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GABAergic RIP-Cre Neuronsin the Arcuate Nucleus SelectivelyRegulate Energy ExpenditureDong Kong,1,6 Qingchun Tong,1,3,6 Chianping Ye,1 Shuichi Koda,1,4 Patrick M. Fuller,2 Michael J. Krashes,1 Linh Vong,1
Russell S. Ray,5 David P. Olson,1 and Bradford B. Lowell1,*1Division of Endocrinology, Department of Medicine2Department of Neurology
Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA3Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA4Asubio Pharma, Kobe 650-0047, Japan5Department of Genetics, Harvard Medical School, Boston, MA 02115, USA6These authors contributed equally to this work
*Correspondence: blowell@bidmc.harvard.edu
http://dx.doi.org/10.1016/j.cell.2012.09.020
SUMMARY
Neural regulation of energy expenditure is incom-pletely understood. By genetically disruptingGABAergic transmission in a cell-specific fashion,and by combining this with selective pharmacoge-netic activation and optogenetic mapping tech-niques, we have uncovered an arcuate-based circuitthat selectively drives energy expenditure. Specifi-cally, mice lacking synaptic GABA release fromRIP-Cre neurons have reduced energy expenditure,become obese and are extremely sensitive to high-fat diet-induced obesity, the latter due to defectivediet-induced thermogenesis. Leptin’s ability tostimulate thermogenesis, but not to reduce feeding,is markedly attenuated. Acute, selective activationof arcuate GABAergic RIP-Cre neurons, whichmonosynaptically innervate PVH neurons projectingto the NTS, rapidly stimulates brown fat andincreases energy expenditure but does not affectfeeding. Importantly, this response is dependentupon GABA release from RIP-Cre neurons. Thus,GABAergic RIP-Cre neurons in the arcuate selec-tively drive energy expenditure, contribute to leptin’sstimulatory effect on thermogenesis, and protectagainst diet-induced obesity.
INTRODUCTION
Neural circuits operating within and extending beyond the
hypothalamus regulate food intake and energy expenditure.
Proopiomelanocortin (POMC)-expressing neurons and adja-
cent agouti-related peptide (AgRP)-expressing neurons, both
located in the arcuate nucleus (ARC) of the hypothalamus, are
key components of this circuitry. The ARC also contains
many other neurons that express neither POMC nor AgRP
(i.e., non-POMC, non-AgRP neurons). Due to their location in
the ARC, these could also regulate energy balance. A subset
of these ‘‘non-POMC, non-AgRP’’ neurons has clear neuroen-
docrine functions, such as those expressing kisspeptin, growth
hormone-releasing hormone, or dopamine. ‘‘Non-POMC, non-
AgRP’’ neurons whose function is something other than neuro-
endocrine also likely exist. These neurons, like POMC and AgRP
neurons, could be additional, important regulators of energy
balance.
In Rip-Cre transgenic mice (Postic et al., 1999), the rat
insulin-2 (Ins2) promoter drives cre expression in both pancre-
atic b cells and the brain (Song et al., 2010; Wicksteed et al.,
2010). Neurons expressing cre activity, hereafter referred to
as ‘‘RIP-Cre neurons,’’ are distributed in many hypothalamic
sites, including the ARC. Within the ARC, RIP-Cre neurons
are intermingled with, but are distinct from, POMC and AgRP
neurons (Choudhury et al., 2005). Deletion of various loxed
alleles in Rip-Cre mice, often with the initial intent of manipu-
lating gene expression in pancreatic b cells, has marked effects
on energy balance (Chakravarthy et al., 2007; Choudhury et al.,
2005; Covey et al., 2006; Kubota et al., 2004; Lin et al., 2004;
Mori et al., 2009). It is generally believed that this is secondary
to altered function of RIP-Cre neurons as opposed to pancre-
atic b cells. Notably, disruption of leptin signaling in Rip-Cre
transgenic mice causes obesity (Covey et al., 2006). Because
RIP-Cre neurons are found in many locations in the brain,
the specific subgroup of RIP-Cre neurons responsible for
regulating energy balance is unknown. Similarly, the relevant
neurotransmitter(s) and downstream circuitry are likewise
unknown.
Synaptic transmission via glutamate and GABA are critical
components of the hypothalamic circuitry regulating energy
balance (Cowley et al., 2001; Liu et al., 2012; Pinto et al., 2004;
Tong et al., 2008; van den Pol, 2003; Vong et al., 2011; Wu
et al., 2009; Yang et al., 2011). Consequently, it is of interest to
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. 645
Figure 1. Generation of Mice Lacking VGAT
in RIP-Cre Neurons
(A) Immunodetection of GFP in the brain of Rip-
Cre, lox-GFP mice. 3V, third ventricle.
(B and C) In situ hybridization for VgatmRNA in the
brain of (B) control (Vgatflox/flox) and (C) Rip-Cre,
Vgatflox/flox littermates. Arrows indicate the regions
with notable reduction of Vgat mRNA signal in
Rip-Cre, Vgatflox/flox mice.
