and Maratos-Flier, 1998;
Mobbs, 2007; Park and Bloom, 2005; Schwartz et al., 2000; Ukropec et al., 2006). All these hypothalamic functions are criticallymediated by various hypothalamic neuropeptides. Several wellappreciated examples of such neuropeptides include a-melanocyte stimulating hormone (a-MSH), cocaine and amphetamine
regulated transcript (CART), neuropeptide Y (NPY), and agoutirelated peptide (AGRP). These neuropeptides have been shown
to be controlled at the gene transcriptional levels (Bates et al.,

2003; Kim et al., 2006; Kitamura et al., 2006; Xu et al., 2005) bynuclear transcription factors that sense nutrient and metaboliccues of the body (Ahima et al., 1996; Air et al., 2002; Friedman
and Halaas, 1998). Interestingly, recent research has begun torecognize the importance of neuropeptide posttranscriptional
modulation (Plum et al., 2009), indicating that the control ofneuropeptide gene expression represents only an initial step inthe whole cascade of neuropeptide regulation. Logically, thisprocess ultimately involves regulation of neuropeptide releaseto precisely control the biological functions of neuropeptides.
However, how hypothalamic neuropeptide exocytosis is regulated and whether it is critical for metabolic physiology anddisease have not been explored.
Recent research in basic science has obtained significant
knowledge regarding the general principles of neuropeptide/
neurotransmitter vesicular exocytosis (Stojilkovic, 2005). Studies
based on synaptic neurotransmitter release have identified vesicular exocytosis as a process that is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE)
complex (Jahn and Scheller, 2006; Südhof and Rothman, 2009)
under the regulation of synaptotagmins (Syts) (Chapman, 2008;
Südhof, 2002). Syts are a group of Ca2+-binding proteins that catalyze the formation of SNARE complex to provide the force andenergy required for exocytosis. The mammalian Syt family iscomposed of 17 members. While most of them are predominantly
present in the CNS, some of them are involved in the vesicularfunctions of endocrine cells such as pancreatic a and b cells(Fukuda and Mikoshiba, 1999; Gao et al., 2000; Gauthier et al.,2008; Iezzi et al., 2005) and glucose-transport metabolic cells(Hudson and Birnbaum, 1995; Li et al., 2007). These interestingstudies, which were mainly based on peripheral endocrine
systems, have raised the recent alarm on the potential implicationof Syts in diabetes (Gauthier and Wollheim, 2008). However,research to date addressing Syts in hypothalamic neuroendocrine
neurons is still missing.
Syt4 is an inducible Syt isoform detectable only in the brain andin the neuroendocrine system (Vician et al., 1995), hinting at
a possible role in neuroendocrine physiology. Notably, comparedto other Syt family members, the puzzling aspect of Syt4 is its lackof a critical Ca2+-binding amino acid (von Poser et al., 1997) andrelated inability to induce Ca2+-dependent exocytosis in biophysical models (Chapman et al., 1998; Thomas et al., 1999). Recent
Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc. 523

Neuron
Synaptotagmin-4 in Controlling Energy Balancebiophysical research reported that Syt4 inhibits exocytotic activities in the posterior pituitary (Zhang et al., 2009) and in cultured
PC12 cells (Machado et al., 2004), suggesting a potential neuroendocrine role of Syt4. In this research, through targeting theregulators of vesicle exocytosis including SNARE complex
proteins and Syt family members, we identified a hypothalamic
program of oxytocin (OXT) release regulated by Syt4 and established the physiological significance of this regulation in the hypothalamic control of energy balance and related diseases.

A
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RESULTS
Abundant Distribution of Syt4 in the Hypothalamic
Paraventricular Nucleus
The hypothalamus regulates energy balance essentially throughneuropeptide release. Therefore, we explored whether the hypothalamus has a regulatory program for neuropeptide release thatis critical for metabolic physiology, and whether alteration of thisprogram causes metabolic disease. We adopted a candidate
screening strategy by targeting neuropeptide release regulators,
including SNARE complex proteins and Syt family proteins in the
hypothalamus. Western blots initially confirmed that Syt4 wasdetectable in the brain and the posterior pituitary, but not in the
peripheral tissues. Immunostaining further revealed that Syt4
was expressed abundantly in a subpopulation of cells in the paraventricular nucleus (PVN) (Figure 1A) and the supraoptic nucleusof the hypothalamus, but only modestly in a few other brain
regions such as hippocampus and the cortex (data not shown).
The specificity of Syt4 antibody (Zhang et al., 2009) was verified
by western blot and immunostaining analyses of hypothalamic
samples from Syt4 knockout (Syt4 / ) mice (Ferguson et al.,2000a) (Figures S1A–S1C available online). In addition, bothmRNA in situ hybridization (Figure S1D) and real-time RT-PCR(Figure S1E) confirmed that Syt4 expression was uniquely enriched in the hypothalamus. High-magnification images of Syt4
immunostaining (Figure 1A) revealed that Syt4 was present in
the cell bodies and projections of a subpopulation of cells withneuronal morphology in the PVN. Subsequent coimmunostainingof Syt4 with NeuN (a neuronal marker) confirmed that Syt4 was
exclusively expressed in neurons, but not other cell types (Figure 1B). High-resolution images of coimmunostaining revealed
that Syt4 colocalized with vesicle-like structures in the cytoplasm
and projections of neurons (Figure 1B, inserts). In support of thisobservation, Syt4 immunogold labeling and electron microscopy
analysis showed that Syt4 was abundantly localized in vesicles ofa subpopulation of PVN neurons (Figure 1C) and in axonal terminals of the PVN neurons that projected into the posterior pituitary(Figure 1D). Thus, in line with a previous report that Syt4 is present
only in the brain and neuroendocrine system (Vician et al., 1995),
our data revealed that Syt4 is abundantly expressed in a specificsubtype of PVN neurons, which may indicate a previously unappreciated role of Syt4 in hypothalamic control of physiology.
Nutritional Excess Promotes Syt4 Expression
and Vesicular Localization
Since the PVN is one of the hypothalamic regions that criticallyregulate energy and metabolic balance, we subsequently
explored whether nutritional excess could affect the expression
524 Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc.