(D and E) Quantitative PCR results of Vgat mRNA
in (D) mediobasal hypothalamus and (E) isolated
pancreatic islets of 2-month-old littermates
(mean ± SEM; n = 4). N.D., nondetectable. *p <
0.05, unpaired t tests.
Scale bars in (A)–(C) represent 100 mm. See also
Table S1.
establish if synaptic release of glutamate or GABA, specifically
by RIP-Cre neurons, regulates energy balance. For the purpose
of addressing such questions, we previously generated mice
with loxed alleles of the vesicular glutamate transporter 2
(VGLUT2, required for synaptic release of glutamate from hypo-
thalamic neurons) and the vesicular GABA transporter (VGAT,
required for synaptic release of GABA from GABAergic neurons)
(Tong et al., 2007, 2008). In the present study, we have deleted
Vglut2 and Vgat specifically from RIP-Cre neurons. This
has uncovered a critical role for synaptic release of GABA,
but not glutamate, from RIP-Cre neurons, in selectively stimu-
lating energy expenditure. Using pharmacogenetic techniques
(Alexander et al., 2009; Krashes et al., 2011), we go on to
establish that RIP-Cre neurons located specifically in the ARC
drive this effect. Finally, using channelrhodopsin-assisted circuit
mapping (Atasoy et al., 2008; Petreanu et al., 2007), we identify
the downstream circuitry selectively engaged by ARC RIP-Cre
neurons.
RESULTS
Neuronal Expression of Cre Recombinase in Rip-Cre
Transgenic MiceRip-Cre transgenicmicewere crossedwith cre-dependent green
fluorescent protein (GFP) reporter mice (Novak et al., 2000) to
generate Rip-Cre, lox-GFP mice, and immunohistochemistry
for GFP was performed. GFP-positive neurons (i.e., RIP-Cre
neurons)wereprimarily found in the hypothalamusandoccasion-
ally in the cortex and striatum (Figure 1A; see Table S1 available
online). Within the hypothalamus, GFP-positive neurons were
observed in the ARC, the ventromedial hypothalamus (VMH),
the medial tuberal nucleus (MTu), the suprachiasmatic nucleus
(SCN), and the dorsomedial hypothalamus (DMH). These obser-
vations are consistent with previous reports by Choudhury et al.
646 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
(2005), Lin et al. (2004), Song et al.
(2010), and Wicksteed et al. (2010).
Generation of Mice Lacking VGATin RIP-Cre NeuronsRip-Cre transgenic mice were crossed
with Vgatflox/flox mice. The resulting Rip-
Cre, Vgatflox/+ mice were then crossed with Vgatflox/flox mice
to obtain Rip-Cre, Vgatflox/flox study subjects and their control
littermates (Vgatflox/flox mice and Rip-Cre, Vgatflox/+ mice). Rip-
Cre, Vgatflox/+ littermates were included as controls to rule
out nonspecific effects of the Rip-Cre transgene (Lee et al.,
2006). In situ hybridization studies for Vgat mRNA were then
performed to assess for GABAergic neurons and also for
sites where the Rip-Cre transgene disrupts Vgat expression.
In Vgatflox/flox control mice, Vgat mRNA signal was detected
in sites known to contain GABAergic neurons. With regard to
sites shown in Figure 1B, these include, within the hypo-
thalamus, the ARC, DMH and MTu; and beyond the hypothal-
amus, the central amygdala (CeA) and the reticular nucleus of
thalamus (RT). In Rip-Cre, Vgatflox/flox mice (Figure 1C), Vgat
signal was substantially reduced in sites containing RIP-Cre
neurons, such as the ARC, DMH, and MTu. In sites where
RIP-Cre neurons are not found, such as the CeA and RT,
Vgat signal, as expected, was unchanged. Finally, we mea-
sured Vgat mRNA levels in the mediobasal hypothalamus,
a region that includes the ARC, DMH, and MTu, which are
three sites suggested by the aforementioned analysis to
contain GABAergic RIP-Cre neurons. This quantitative analysis
confirmed that Vgat mRNA, but not that of a control transcript
(Ucp2 mRNA), was substantially reduced in Rip-Cre, Vgatflox/flox
mice (Figure 1D).
Vgat mRNA was undetectable in both control and Rip-Cre,
Vgatflox/flox pancreatic islets (Figure 1E), whereas, as noted
above, using the identical assay, Vgat mRNA was readily de-
tected in the hypothalamus (Figure 1D). Confirming the intact
nature of pancreatic islet RNA, Ucp2, a gene expressed in islets
(Zhang et al., 2001), was readily detected in the same samples
(Figure 1E). Thus, mouse islets express little or no Vgat mRNA.
Consequently, deletion of the Vgat gene in pancreatic b cells
should produce no effects.
Figure 2. Energy Balance in Mice Lacking VGAT in Rip-Cre Neurons
(A–D) (A) Body weight, (B) body fat mass (3 months old), (C) daily food intake
(2months old), and (D) locomotor activity (2 months old) of ad libitum chow-fed
male littermates (n = 8–10). Black bars in (D) indicate dark cycles.
(E and F) Oxygen consumption of 2-month-old male littermates expressed (E)
per body weight and (F) per animal (n = 8).
(G) H&E of iBAT from 2-month-old littermates.
(H and I) (H) Temperature (n = 12) and (I)Ucp1mRNA expression (n = 4) in iBAT.
Data are presented asmean ±SEM. *p < 0.05 and ***p < 0.001, unpaired t tests
compared with Vgatflox/flox group. See also Figures S1 and S2.
Energy Balance in Mice Lacking VGAT in RIP-CreNeuronsWhen fed a standard chow diet, Rip-Cre, Vgatflox/flox mice have
modestly increased body weight (Figure 2A) and, at 3 months
of age, markedly increased fat stores (Figure 2B). Lean body
mass, on the other hand, was unchanged (data not shown).
The possible causes of positive energy balance were then as-
sessed in 2-month-old animals. Food intake (Figure 2C) and
locomotor activity (Figure 2D) were found to be unchanged.
Obesity, in the face of normal food intake, strongly implicates
reduced energy expenditure in Rip-Cre, Vgatflox/flox mice. This
was confirmed by direct assessments of energy expenditure.
Oxygen consumption was markedly reduced in Rip-Cre,
Vgatflox/flox mice, and this was apparent when data were ex-
pressed per body weight (Figure 2E) or per animal (Figure 2F).
Thus, obesity in Rip-Cre, Vgatflox/flox mice is due entirely to a
selective reduction in energy expenditure.
We next analyzed brown adipose tissue (BAT), a well-estab-
lished mediator of thermogenesis (Cannon and Nedergaard,
2004). Interscapular BAT (iBAT) of Rip-Cre, Vgatflox/flox mice
was markedly enlarged and pale in comparison with that from
control littermates. As shown in Figure 2G, BAT from Rip-Cre,
Vgatflox/floxmice contained larger cells with unilocular triglyceride
deposits, similar to that observed in animals with defective
sympathetic activation of BAT (Bachman et al., 2002). As as-
sessed by biotelemetry probes implanted subcutaneously in
the interscapula fossa beneath iBAT (Enriori et al., 2011), iBAT
temperature, an index of BAT thermogenesis, was reduced in
Rip-Cre, Vgatflox/flox mice (Figure 2H). In contrast, subcutaneous
temperature of a flank site devoid of BAT was similar in the
two groups (Figure S1A). Finally, expression of Ucp1 mRNA,
which encodes the BAT-specific thermogenic molecule, uncou-
pling protein 1, was significantly lower in Rip-Cre, Vgatflox/flox
mice (Figure 2I). These results indicate that GABA release from
RIP-Cre neurons regulates BAT thermogenic function and
suggest that decreased BAT activity is, at least in part, respon-
sible for reduced energy expenditure inRip-Cre,Vgatflox/floxmice.
Given that Rip-Cre transgenic mice express cre in some SCN
neurons (Figure S1B) and that virtually all SCN neurons are
GABAergic (Figure S1C), Rip-Cre, Vgatflox/flox mice could have
altered circadian regulation, which could in turn affect energy
balance (Bass and Takahashi, 2010). To address this possibility,
body temperature (Tb), a reliable indicator of circadian clock
activity (Fuller et al., 2008), was measured using implanted
biotelemetry. As shown in Figures S1D and S1E, Rip-Cre,
Vgatflox/flox mice displayed diurnal and circadian Tb patterns
(phasing, amplitude, or period) that were comparable to controls.
Thus, the circadian clock does not appear to be altered or other-
wise dysfunctional in Rip-Cre, Vgatflox/flox mice and unlikely con-
tributes to the metabolic phenotypes of the Rip-Cre, Vgatflox/flox
mice. Likewise, serum T4 and corticosterone levels, two other
potential regulators of energy balance, were found to be
unchanged in Rip-Cre, Vgatflox/flox mice (Figures S1F and S1G).
Energy Balance in Mice Lacking VGLUT2in RIP-Cre NeuronsSome RIP-Cre neurons are glutamatergic, such as those in
the VMH. To assess if glutamatergic RIP-Cre neurons regulate
energy homeostasis, mice lacking VGLUT2 in RIP-Cre neurons
(Rip-Cre, Vglut2flox/flox mice) were generated as was done for
Rip-Cre, Vgatflox/flox mice. In situ hybridization analyses revealed
that, as expected, Vglut2 mRNA was dramatically reduced in
the VMH of Rip-Cre, Vglut2flox/flox mice (Figures S2A and S2B).
Of note, Rip-Cre, Vglut2flox/flox mice had normal body weight,
oxygen consumption, and food intake (Figures S2C–S2E),
indicating that glutamate release from RIP-Cre neurons is not
required for regulation of energy balance.
Diet-Induced Obesity in Mice Lacking VGATin RIP-Cre NeuronsIncreased energy expenditure following the ingestion of highly
palatable, calorically dense diets, a phenomenon often referred
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. 647
Figure 3. Diet-Induced Obesity in Mice
Lacking VGAT in RIP-Cre Neurons
(A and B) Body weight on HFD (A) and daily food
intake (B) averaged over the first 2 weeks on HFD
(n = 8–10).