3V

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NeuN

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Dendrites (PVN)

Axonal terminals (PP)

Figure 1. Syt4 Distribution in Hypothalamic PVN(A) Distribution of Syt4 in the hypothalamic PVN. Immunohistochemical staining of Syt4 in hypothalamic sections across the PVN was examined undera light microscope. Scale bar = 100 mm.
(B) Neuronal expression of Syt4 in the PVN. PVN sections were coimmunostained for Syt4 (green) and neuronal marker NeuN (red). Colocalization of
two fluorescent signals within the same cells indicates Syt4 expression inneurons. Scale bar = 50 mm. Inserts: Intracellular distribution of Syt4 in neurons
by coimmunostaining at high magnification (insert scale bar = 5 mm).
(C and D) Syt4 immunogold labeling in hypothalamic PVN (C) and posterior
pituitary (D) sections was examined by electron microscopy. Yellow arrows
indicate Syt4 immunogold labeling. Scale bar = 100 nm.
All experimental mice were adult males, chow-fed, and in the C57BL/6 background. AMY, amygdala; COR, cortex; LH, lateral hypothalamus; OP, optic
tract; PVN, paraventricular nucleus; PP, posterior pituitary; TH, thalamus;
3V, third ventricle.

and vesicular profiles of Syt4 in the hypothalamus-pituitary axis.
First, quantitative real-time RT-PCR was used to measure Syt4
mRNA levels in the hypothalamus of C57BL/6 mice that werechronically maintained on a normal chow diet versus a high-fat
diet (HFD). Results showed that HFD feeding increased Syt4
mRNA levels in the hypothalamus, but not in other brain regions

Neuron
Synaptotagmin-4 in Controlling Energy Balance

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Figure 2. Metabolic Profiles of Mice with Syt4 Ablation

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0Body Weight (g)A(A and B) Syt4 / mice (blue and green curves/bars) and WT
littermate controls (black and red curves/bars) were maintained on either a normal chow (black and blue curves/bars)
or an HFD (red and green curves/bars) since weaning (3 weeksold) and were longitudinally monitored for body weight (A) andfood intake (B). Data in (B) represent the average daily foodintake over a 6 month follow-up period.
(C) Normal chow-fed Syt4 / mice (pink curves) and WT littermate controls (black curves) at 12 weeks of age were
measured for O2 consumption using metabolic chambers.
Data represent real-time (left panel) and daytime average (rightpanel) O2 consumption over a 64 hr period. The levels of O2
consumption were normalized by lean body mass of individual
mice.
(D) Representative DEXA scanning images of Syt4 / miceversus WT littermate controls that received 4 months of HFD
feeding since weaning.
(E) Normal chow-fed Syt4 / mice (green curve) and WT littermate controls (red curve) at 6 weeks of age were switched to
HFD pair feeding (food supply based on the average ad libitum
HFD intake of Syt4 / mice), and body weight (BW) gains ofthese mice were followed up for 4 weeks.
(F–I) HFD-fed C57BL/6 mice received bilateral PVN injectionsof Syt4 shRNA lentiviruses or matched control shRNA lentiviruses and were subsequently subjected to HFD pair feeding
after injection. Lentiviral shRNA-mediated Syt4 ablation wasverified by Syt4 immunostaining (green fluorescence) in the
PVN sections (F) and by counting Syt4-positive neurons
across serial PVN sections (G). Mice were measured for O2
consumption at week 1 after injection (H) and followed up
for body weight (BW) gain over a 3 week period (I). Scalebar = 50 mm.
Data in these experiments were based on male mice. Data in(A)–(D) also represented similar observations in female mice.
*p < 0.05, **p < 0.01, ***p < 10 3, compared to genotype/treatment-matched controls; n = 6–10 per group (A and B),
and n = 4–6 per group (C, E, and G–I). Error bars reflectmean ± SEM.

0.0
HFD Pair Feeding(Figure S1E), suggesting that Syt4-directed vesicular exocytosis
could be involved in the hypothalamic control of HFD-induced
metabolic disorders. To further evaluate this possibility, Syt4 immunogold labeling and electron microscopy were employed to
examine whether HFD feeding could affect vesicular localizationof Syt4 in PVN neurons. Results showed that HFD feeding nearly
doubled the number of vesicle-bound Syt4 particles in PVN
dendrites and axonal terminals (Figures S1F and S1G). Thus,
obesity development under nutritional excess is associatedwith hypothalamic changes of Syt4 expression and in particular
Syt4 vesicular distribution.
Syt4 Inhibition Prevents Obesity by Normalizing Energy
Balance
Syt4 / mice were then employed to test if Syt4 ablation could
affect obesity and related metabolic diseases. Based on ourobservation as well as the literature (Vician et al., 1995), Syt4 isexpressed in the brain, but not in peripheral tissues; therefore,
Syt4 knockout functionally targets the brain. We first confirmedthat Syt4 / mice have normal growth, appearance, viabilities,

and physical activities, and thus represent a suitable model for
metabolic research without involving confounding developmental changes. Syt4 / mice and wild-type (WT) littermate
controls were maintained on a normal chow upon weaning. At
young ages, Syt4 / mice displayed similar body weights
compared to WT controls (Figure 2A) despite slightly reduced
food intake (Figure 2B) and evidently increased energy expenditure (Figure 2C). On the other hand, a long-term follow-up revealed that Syt4 / mice were completely protected from thedevelopment of postadult weight gain (Figures S2A and S2B).
Therefore, the catabolic effects of Syt4 ablation on body weight
were accumulative and required an adequate age to manifest.
Taken together, Syt4 inhibition is catabolic; while its impact onbody weight is minor at young ages, it can provide accumulative
benefits against age-related fat expansion and weight gain at
postadult ages.
To better elucidate the potential antiobesity effect of Syt4 ablation, the paradigm of HFD feeding was applied to Syt4 / miceand WT littermate controls after weaning. Data revealed that while
WT controls developed profound obesity over a 6 month period,
Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc. 525

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Synaptotagmin-4 in Controlling Energy Balance

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(A–E) Syt4 / mice (blue and green curves/bars) and WT littermate controls (black and red curves /bars) were maintained ona chow (black and blue curves/bars) versus an HFD (red andgreen curves/bars) since weaning. At 4 months of age, mice
were analyzed with the glucose tolerance test (GTT) (A andB), and random blood glucose levels (C), and fasting blood
insulin (D) and leptin (E) levels were also analyzed. AUC,
area under curve of GTT. *p < 0.05, **p < 0.01, n = 5–7 pergroup.
(F and G) Syt4 / mice and WT littermate controls were maintained on an HFD for 4 months since weaning. Pancreases
were collected and sectioned for H&E staining (F) and coimmunostaining of insulin (green) and glucagon (red) (G). Scalebar = 100 mm.
Data presented were based on male mice but representativesof both sexes. Error bars reflect mean ± SEM.

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Figure 3. Counteraction against Insulin and Leptin
Resistance in Syt4–/– Mice30

Fasting Blood
Leptin (ng/ml)* WT, HFD
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AUC of GTT(x 103 mg/dl*min)

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Syt4 / mice maintained completely normal body weight (Figures
2A and 2D). A prolonged follow-up of 12 month HFD feeding
showed that the body weight of WT mice increased to a morbid
level, but the matched Syt4 / mice remained intact (Figures
S2C and S2D). DEXA scanning and histological analyses (Figures
S2E–S2H) confirmed that the antiobesity phenotype of Syt4 /mice was due to the resistance to fat mass expansion without
loss of lean body mass. Daily HFD intake of these mice was longitudinally monitored, revealing that Syt4 / mice consumed
a reduced amount of HFD compared with WT mice (Figure 2B).
Indirect calorimetry was applied to Syt4 / and WT mice during
the initial stage (1–2 weeks) of HFD feeding when mice of bothgroups still had comparable body weight. Syt4 / mice, uponswitching to an HFD, had higher energy expenditure levels thanmatched WT mice (data not shown), which was similar to theenergy expenditure pattern under the normal chow feeding condition (Figure 2C). To further assess whether increased energy
expenditure contributed to the antiobesity phenotype of Syt4 /mice, HFD pair feeding was provided to adult Syt4 / and WT
mice. Although the total calories provided through HFD pairfeeding were reduced compared with ad libitum feeding, adult
526 Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc.

WT mice still gained weight over a 4 week
period—an outcome of impaired energy expenditure resulting from HFD-associated malnutrition. In
contrast, a significantly smaller magnitude of weightgain was observed in Syt4 / mice under the same
HFD feeding regime (Figure 2E), and such weightgain reduction can be attributed to elevated energy
expenditure induced by Syt4 ablation.
Finally, we explored whether obesity development could be acutely attenuated by Syt4 ablation
through the control of energy expenditure in addition to food intake. HFD-fed C57BL/6 micereceived bilateral intra-PVN injections of Syt4
shRNA lentiviruses or control lentiviruses (Figures
2F and 2G). HFD pair feeding was then provided
to both groups after injection (Figure S2I).
Compared with the control mice, Syt4 shRNA-injected mice had higher energy expenditure levels
at week 1 postinjection (Figure 2H), a time point when bodyweights of both groups were still comparable. Further followup revealed that Syt4 shRNA-injected mice gained less bodyweight than control mice (Figures 2I and S2J). Such an antiweight gain effect of Syt4 ablation can be specifically related
to increased energy expenditure, since HFD intake wascontrolled at the same level via pair feeding. Therefore, in conjunction with the food intake data in Figure 2B, it can beconcluded that the antiobesity effect of Syt4 inhibition undernutritional excess is mediated by both energy expenditure
increase and energy (food) intake restriction.
Syt4 Inhibition Prevents against Obesity Comorbidities
The frequent disease consequences of obesity include glucose
intolerance, insulin resistance, and tissue lipid infiltrations. In
the context of Syt4’s relevance to obesity, we additionally assessed whether Syt4 inhibition was sufficient to prevent against
obesity-associated diseases. Under HFD feeding, WT micedeveloped severe glucose intolerance (Figures 3A and 3B) andslight hyperglycemia (Figure 3C); in contrast, Syt4 / mice
were completely normal. Consistent with these metabolicNeuron
Synaptotagmin-4 in Controlling Energy Balance

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Figure 4. Colocalization of Syt4 with OXT in Hypothalamic PVN

Merge

Syt4

(A) OXT (upper panels, green) or AVP (lower panels, green) in
the PVN was coimmunostained with Syt4 (all panels, red)and merged to display their colocalization (indicated by yellow
color). Scale bar = 50 mm.
(B) High-magnification images of Syt4 (red) and OXT (green)coimmunostaining. Yellow color in merged images indicates
intracellular colocalization of Syt4 and OXT. DAPI staining(blue) revealed nuclei of all cells in the sections. Scale bar =5 mm.
(C) OXT and Syt4 coimmunogold labeling in OXT versus AVP
axonal terminals. The posterior pituitaries from normal
C57BL/6 mice were sectioned and coimmunogold-labeledwith OXT (small particles) and Syt4 (large particles). The image
represents a junction region that contains both OXT axonal
terminals and AVP axonal terminals (separated by a blue
dotted line). Red arrows indicate dense-core vesicles, andgreen arrows indicate microvesicles. Scale bar = 100 nm.
All experimental mice were adult males, chow-fed, and in the
C57BL/6 background.

AVP axonal
terminal

Merge

OXT axonal
terminal

profiles, HFD-treated WT mice, but not Syt4 / mice, developed
profound insulin resistance and leptin resistance, presented in
the forms of hyperinsulinemia (Figure 3D), hyperleptinemia (Figure 3E), and pancreatic islet hypertrophy (Figure 3F). Double immunostaining of insulin and glucagon in pancreatic sections
confirmed that pancreatic islets of HFD-treated WT mice displayed centralization of glucagon-producing cells, a morphological indicator of islet damage, but such pathologic changes were
not seen in HFD-treated Syt4 / mice (Figure 3G). Finally, themice were assessed for lipid infiltration profiles in the liver andskeletal muscles. HFD-treated WT mice showed severe hepatic
steatosis (Figure S3A) and muscular lipid deposits (Figure S3B);
in contrast, HFD-treated Syt4 / mice were completely or significantly protected from developing these lipid abnormalities. All
these metabolic benefits to Syt4 / mice were predicted to bemainly secondary to obesity prevention, but it is also possiblethat Syt4 ablation may directly improve obesity-related glucose
and lipid homeostasis.
Colocalization of Syt4 and OXT in the PVN Neurons
Next our study aimed to understand which neuropeptide orneuropeptides were directed by hypothalamic Syt4 to accountfor the physiological effects of Syt4 knockout.
Since Syt4 is abundantly expressed in the PVN
neurons (Figure 1), and PVN importantly integrates many other hypothalamic nuclei to control
energy balance, we focused our study on thishypothalamic site. Coimmunostaining of Syt4
with various PVN neuropeptides revealed that
Syt4 was enriched in OXT-expressing neurons in
the PVN (Figure 4A, upper panels). For comparison, PVN sections were coimmunostained for
Syt4 and arginine vasopressin (AVP), a neuropeptide structurally closely related to OXT. Surprisingly, Syt4 was undetectable in the majority of
AVP neurons except for a few that coexpressed
OXT and AVP (Figure 4A, lower panels). These
results indicate that OXT and AVP probably employ different
Syt-directed regulatory systems to control their exocytosis,
and such a difference may form a basis for their different physiological functions.
Subsequently, high-magnification imaging of Syt4 and OXT
coimmunostaining was used to examine the morphology of
Syt4-containing subcellular structures in detail. As shown in Figure 4B, the subcellular organelles recognized by Syt4 immunostaining displayed the appearance of aggregated vesicles thatwere also encompassed by OXT, suggesting that Syt4 is physically present in OXT vesicles. To prove this hypothesis, immunogold colabeling of Syt4 with OXT and electron microscopy
analysis were performed, and data confirmed that Syt4 wasselectively present in the dendritic and axonal vesicles of OXT
neurons (Figure 4C). In comparison, Syt4-positive vesicles
were not detected in adjacent AVP neurons (Figure 4C). In addition to the dense-core vesicles, Syt4 particles appeared to bemodestly present in microvesicles, suggesting that regulationof neurotransmitter release by Syt4 in these neurons is also
possible. Overall, Syt4 is expressed specifically in OXT neurons
and might critically regulate OXT release to modulate the biological functions of hypothalamic OXT.
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Figure 5. Metabolic Effects of Syt4 in Hypothalamic
OXT Neurons