(C) Oxygen consumption expressed per body
weight during the transition from chow to HFD
(n = 8). CD, averaged oxygen consumption over
3 days on chow; HD1, HD2 and HD3, oxygen
consumption during day 1, 2, and 3, respectively,
on HFD. The percent increase in oxygen con-
sumption on HFD above that on chow diet is
indicated above each bar.
(D) Ucp1 mRNA level in iBAT of HFD-treated
littermates (n = 6–8).
Data are presented as mean ± SEM. *p < 0.05
and **p < 0.01, unpaired t tests compared with
Vgatflox/flox group.
to as diet-induced thermogenesis, plays an important role in
resisting diet-induced obesity (Bachman et al., 2002). To deter-
mine if GABAergic RIP-Cre neuron-driven energy expenditure
is involved in this adaptive response, Rip-Cre, Vgatflox/flox mice
and control animals were fed a high-fat, high-sucrose diet
(HFD) from between 6 and 26 weeks of age. As shown in
Figure 3A, compared to control mice, Rip-Cre, Vgatflox/flox mice
developed massive obesity. Of interest, this diet-induced
obesity was not caused by increased food intake (Figure 3B),
strongly implicating impaired diet-induced thermogenesis. To
directly assess this, oxygen consumption was measured during
the transition from chow to HFD (3 days on chow followed by
3 days on HFD) in 7-week-old mice (note, body weight is normal
at this early age). As shown in Figure 3C, oxygen consumption of
Rip-Cre,Vgatflox/flox mice, on both chow and HFD, was markedly
lower than that observed in control animals. Importantly,
whereas HFD increased energy expenditure by 7%–12% in
control mice, this response was markedly blunted in Rip-Cre,
Vgatflox/flox mice (increased by only 3%–5% in response to
HFD). In addition, HFD-treated Rip-Cre,Vgatflox/flox mice
(6 months old and 20 weeks on HFD) exhibited reduced
Ucp1 mRNA expression in BAT (Figure 3D). In summary, Rip-
Cre, Vgatflox/flox mice are extremely sensitive to diet-induced
obesity, and this is due entirely to a defect in diet-induced
thermogenesis.
Effects of Leptin Treatment in Mice Lacking VGATin RIP-Cre NeuronsGenetic deletion of leptin receptors (LEPRs) from RIP-Cre
neurons causes marked obesity without affecting food intake
(Covey et al., 2006), suggesting a defect in energy expenditure.
Given that Rip-Cre,Vgatflox/flox mice also have defective energy
648 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
expenditure, this raises the possibility
that GABA release from RIP-Cre neurons
mediates leptin’s effects on energy
expenditure. To test this, changes in
body weight and food intake were moni-
tored following injection of saline or leptin.
In control mice, treatment with leptin
reduced body weight (Figures 4A and 4B) and food intake
(Figures 4C and 4D). Of note, whereas the ability of leptin to
reduce food intake in Rip-Cre,Vgatflox/flox mice was completely
intact (Figure 4D), its ability to reduce body weight was markedly
attenuated (Figure 4B). Attenuation of body weight loss in
the face of intact inhibition of food intake indicates that leptin’s
ability to increase energy expenditure is impaired in Rip-Cre,
Vgatflox/flox mice. To directly assess leptin action on energy
expenditure, in particular, in stimulating BAT activity, iBAT
temperature and Ucp1 mRNA were measured. In control mice,
leptin but not saline dramatically and rapidly increased the
temperature of iBAT (as previously described by Enriori et al.,
2011) (Figures 4E and 4F), but not the temperature of a subcuta-
neous flank site devoid of BAT (Figures S3A and S3B). Leptin
also markedly increased Ucp1 mRNA levels (Figure 4G). Of
note, these stimulatory effects of leptin on iBAT temperature
and Ucp1 mRNA levels were attenuated in Rip-Cre,Vgatflox/flox
animals (Figures 4E–4G). Thus, GABA release from RIP-Cre
neurons is required for leptin to fully stimulate energy expendi-
ture, but not for leptin to inhibit feeding.
To determine which subset of RIP-Cre neurons in the hypo-
thalamus expresses LEPRs, leptin-induced phosphorylation of
STAT3 (Tyr705, pSTAT3), a marker for LEPR activity (Munzberg
et al., 2004), was assessed in Rip-Cre, lox-GFP mice. The
neurons double positive for pSTAT3 and RIP-Cre activity were
mainly observed in the ARC and the VMH (Figures 4H, 4I, and
S3C–S3N). The DMH, which contained both RIP-Cre neurons
and pSTAT3-positive neurons, exhibited negligible colocali-
zation (Figures 4I and S3I–S3K). Because VMH neurons are
glutamatergic and not GABAergic (Vong et al., 2011), all leptin-
responsive, GABAergic RIP-Cre neurons appear to be located
in the ARC. Collectively, given the aforementioned observations,
Figure 4. Response to Leptin in Mice Lack-
ing VGAT in RIP-Cre Neurons
(A–F) The effects of saline or leptin on (A and B)
body weight, (C and D) daily food intake, and
(E and F) iBAT temperature in 2-month-old male
littermates (n = 8–12). *p < 0.05, paired t tests
compared with animals of the same genotype
before leptin injection (i.e., time point 0); #p < 0.05,
unpaired t tests compared with control animals
at given time point.