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GFP

Gene Induction(% of OXT neurons)A(A–E) Chow-fed C57BL/6 mice received bilateral intra-PVN
injections of OXT promoter-driven Flag-tagged Syt4 lentiviruses (OP-Syt4) versus OXT promoter-driven GFP lentiviruses(OP-GFP). (A) Lentivirus-induced GFP expression (green) was
coimmunostained with endogenous OXT expression (red) in
the PVN. Merged image indicates GFP expression in OXT
neurons. DAPI staining (blue) revealed the nuclei of all cells
in the section. Scale bar = 50 mm. (B) The percentage of OXT
neurons immunoreactive for GFP versus Flag in serial PVN
sections that were coimmunostained for GFP or Flag with
OXT. (C and D) Mice received daily injections of OXT (+) versus
control vehicle ( ) via preimplanted third ventricle cannula for
1 week, and daily food intake and weekly body weight (BW)gain were measured. (E) Lentivirus-injected mice were longitudinally monitored for BW gain over a 20 week period.
(F–H) Chow-fed Syt4 / mice and WT littermate mice werebilaterally injected with OP-Syt4 versus OP-GFP lentivirusesin the PVN. (F) Syt4 mRNA in the hypothalamus was measured
at week 1 after injection. (G and H) Lentivirus-injected mice
were placed on an HFD, and monitored for BW gain (G) and
HFD intake (H) over a 20 week period. AU, arbitrary unit.
Mice in these experiments were males. *p < 0.05, **p < 0.01;
NS, nonsignificant; n = 5–7 per group (A–D), and n = 6–8 pergroup (E–H). Error bars reflect mean ± SEM.

HFD
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analysis revealed a 42% restoration of Syt4 mRNAin the PVN of Syt4 / mice (Figure 5F). Longitudinal
food intake and body weight monitoring indicated1.0
2that the delivery of Syt4 significantly abrogated4
0.5
the antiobesity phenotype of Syt4 / mice (Fig0
ure 5G), and these mice regained hyperphagia (Fig00
WT Syt4-/ure 5H). The reversal of metabolic changes was
Syt4-/WT Syt4-/partial, which might be related to the technical limitation of a viral injection approach, or it might point
to the possibility that other neurons in the brain also
contribute to the metabolic phenotype of Syt4 / mice. AltoSyt4 in OXT Neurons Mediates Metabolic Actions
The connection between Syt4 and OXT prompted further inves- gether, based on the gain-of-function study using conventional
tigation of whether Syt4 acts in OXT neurons to mediate the anti- C57BL/6 mice and the rescue experiment using Syt4 / mice,obesity benefit of Syt4 / mice. To do so, we examined whether it can be concluded that OXT neurons are important for the metaan exogenous induction of Syt4 in OXT neurons within the PVN bolic effects of Syt4.
could recapitulate the effects of HFD feeding to cause weightgain. OXT gene promoter (Zhang et al., 2002) was used to Syt4 Negatively Regulates OXT Release in the PVN
generate OXT neuron-specific Syt4 or control (GFP) lentiviruses. Following the above physiological studies, we aimed to investiThe cell specificity of these lentiviruses was verified by exam- gate if and how Syt4 can regulate OXT exocytosis. Experiments
ining the PVN of virus-injected mice (Figures 5A and 5B). As ex- were designed to test the effects of Syt4 loss of function on OXT
pected, delivery of Syt4 into OXT neurons within the PVN release via OXT ex vivo release assay, an established method tomimicked HFD feeding to promote food intake (Figure 5C) and study OXT release in the PVN (Jin et al., 2007; Sladek and Somweight gain (Figures 5D and 5E) in C57BL/6 mice despite normal ponpun, 2008). Live PVN slices from the hypothalamus of chowchow feeding condition. Importantly, both effects were abol- fed Syt4 / mice or WT controls were used for OXT release
ished by daily OXT injections via the third ventricle (Figures 5C measurement. Results revealed that Syt4 ablation significantly
and 5D), indicating that OXT in the brain can antagonize the enhanced OXT release in the PVN slices under both basal andmetabolic action of Syt4. In parallel, we examined whether the KCl-depolarized conditions (Figure 6A). This finding wasantiobesity phenotype of Syt4 / mice could be reversed by confirmed by an independent assay that analyzed the 60 min
restoring Syt4 in OXT neurons. OXT neuron-specific Syt4 or dynamics of OXT release in the PVN tissues from Syt4 / versus
control GFP lentiviruses were injected into the PVN of HFD-fed WT mice (Figures S4A and S4B). Hypothalamic OXT mRNA
Syt4 / mice and matched WT controls. All mice were main- levels were comparable between chow-fed Syt4 / and WT
tained on an HFD after injection. Quantitative real-time RT-PCR mice (Figure S5A), which excluded the involvement of OXT
4NeuronB

18

O2 Consumption(ml/g/4h)
Basal

KCl

KCl

Basal

KCl

Basal

Basal

0
Mice: WT Syt4-/- Syt4-/- Syt4-/OP-GFP OP-Syt4
Lentivirus: -

OXT
*

HFD
*

1.5
1.0
0.5
0

OP-Syt4

*

2.0

WT

*

*

16

C57BL/6

F

OP-GFP

*

Veh

0

KCl

OXT Release (pg/min)

6

HFD

20

4

Chow

OXT Release Efficiency(fold of WT control)

0.5

C57BL/6

8

HFD

12

1.0

12

Chow

*

1.5

Veh

Food Intake (g/4h)

D

2.0

0.0

E

50
0

Chow
**

2.5

100

Syt4-/-

OXT

C

**

150

Syt4-/-

WT

200

WT

0

Basal

10

Basal

20

KCl

*

*

**

250

WT

30

(Zhang et al., 2009) characteristics of Syt4. We further inferredthat the blood OXT levels should be higher in Syt4 / micethan in WT controls regardless of dietary conditions. Measurements of circulating OXT levels in Syt4 / and WT mice on either
a normal chow or an HFD confirmed this prediction (Figure 6B).
We also measured hypothalamic OXT mRNA levels of HFD-fed
Syt4 / and WT mice. Compared with chow feeding, HFD
enhanced OXT mRNA levels in WT mice (Figure S5A), which
was likely a compensatory response to suppressed OXT release
under HFD. In contrast, hypothalamic OXT mRNA levels werecomparable between HFD-fed and chow-fed Syt4 / mice (Figure S5A), indirectly indicating that HFD-fed Syt4 / mice did not
suffer OXT release impairment. To summarize, exocytosis, but
not gene expression, of OXT is negatively regulated by Syt4.

**

300

Serum OXT (pg/ml)

*

40

350

Syt4-/-

Chow

50

KCl

A

OXT Release (pg/min)

Synaptotagmin-4 in Controlling Energy Balance

Syt4-/-

Figure 6. Regulation of OXT Release by Syt4 and OXT’s Metabolic
Action(A) PVN slices were prepared from chow-fed Syt4 / mice and WT littermate
controls at 3 months of age. Tissues were incubated in Locke’s solution,
washed every 5 min for 10 times, and incubated for 5 min in Locke’s solution
in the presence or absence of 70 mM KCl. Aliquots of the final solution wereused to measure OXT release. *p < 0.05, n = 6–8 per group.
(B) Syt4 / mice and WT littermate controls were maintained on a normal chow
versus an HFD for 4 months since weaning, and serum samples were collected
to measure serum OXT concentrations. **p < 0.01, n = 8–10 per group.
(C and D) Adult C57BL/6 mice received third ventricle injection of OXT (4 mg)
versus vehicle (Veh) and were monitored for food intake (C) and O2 consumption (D) over a 4 hr period after injection. The levels of O2 consumption werenormalized by lean body mass of individual mice. *p < 0.05, **p < 0.01,
n = 11 per group (C), and n = 4 per group (D).
(E and F) Three-month-old Syt4 / mice and WT littermate controls received
HFD feeding for three months. A subgroup of mice received bilateral injectionsof OP-Syt4 or OP-GFP lentiviruses in the PVN prior to the 3 month HFD treatment (E, right panel). The PVN slices from these mice were prepared and incubated in Locke’s solution, and were measured for OXT release under basal andKCl-stimulated conditions. (F) OXT release efficiency of PVN slices from thevirus-injected Syt4 / mice was calculated as the ratio of KCl-stimulated
OXT release to the basal OXT release, and data are presented as fold changes
compared with WT control. *p < 0.05, n = 4–8 per group.
Mice in all these experiments were males. Error bars reflect mean ± SEM.

gene expression. These data indicate that Syt4 negatively regulates OXT exocytosis, which is supported by the inhibitory structural (Chapman et al., 1998; Thomas et al., 1999) and biophysicalBrain OXT Restricts Food Intake and Promotes Energy
Expenditure
OXT is a hypothalamic neuropeptide that is synthesized by OXT
neurons and released from both axon terminals and somatodendritic regions. In addition to its classical role in reproductive
physiology, OXT also regulates various social behaviors suchas care, love, emotion, and trust (Ferguson et al., 2000b; Keverne
and Curley, 2004; Kosfeld et al., 2005). Of note, many of theseclassical and nonclassical actions of OXT were associated withfeeding changes (Douglas et al., 2007; Leng et al., 2008). Morerecently, genetic studies reported that hyperphagia and obesity
developed in mice that were genetically deficient in OXT (Amico
et al., 2005; Kublaoui et al., 2008) or OXT receptor (Takayanagi
et al., 2008). While these genetic approaches targeted the whole
body, the brain-specific role of OXT in metabolic physiology has
not yet been defined. To study the central action of OXT, we injected OXT into the brains of normal C57BL/6 mice via thirdventricle cannula. Central administration of OXT readily suppressed food intake (Figure 6C) and elevated energy expenditure(Figure 6D). Importantly, brain injection of OXT did not yield side
effects on the general health, as confirmed by multiple behavioral tests including kaolin intake test (Figure S5B), conditioned
taste aversion test (Figure S5C), elevated plus maze (Figure S5D–S5F), and open field test (Figure S5G). In conclusion,
OXT in the brain can exert a catabolic regulatory effect on energybalance by restricting food intake and promoting energy
expenditure.
Obesity Is Associated with Impaired OXT Release
Following the observations that HFD-induced obesity is associated with increased hypothalamic Syt4 mRNA levels and Syt4
vesicular distribution (Figures S1E–S1G), we predicted that
dietary obesity might be causally related to altered OXT release.
This hypothesis was also suggested by the data in Figure 6B
showing that circulating levels of OXT in HFD-fed mice werereduced by 40% compared with those of chow-fed mice. To
test this hypothesis, we employed an ex vivo OXT release assay
to determine if HFD feeding could affect OXT release of the hypothalamic PVN. KCl-induced depolarization elicited an OXT
release response in the PVN slices of chow-fed WT mice (Figure 6A), but this effect was blunted by HFD feeding (Figure 6E,
left panel). In contrast, HFD did not blunt depolarization-induced
OXT release in the PVN slices of Syt4 / mice (Figure 6E, left
Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc. 529

Neuron
Synaptotagmin-4 in Controlling Energy Balance

PVN

1.5

0

3V

PVN

PVN PVN

PVN

Veh
OVT
Veh
OVT

0.8
0.4
0

WT Syt4-/-

J

HFD

3

2.0

*
*
2

1.6

Chow
*

1.2

I
Food Intake (g/day)

AVP

AVP

3V

PVN

Chow
*
*

BW Gain (g/week)

OXT
Food Intake (g/day)

OXT

3.0

PVN

WT Syt4-/-

H

4.5
3V

0

WT Syt4-/-G

OXT shRNA

3V

Veh
OVT

0

0.5

*
Ctr-s
OXT shRNA
Ctr-s
OXT shRNA

0.2

BW Gain (g/week)

Control shRNA

WT Syt4-/-

*

1.0

Veh
OVT

0

Veh
OVT

0.2

WT Syt4-/-

PVN

1.5

0.4

0.4

BW Gain (g/week)

Daytime Food (g/12h)

0.6*

Figure 7. Hypothalamic Syt4-OXT Links HFD Feeding
to Disease

HFD
*

2.0

*

0.6

*

D

HFD

0.8

0.8

Veh
OVT

0

Veh
OVT

0.3

BW Gain (g/week)

1.0

0.6

C

Chow
*

Control shRNAOXT shRNA
Control shRNAOXT shRNA

E

0.9

B

Chow
*
*

Veh
OVT

Daytime Food (g/12h)

A

WT Syt4-/-(A–D) Syt4 / mice and WT littermate controls were maintained on a normal chow (A and B) versus an HFD (C and D)
for 3 months since weaning, and were subsequently implantedwith cannula into the third ventricle. After 2 weeks of postoperative recovery, mice received daily third ventricle injectionsof OVT (4 mg) versus vehicle (Veh) for 2 weeks, and were monitored for daily 12 hr daytime food intake after injection (A andC) and weekly body weight (BW) gain (B and D) during the
2 week treatment period. *p < 0.05, n = 5–6 per group. Micein all these experiments were males.
(E–J) Syt4 / mice and WT littermate controls were maintained on a normal chow (E–H) versus an HFD (I and J) for
5 months since weaning, and subsequently received bilateral
intra-PVN injection of OXT shRNA (OXT-s) lentiviruses orcontrol shRNA (Ctr-s) lentiviruses. (E and F) Lentivirus-mediated OXT ablation was assessed by OXT immunostaining(E, upper panels) and quantitated by counting OXT-expressing
neurons in the serial PVN sections (F). AVP immunostaining ofmatched PVN sections (E, lower panels) was used as a control.
3V: third ventricle; scale bar = 50 mm. (G–J) Mice were monitored for daily food intake (G and I) and weekly BW gain
(H and J) during a 3 week follow-up. *p < 0.05, n = 4–7 pergroup. Mice in all these experiments were males. Error bars
reflect mean ± SEM.