(G) Ucp1 mRNA level in iBAT 4 hr after saline (Sal)
or leptin (Lep) injection (n = 6). *p < 0.05 and
**p < 0.01, unpaired t tests compared with saline-
injected animals of the same genotype; #p < 0.05,
unpaired t tests compared with Vgatflox/flox animals
of the same treatment.
(H) Double immunohistochemistry for GFP (green)
and leptin-induced phosphorylation of STAT3
(Tyr105, pSTAT3, magenta) in the ARC of Rip-Cre,
lox-GFP mice. Arrows indicate the neurons with
coexpression of GFP and pSTAT3.
(I) Quantification of the neurons that expressed
GFP, pSTAT3, or both in the hypothalamic nuclei
(n = 3 mice).
Data are presented as mean ± SEM. See also
Figures S3 and S5.
it is likely that GABAergic RIP-Cre neurons in the ARC mediate
leptin’s stimulatory effect on energy expenditure.
Pharmacogenetic Activation of RIP-Cre Neuronsin the ARCTo directly test the ability of ARC RIP-Cre neurons to drive
energy expenditure, we used the pharmacogenetic approach
referred to as designer receptors exclusively activated by
designer drugs (DREADD). The stimulatory DREADD, hM3Dq,
is activated by the otherwise inert, brain-penetrable compound,
clozapine-N-oxide (CNO) (Alexander et al., 2009). The cre-
Cell 151, 645–657,
dependent adeno-associated virus, AAV-
Flex-hM3Dq-mCherry (Krashes et al.,
2011), was stereotaxically injected into
the ARC of 3- to 4-week-old Rip-Cre
transgenic mice (Figure 5A), and studies
were performed 2–3 weeks after injec-
tion. Brain slice electrophysiology studies
confirmed that CNO depolarizes and
increases the firing rate of hM3Dq-ex-
pressing RIP-Cre neurons (Figure 5B),
but not control non-hM3Dq-expressing
RIP-Cre neurons (Figure S4A). hM3Dq
virus was then bilaterally injected into the
ARC of 5- to 6-week-old Vgatflox/flox mice,
Rip-Cre mice, or Rip-Cre, Vgatflox/flox
mice. Studies were performed 3 weeks
after injection. The mCherry fusion tag
was exclusively detected in the ARC of
Rip-Cre mice and Rip-Cre, Vgatflox/flox
mice and was absent in the ARC of
Vgatflox/flox mice (because these mice
lack cre activity that enables hM3Dq expression) (Figure 5C).
When CNO was injected in vivo, c-fos immunoreactivity was
markedly increased in the ARC of Rip-Cre mice and Rip-Cre,
Vgatflox/flox mice, but not in the ARC of Vgatflox/flox mice (Fig-
ure 5D). Thus, in vivo treatment with CNO activates hM3Dq-
expressing RIP-Cre neurons in the ARC.
To assess effects on energy expenditure, virus-injected
animals were housed individually in metabolic cages, and
oxygen consumption was monitored following injection with
saline or CNO. After an acclimation period, each mouse was
injected with saline on the first day followed by CNO on the
October 26, 2012 ª2012 Elsevier Inc. 649
Figure 5. Pharmacogenetic Activation of
ARC RIP-Cre Neurons
(A) Diagram of AAV-Flex-hM3Dq-mCherry (left)
and schematic indication of the stereotaxic injec-
tion into the ARC of Rip-Cre transgenic mice
(right).
(B) Representative whole-cell, current-clamp
recording from an ARC RIP-Cre neuron marked by
mCherry fluorescence from a Rip-Cre, lox-GFP
mouse injected with AAV-Flex-hM3Dq-mCherry
virus.
(C and D) Immunohistochemistry for (C) mCherry
and (D) CNO-induced c-Fos (DAB, black stain) in
the ARC of virus-injected Vgatflox/flox (top), Rip-Cre
(middle), and Rip-Cre, Vgatflox/flox (bottom) male
mice.
(E–G) Oxygen consumption over a 24 hr period
(left) and during the first 4 hr (right) following i.p.
injection of saline or CNO (n = 8). *p < 0.05, **p <
0.01, ***p < 0.001, paired t tests compared to
saline groups.
(H–J) iBAT temperature (Temp) over 4 hr following
saline or CNO injection (n = 6–8). *p < 0.01, paired
t test compared to animals of the same genotype
before CNO injection; #p < 0.01, paired t tests
compared to saline-injected animals at given time
point.
(K–M) Ucp1 mRNA in iBAT 6 hr after saline or
CNO injection (n = 4–6). *p < 0.05, unpaired t tests
compared with saline-injected animals of the
same genotype.