HFD
*

1.5

**

40

30

20

10

0

530 Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc.

*

Ctr-s
OXT-s
Ctr-s
OXT-s

OXT shRNA

Control shRNAOXT shRNA
Control shRNAOXT shRNA

Syt4 / mice was significantly abrogated (Figure
7D). Next, OXT shRNA lentivirus was used to
1.0
F
OXT shRNAevaluate the Syt4-OXT connection in the brain
1
Control shRNA
control of feeding and body weight. Syt4 / mice
0.5
and WT controls, maintained on either chow or
HFD feeding, received bilateral intra-PVN injections
0
0
OXT-Positive
WT Syt4-/WT Syt4-/of OXT shRNA lentiviruses or control lentiviruses.
PVN Neurons (%)
Immunostaining confirmed that OXT shRNA
delivery sufficiently ablated the expression of
OXT, but not the control neuropeptide AVP, in thepanel). Moreover, when Syt4 expression was restored into OXT PVN (Figures 7E and 7F). As shown in Figures 7G–7J, shRNAneurons by lentivirus-mediated gene delivery, depolarization mediated OXT ablation increased food intake and weight gain
failed to elicit OXT release in the PVN slices of HFD-fed Syt4 /similarly in WT mice and Syt4 / mice under either chow ormice (Figure 6E, right panel, and Figure 6F). In sum, while obesity HFD condition. Compared with OVT injection, the obesogenic
causes impaired OXT release of the PVN, the antiobesity pheno- effects of OXT shRNA were stronger, consistent with the obsertype of Syt4 / mice is associated with retained sensitivity of vation that OXT shRNA affected 24 hr food intake while the effectOXT release.
of OVT injection lasted only 12 hr after injection. In summary,
results from both pharmacological and shRNA lentiviral injection
Obesogenic Effects of OXT Antagonists in WT
experiments indicate that OXT critically mediates the role of Syt4
and Syt4–/– Micein hypothalamic control of weight gain and obesity.
To further analyze the significance of the hypothalamic Syt4-OXT
pathway in obesity development, we examined whether OXT Therapeutic Potential of OXT in Preventing Obesity
antagonists could be obesogenic. First, a pharmacological The overall results in Figures 1–7 demonstrated that chronic
approach was employed by daily injections of OVT (an OXT nutritional excess can promote hypothalamic Syt4 to suppress
antagonist) into the third ventricle of WT and Syt4 / mice under OXT release, leading to energy imbalance and obesity. Following
a chow or HFD feeding condition. Compared with vehicle injec- these findings, we explored the therapeutic relevance of thistions, OVT increased food intake in chow-fed WT and Syt4 /conceptual model by testing if central administration of OXT
mice (Figure 7A), resulting in increased weight gain over could treat dietary obesity in mice. C57BL/6 mice were firsta 2 week follow-up period (Figure 7B). Similarly, OVT increased allowed to develop obesity via 5 month HFD feeding, and were
food intake in HFD-fed WT and Syt4 / mice (Figure 7C). As subsequently implanted with third ventricle cannula. Following
a result, the effect of HFD in promoting weight gain was exacer- postsurgical recovery, mice received daily ICV injections ofbated in WT mice, and the antiobesity phenotype in HFD-fed OXT or vehicle control for a period of 2 weeks. Injections wereControl shRNANeuron
Synaptotagmin-4 in Controlling Energy Balance

DISCUSSION
Neuropeptide Exocytosis in Hypothalamic Control
of Energy Balance
The CNS regulates whole-body energy balance primarily via themediobasal region and the PVN of the hypothalamus (Elmquist
and Flier, 2004; Schwartz and Porte, 2005). Recent researchadvances have significantly elucidated the neuronal subtypes
and molecular pathways in these hypothalamic regions that
direct the central control of feeding and energy expenditure(Balthasar et al., 2005; Bouret et al., 2004; Cone, 2005; Cowley
et al., 2001; Elmquist and Flier, 2004). The underlying molecularbasis involves transcriptional regulation of neuropeptide genes
in response to dynamic changes of the body’s energy status(Brüning et al., 2000; Coll et al., 2007; Cota et al., 2006; Flierand Maratos-Flier, 1998; Friedman and Halaas, 1998; Minokoshi
et al., 2004; Mobbs, 2007; Münzberg and Myers, 2005; Park andBloom, 2005; Schwartz et al., 2000). However, compared withthe appreciated neuropeptide gene regulation, the role of neuropeptide exocytotic regulation in metabolic actions remains unexplored, despite recent basic research advances showing thatneuropeptide release from secretory granules is a cellular eventwith sophisticated regulation (Stojilkovic, 2005). The present
work discovered that exocytosis regulator Syt4 is expressed
predominantly in a neuronal subtype of hypothalamic PVN,OXT neurons. Moreover, Syt4 is revealed to negatively regulate
OXT release from OXT neurons. The physiological role of thisnegative regulation by hypothalamic Syt4 is anabolic, and underthe environment of chronic nutritional excess, this Syt4 programis further enhanced by an unidentified mechanism or mechanisms to become obesogenic. While forcefully suppressing thisSyt4 program does not evidently impact normal body weighthomeostasis—which might be due to rebalance by otheranabolic/catabolic systems—it provides a remarkable andnearly complete protection against the development of obesityand various other metabolic diseases under nutritional excess.
These findings suggest that the significance of hypothalamic
neuropeptide exocytotic regulation in obesity development andcontrol is substantial. Along this line, further research is neededA

Body Weight Gain (g)given every night before the light was off so that the duration(4 hr) of OXT action overlapped the peak food-consuming
period (6 hr) of mice. Data revealed that OXT treatment rapidly
reduced the magnitude of obesity during the first 3 days of thetreatment and subsequently prevented HFD from causing weightgain (Figure 7A). Comparatively, we assessed whether OXT
treatment might affect the body weight of chow-fed mice—
which is mainly composed of lean body mass. Three days of
OXT injections were found to only slightly reduce the bodyweight of chow-fed mice, while the same treatment had
a more evident antiobesity effect in age- and sex-matched
HFD-fed mice (Figure S5H). Thus, OXT has a strong antiobesity
effect, but its impact on lean body mass was minimal, highlighting the potential value of OXT analogs in obesity treatment.
To summarize this study, Syt4-directed OXT release in the hypothalamus is critical for obesity development (Figure 8B), and Syt4
inhibitors or OXT analogs bear clinical potentials for treating
obesity and related health problems.