Data are presented as mean ± SEM. See also
Figure S4.
second day. Selective activation of ARC RIP-Cre neurons with
CNO rapidly increased oxygen consumption (Figure 5F), and
this effect lasted for approximately 9 hr. Importantly, CNO
had no effect on oxygen consumption in Rip-Cre, Vgatflox/flox
mice (Figure 5G), which are notable for their inability to release
GABA. Similarly, CNO had no effect on oxygen consumption
in control mice (i.e., Vgatflox/flox mice), which do not express
hM3Dq (Figure 5E). Remarkably, and consistent with our earlier
findings suggesting that GABAergic RIP-Cre neurons selec-
tively control energy expenditure, food intake was unaltered
650 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
by CNO treatment (Figure S4B). We
next assessed BAT activity during
stimulation of ARC RIP-Cre neurons. In
Rip-Cre mice, CNO significantly in-
creased iBAT temperature (Figure 5I),
but not subcutaneous flank tempera-
ture (Figure S4C), and also markedly
increased Ucp1 mRNA (Figure 5L).
CNO did not stimulate iBAT temperature
or Ucp1 mRNA in Vgatflox/flox mice (which
do not express hM3Dq) or in Rip-Cre,
Vgatflox/flox mice (which are unable to
release GABA) (Figures 5H, 5J, 5K, and
5M). These results demonstrate that
synaptic GABA release from ARC RIP-
Cre neurons selectively stimulates BAT activity and energy
expenditure.
Electrophysiologic Effects of Leptin on ARCRIP-Cre NeuronsWhole-cell current-clamp recordings were performed as previ-
ously described by Dhillon et al. (2006) on arcuate RIP-Cre
neurons visualized by expression of GFP in Rip-Cre, lox-GFP
mice. In data not shown, ARC RIP-Cre neurons exhibited
heterogeneous responses to leptin: 30% of neurons (6 of 20)
Figure 6. Projection of ARC RIP-Cre
Neurons
(A) Diagram of AAV-Flex-ChR2(H134R)-mCherry
(left) and schematic indication of the stereotaxic
injection into the ARC of Rip-Cre transgenic mice
(right).
(B) Representative voltage tracing showing light-
driven spikes in a current-clamped arcuate neuron
marked by mCherry fluorescence. Blue tick marks
represent 0.5 ms light flashes at 0.5 Hz.
(C–E) Immunohistochemistry for mCherry in the
hypothalamus of virus-injected Rip-Cre transgenic
mice. mCherry-expressing RIP-Cre neurons in the
ARC are shown in (C) and in a zoomed view in (D).
(E) mCherry-expressing RIP-Cre neuron fibers in
the PVH. ME, median eminence; fx, fornix; opt,
optic tract.
(F) (i) Light-evoked IPSCs in a PVH neuron before
(left) and after (right) the addition of 20 mM bicu-
culline in response to clusters of light pulses.
Blue tick marks represent 0.5 ms light flash at
0.5 Hz. (ii) Zoomed in view of response to a single
pulse of light.
See also Figure S6 and Table S2.
were excited (depolarized membrane potential and increased
firing rate), 35% (7 of 20) were inhibited (hyperpolarized
membrane potential and decreased firing rate), and 35% (7 of
20) were not affected by leptin. As discussed in the following
section, the paraventricular hypothalamus (PVH) is the likely
downstream site that mediates the thermogenic effects of
GABAergic ARC RIP-Cre neurons. To assess the effects of leptin
on PVH-projecting ARC RIP-Cre neurons, retrograde red fluo-
rescent beads were stereotaxically injected into the PVH of
Rip-Cre, lox-GFP mice (Figures S5A and S5B). Retrogradely
transported beads were observed in sites known to innervate
the PVH, including the ARC, DMH, SCN, nucleus of the solitary
tract (NTS), and posterodorsal medial amygdala (MEpd) (data
not shown). In the ARC, 62% (194 of 312) of RIP-Cre neurons
contained red beads (Figure S5C). Recordings were then per-
formed on PVH-projecting (GFP+/beads+) and in non-PVH-pro-
jecting (GFP+/beads�) ARC RIP-Cre neurons. Ionotropic gluta-
mate (kynurenate) and GABA (PTX) receptor blockers were
added to minimize indirect effects of leptin. As shown in Fig-
ure S5D, 9 of 15 PVH-projecting ARC RIP-Cre neurons were
directly excited by leptin, 6 of 15 were unaffected by leptin,
Cell 151, 645–657,
and 0 of 15 were inhibited by leptin. In
contrast, 0 of 12 non-PVH-projecting
ARC RIP-Cre neurons were excited by
leptin, 9 of 12 were unaffected by leptin,
and 3 of 12 were directly inhibited by lep-
tin (Figure S5E). Thus, leptin excites the
majority of PVH-projecting ARC RIP-Cre
neurons.