HFD feeding
2

Vehicle

1
0
-1

OXT

-2
-3
-4

**
** ** **
* * ** **
*
* * *
*
2 4
6 8 10 12 14 16*0Treatment (days)
B

Chronic Challengeof Nutritional Excess
Syt4-Vesicle Binding
in OXT Neurons (↑)
Hypothalamic
OXT Release (↓)
Food Intake (↑)
Energy Expenditure (↓)
Obesity and Related
Metabolic Diseases

Figure 8. Antiobesity Therapy by OXT and the Syt4-OXT Model in
Disease(A) Male C57BL/6 mice with dietary obesity (via 5 months of HFD feeding)received daily third ventricle injections of OXT (1 mg/day) versus vehicle for
15 days, and were monitored for body weight gain during the treatment period.
*p < 0.05, **p < 0.01, n = 5–10 per group. Error bars reflect mean ± SEM.
(B) Model of hypothalamic Syt4-OXT in the neural mechanism of obesity andrelated diseases. Chronic nutritional excess sensitively upregulates Syt4 in
the hypothalamus to suppress OXT release, leading to energy imbalance
and the development of obesity and comorbidities. Syt4 and OXT represent
two molecular targets for the intervention of obesity and related diseases.

to explore the exocytotic regulations of other hypothalamic
neuropeptides/neurotransmitters and the implications of thesepotential regulations in hypothalamic control of physiology anddisease.
Inhibitory Regulation of Syt4 on OXT Exocytosis
The identification of the hypothalamic Syt4-OXT pathway by thiswork can provide an answer to the question regarding the physiological function of Syt4. Unlike other Syt members, Syt4
contains an atypical C2A domain in a midacid sequence (vonPoser et al., 1997) and fails to catalyze the Ca2+-dependent
exocytosis (Chapman et al., 1998; Thomas et al., 1999). The
physiological relevance of Syt4 became more puzzling with therelatively normal phenotype of Syt4 / mice under normal
feeding condition (Ferguson et al., 2000a), calling into question
whether Syt4 can really regulate vesicle exocytosis in vivo.
Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc. 531

Neuron
Synaptotagmin-4 in Controlling Energy Balance

A recent electrophysiological study pointed to the neuroendocrinic relevance of Syt4 (Zhang et al., 2009); however, it offered
only a biophysical characterization rather than a physiologicalstudy of Syt4 function in neuroendocrine and related diseases.
In the current work, we obtained results establishing Syt4 as
an important negative regulator of OXT exocytosis in the hypothalamic neuroendocrine system. Considering that Syt4 ispresent not only in dense-core vesicles but also in microvesicles,
we speculate that Syt4 may also contribute to the regulation ofneurotransmitter release. While such a possibility was not examined in the current study, it represents an interesting subject for
the next-step investigation. Other immediate questions stemming from the current research include whether and how other
Syt members might be involved in the hypothalamic control ofneuropeptide/neurotransmitter release to direct metabolic physiology and disease. Studies toward understanding these questions could form an interesting research area that addresses
the role of vesicular exocytosis in the hypothalamic control ofendocrine and metabolic physiology.
Central Actions of OXT in Controlling Energy Balance
OXT, a hypothalamic neuropeptide known for its role in mediating reproductive activities, is synthesized by neurons thatare localized predominantly in the PVN of the hypothalamus.
OXT is released on demand to the blood stream from neuronalaxon terminals that innervate the posterior pituitary. Recent
attention has been drawn to OXT’s role in regulating social
behaviors, including care, love, emotion, and trust (Ferguson
et al., 2000b; Keverne and Curley, 2004; Kosfeld et al., 2005),
and local release of OXT in the brain seems to underlie theseregulations (Ludwig et al., 2002; Ludwig and Leng, 2006). In
addition, OXT has catabolic effects through suppressing foodintake and promoting physical activities (Douglas et al., 2007;
Leng et al., 2008). More recently, several genetic studies reported the development of overeating and obesity in mice thatwere deficient in OXT (Amico et al., 2005; Kublaoui et al.,2008) or OXT receptor (Takayanagi et al., 2008). The current
research focused on OXT exocytosis regulation and provided
evidence supporting the notion that regulation of OXT’s local
release is critical for hypothalamic control of energy and bodyweight balance. This concept is in line with recent literaturethat linked OXT to the hypothalamic actions of leptin (Kutlu
et al., 2010) and nesfatin (Maejima et al., 2009). Moreover, ourresearch revealed a reverse relationship between OXT andfeeding, i.e., excessive nutrient intake through HFD feeding
suppresses OXT release. This observation is supported bya recent study showing that chronic sugar intake dampened
the feeding-related c-Fos expression (an indicator of neuronal
activities) in OXT neurons (Mitra et al., 2010). Therefore, compromised action of OXT represents a significant hypothalamicmechanism for the development of dietary obesity and comorbidities. In addition to the mechanistic understanding, ourexperiments further confirmed that brain injection of OXT was
effective in treating mouse obesity without causing appreciablenonspecific or side effects. However, given that OXT has a shorthalf-life, the use of OXT for human obesity treatment remains
challenging until appropriate OXT analogs and practical delivery
methods can be developed.
532 Neuron 69, 523–535, February 10, 2011 ª2011 Elsevier Inc.

Syt4: A Potential Antiobesity Target
The hypothalamus has been known as a pathogenic culprit forovereating, obesity, and related diseases. The underlying molecular mechanisms have been related to hypothalamic leptin andinsulin resistance, which involve SOCS3, PTP1B, IKK/NF-kB,
MyD88, and ER stress (Bence et al., 2006; Howard and Flier,
2006; Kievit et al., 2006; Kleinridders et al., 2009; Zhang et al.,2008). The current research demonstrated that chronic nutritional excess suppresses OXT release to promote obesity development, and more importantly, Syt4 is identified as the mediator
for this dysregulation and hence a molecular target to prevent/reverse the pathogenesis of obesity. Such potentials of Syt4
were verified in animals by the current research showing that
Syt4 inhibition was sufficient to prevent or reverse obesity.
Notably, the catabolic effect of Syt4 inhibition on body weightin lean animals was modest, reflecting only a ‘‘miniature’’ levelof negative energy balance; such a small quantity of negative
energy balance is insufficient to cause deleterious effects on
health, but accumulatively is sufficient to prevent or treat obesity.
Thus, in addition to OXT analogs, developing Syt4 modulators
could be another avenue for targeting the Syt4-OXT pathway
to combat obesity and related diseases.
EXPERIMENTAL PROCEDURES
Animals and Phenotyping
C57BL/6 mice were purchased from Jackson Laboratory. Syt4 / mice werepreviously described (Ferguson et al., 2000a) and backcrossed into C57BL/6
for more than five generations. All mice were housed in standard conditions.
HFD was from Research Diets, Inc. The Institutional Animal Care and Use
Committee approved all the procedures. Mouse body weight was regularly
measured and food intake was determined on a daily basis by individual housing.
For pair feeding, individually housed mice were provided daily with a defined
amount of food, which was based on mouse group with lower ad libitum foodintake prior to pair feeding. Mice that did not consume the whole amount of
supplied food were excluded from the final analysis. Energy expenditure wasdetermined using metabolic chambers (Columbus Instrument, Inc.) at DRTC
core facility of Albert Einstein College of Medicine. For the glucose tolerance
test (GTT), overnight-fasted mice were IP injected with glucose (2 g/kg bodyweight). Blood glucose was measured using Glucometer Elite (Bayer). Blood
insulin and leptin were measured using ELISA kits (Crystal Chem.). Serum OXT
levels were measured using Oxytocin EIA kit (Assay Design).
Third Ventricle Cannulation and Animal Treatment
As previously described (Zhang et al., 2008), an ultraprecise small animal stereotactic apparatus (Kopf Instruments) was employed to implant a guide cannula
into the third ventricle of anesthetized mice at the midline coordinates of
1.8 mm posterior to the bregma and 5.0 mm below the skull surface. Mice
were allowed 1–2 weeks for postsurgical recovery. Individually housed micereceived OXT injection (Bachem California, Inc.) via preimplanted cannula.
Lentiviruses and Intra-PVN Injection
Lentiviral vectors using mouse OXT gene cassette (Zhang et al., 2002) to direct
OXT neuron-specific expression of Flag-tagged Syt4 or GFP were created aspreviously described (Zhang et al., 2008). Briefly, DNA of mouse OXT gene
cassette (AI-03) was provided by Dr. H. Gainer (Zhang et al., 2002), and thetranslation start of exon I was mutated and inserted with Flag-tagged Syt4
or GFP cDNA in the truncated exon III. Lentiviral shRNA against mouse OXT
or Syt4 and the matched control lentiviral vector were purchased from Sigma.
Lentiviruses were produced from HEK293T cells through cotransfection oftarget plasmids with their packaging plasmids using Ca3(PO4)2. Lentiviruses
were purified by ultracentrifugation. As previously described (Zhang et al.,2008), an ultraprecise stereotax was employed to bilaterally inject lentiviruses

Neuron
Synaptotagmin-4 in Controlling Energy Balance

into the PVN at the coordinates of 0.85 mm posterior to the bregma, 0.15 mm
lateral to the midline, and 4.8 mm below the skull surface.
Ex Vivo OXT Release Test
The protocol has been described previously (Jin et al., 2007). Briefly, the PVN slices were dissected from the hypothalamus. In some experiments, the PVN slices
were cut into small pieces at the size of 0.5 mm. The PVN slices were immediately incubated in Locke’s solution constantly supplied with 95% O2 and 5%
CO2 at 37 C. The solution was changed every 5 to 10 min during the 60 min
experimental period. An aliquot of Locke’s solution was collected to measure
the basal levels of OXT release. Depolarization was induced during the final 5
to 10 min by adding KCl to Locke’s solution at a final concentration of 70 mM,
and an aliquot of Locke’s solution was collected to measure KCl-stimulated
release of OXT. PVN slices were also collected in some experiments, washed,
and lysated for the measurement of tissue OXT content. Oxytocin EIA kit (Assay
Design) was used to determine the OXT levels in the solution and tissues.
Heart Perfusion and Brain Immunostaining
Mice under anesthesia were trans-heart perfused with 4% PFA, and brains
were removed, postfixed in 4% PFA for 4 hr, and infiltrated with 20%–30%
sucrose. Brain sections (20 mm thickness) were made using a cryostat at–20 C. Fixed brain sections were blocked with serum of the appropriate
species, penetrated with 0.2% Triton X-100, and treated with primary antibodies including rabbit anti-Syt4 (Synaptic Systems), mouse anti-NeuN(Chemicon), guinea pig anti-OXT, and anti-AVP (Peninsula Lab) IgGs, and followed by either a reaction with Alexa Fluor 488 or 555 secondary antibody(Invitrogen) or a reaction using an ABC kit (Vector Lab). Control IgGs of theappropriate species were used as negative controls. A light microscope wasused to detect color staining, and a confocal microscope, to detect
fluorescence.
Immunogold Labeling and Electron Microscopy
Mice under anesthesia were trans-heart perfused with 1% PFA and 1% glutaraldehyde, and tissues were isolated, postfixed, dehydrated, embedded in
London Resin White, and sectioned at 100 nm. For immunogold labeling,tissue sections on nickel grids were quenched, blocked, and incubated withrabbit anti-Syt4 antibody and guinea pig anti-OXT antibody overnight at 4 C,
followed by incubation with gold particle (size: 10 nm or 20 nm)-conjugatedsecondary antibodies (Ted Pella) for 2 hr at room temperature. Sections
were poststained with uranyl acetate and lead citrate, examined on an electron
microscope (Hitachi 7500), and imaged using AMT digital imaging equipment.
Quantitative RT-PCR
Total RNA from the homogenized tissue samples was extracted using TRIzol(Invitrogen). Complementary DNA was synthesized using the Advantage RTfor PCR kit (Clontech). PCR amplification of Syt4 mRNA was quantified using
SYBRGreen PCR Master Mix (Applied Biosystems). Results were normalizedagainst the expression of house-keeping genes including TATA box-binding
protein (TBP) or b-actin.
Statistical Analyses
Two-tailed Student’s t tests were used for two-group comparisons. ANOVA
and appropriate post hoc analyses were used for comparisons of more than
two groups. Data were presented as mean ± SEM. p < 0.05 was considered
statistically significant.
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes five figures and Supplemental Experimental Procedures and can be found with this article online at
doi:10.1016/j.neuron.2010.12.036.

assistance in confocal microscopy, E. Chapman and M. Jackson for suggestions, and Cai lab members for general assistance. This study was supported
by NIH R01 DK078750 and R01 AG031774, and by American Diabetes Association Junior Faculty Award 1-07-JF-09 (all to D.C.).
Accepted: November 30, 2010
Published: February 9, 2011
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