Downstream NeurocircuitryEngaged by ARC RIP-Cre NeuronsWenext used channelrhodopsin2 (ChR2)-
assisted circuit mapping (Atasoy et al.,
2008; Petreanu et al., 2007) to identify proximal downstream
neurons that could mediate stimulation of energy expenditure
by GABAergic ARC RIP-Cre neurons. A virus that conditionally
expresses ChR2-mCherry fusion protein (AAV-Flex-ChR2
(H134R)-mCherry) (Zhang et al., 2007) in the presence of cre re-
combinase was unilaterally injected into the ARC of 3- to 4-
week-oldRip-Cremice (Figure 6A).Micewere studied 2–3weeks
after injection. Expressed ChR2 was functional as evidenced by
light-evoked action potentials in RIP-Cre neurons (Figures 6B
and S6A–S6C). mCherry-positive dendrites and soma were
abundantly and exclusively detected in the ARC (Figures 6C
and 6D). mCherry-expressing axons were primarily observed in
the PVH (Figure 6E). By comparison, much less abundant, scat-
tered mCherry-expressing axons were seen in the DMH, the
bed nuclei of the stria terminalis (BST), and the medial preoptic
area (MPO) (Figure 6E; Table S2). Thus, the PVH is the dominant
target of ARC RIP-Cre neurons. This is of interest because it
has been suggested that release of GABA in the PVH stimulates
BAT thermogenesis (Madden and Morrison, 2009).
To determine if GABAergic RIP-Cre neurons are functionally,
synaptically connected to PVH neurons, inhibitory postsynaptic
October 26, 2012 ª2012 Elsevier Inc. 651
Figure 7. Downstream Neurocircuitry
Engaged by ARC RIP-Cre Neurons
(A–D) (A) Diagram illustrating ChR2/retrobead
double-injection experiment in Rip-Cre trans-
genic mice. AAV-ChR2, AAV-Flex-ChR2(H134R)-
mCherry; Green Beads, fluorescent retrograde
green beads. (B) Immunohistochemistry against
mCherry (red) and native fluorescence of retro-
grade green beads (green) in the PVH. (C and D)
Representative tracings of light-driven IPSCs
recorded in (C) a PVH neuron with green beads
(19 of 23) and in (D) an adjacent PVH neuron
without green beads (13 of 14).
(E–H) (E) Diagram illustrating ChR2/retrobead
double-injection experiment in Agrp-ires-Cre
mice. (F) Immunostaining against mCherry (red)
and native fluorescence of retrograde green beads
(green) in the PVH. (G and H) Representative
tracings of light-driven IPSCs recorded in (G)
a PVH neuron with green beads (16 of 16), and in
(H) an adjacent PVH neuron without green beads
(2 of 9).
(I and J) (I) Diagram illustrating retrograde tracing
assay with green beads injected into the RPa of
Vgat-ires-Cre, lox-tdTomato mice. (J) Native fluo-
rescence of green beads (green) and immunore-
activity of tdTomato (red) in the NTS. Arrows
indicate the neurons labeled with both green
beads (i.e., the neurons projecting to the RPa)
and tdTomato (i.e., GABAergic neurons); two such
neurons are zoomed in the dashed squares.
(K–M) (K) Diagram illustrating AAV-Flex-
ChR2(H134R)-mCherry virus injected into the NTS
of Vgat-ires-Cre mice. (L) Representative voltage
tracing showing light-driven spikes in a current-
clamped NTS neuron marked by mCherry fluo-
rescence. (M) Representative tracing of light-
driven IPSCs recorded in RPa neurons (five of
eight).
Blue tick marks represent 0.5 ms light flashes of
0.5 Hz.
See also Figure S7.
currents (IPSCs) were assessed in PVH neurons following
illumination of ChR2-expressing RIP-Cre terminals. Light-driven
IPSCs were reliably evoked in a small subset of randomly
selected PVH neurons that were surrounded by mCherry fluo-
rescent terminals (2 out of 14), and these were completely
blocked by bicuculline, a GABAA receptor antagonist (Fig-
ure 6F). The latency between onset of light and onset of IPSC
in these two neurons was 1.2 and 2.3 ms. The low frequency
of responders (i.e., 2 out of 14) likely relates to complexity
within the PVH, which is composed of numerous subsets of
652 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
functionally distinct neurons (Biag et al.,
2012; Simmons and Swanson, 2009).
As explained below, we have used site
of projection to enrich for PVH neurons
likely to control energy expenditure
and, therefore, likely to receive mono-
synaptic input from GABAergic RIP-Cre
neurons.
Given that the NTS receives abundant projections from
the PVH (Geerling et al., 2010), contains neurons in polysyn-
aptic contact with BAT (Bamshad et al., 1999; Cano
et al., 2003; Oldfield et al., 2002), is known to regulate sym-
pathetic outflow (Spyer, 1994), and inhibit BAT function (Cao
et al., 2010), we hypothesized that PVH neurons projecting
to the NTS drive RIP-Cre neuron-mediated energy expenditure.
To test if NTS-projecting PVH neurons receive monosynaptic
input from GABAergic ARC RIP-Cre neurons, dual-injection
studies were performed as illustrated in Figure 7A, with
AAV-Flex-ChR2(H134R)-mCherry virus injected into the ARC
and retrograde green fluorescent beads injected into the NTS
of Rip-Cre mice. Histologic studies confirmed injection of
beads into the NTS and, unavoidably, the nearby dorsal motor
nucleus of the vagus nerve (DMV) (Figure S7A). Correct target-
ing of the NTS is further confirmed by the presence of retro-
gradely transported beads in sites known to project to the
NTS, including the PVH, lateral hypothalamus, CeA, and dorsal
raphe (Geerling and Loewy, 2006; Saper et al., 1976; Saw-
chenko and Swanson, 1982) (Figure S7B). As expected, PVH
neurons labeled by NTS-injected beads are not neuroendocrine
cells as evidenced by their lack of colabeling following periph-
eral administration of the retrograde tracer, Fluoro-Gold, which
is taken up by median eminence- and posterior pituitary-pro-
jecting neurons (Luther et al., 2002) (Figure S7C).
In agreement with previous results from Biag et al. (2012),
retrogradely transported beads were found in the ventral zone
of the medial parvicellular part of the PVH (PVHmpv) (Figure 7B).
Notably, ChR2-mCherry-expressing ARC RIP-Cre fibers were
found commingling with bead-positive neurons in the PVHmpv
(Figure 7B). Light-evoked IPSCs were then assessed in bead+
or adjacent bead� PVH neurons. Light-evoked IPSCs were
observed in most (19 of 23) bead+ PVH neurons, i.e., neurons
that project to the NTS (Figure 7C). The latency between onset
of light and onset of IPSC was 3.4 ± 1.5 ms (mean ± SD, 19
neurons). In contrast, light failed to evoke IPSCs in nearly all
(13 of 14) bead� PVH neurons (Figure 7D). Thus, GABAergic
ARC RIP-Cre neurons are selectively connected to NTS-projec-
ting PVH neurons.
To test the specificity of NTS-projecting PVH neurons with
respect to afferent input, we next determined if AgRP neurons,
like RIP-Cre neurons, provide monosynaptic input. AgRP neu-
rons are a useful comparator because, like RIP-Cre neurons,
they release GABA, originate in the arcuate, and project to the
PVH, but in striking contrast with RIP-Cre neurons, AgRP
neurons inhibit energy expenditure (Krashes et al., 2011), and
in addition, they also stimulate food intake (Aponte et al.,
2011; Krashes et al., 2011). Connectivity between AgRP neurons
and NTS-projecting PVH neurons was determined using similar
methods to those described above except that Agrp-ires-Cre
mice (Tong et al., 2008) were used to enable ChR2 expression
(Figure 7E). Expressed ChR2 was functional as evidenced by
light-evoked action potentials in AgRP neurons (Figure S7D).
Consistent with previous reports (Cone, 2005), ChR2-mCherry-
expressing AgRP fibers were found to heavily innervate the
PVH and to commingle with bead-positive neurons in the
PVHmpv (Figure 7F). However, unlike the situation with RIP-
Cre neuron ChR2-assisted circuit mapping, light-evoked IPSCs
were absent in all (16 of 16) bead+ PVH neurons, i.e., neurons
that project to the NTS (Figure 7G). In contrast, light-evoked
IPSCs were observed in two out of nine bead� PVH neurons
(Figure 7H), with a latency between onset of light and IPSC in
these two neurons of 3.5 and 5.7 ms. Afferent control of
NTS-projecting PVH neurons by RIP-Cre but not AgRP neurons
is consistent with the opposite and different functions of
these two arcuate neurons and demonstrates the specificity
of the ARC RIP-Cre neuron / NTS-projecting PVH neuron
connection.
The neural pathway by which NTS neurons regulate sym-
pathetic outflow to BAT has yet to be established. To address
this, we tested for connections between the NTS and the
raphe pallidus (RPa), a physiologically important hindbrain site
where numerous BAT sympathetic preautonomic neurons are
located (Morrison and Nakamura, 2011). Retrograde beads
were stereotaxically injected into the RPa of Vgat-ires-Cre,
lox-tdTomato reporter mice (to allow for identification of
GABAergic neurons; Vong et al., 2011) (Figure 7I). Consistent
with previous studies by Hermann et al. (1997) and Yoshida
et al. (2009), RPa-projecting neurons were found to be located
in the MPO, DMH, PAG, DR, and NTS (Figures S7E–S7G). Of
note, 68 of the 143 RPa-projecting NTS neurons were
GABAergic, as evidenced by colocalization of beads with
tdTomato (Figure 7J). In contrast, few or no GABAergic RPa-
projecting neurons were detected in other sites (MPO, DMH,
PAG, and DR) (Figure S7H). Thus, the NTS sends dense pro-
jections to the RPa, and notably, GABAergic input to the RPa
comes predominantly from the NTS. ChR2-assisted circuit
mapping was then used to confirm a GABAergic NTS / RPa
synaptic connection. AAV-Flex-ChR2(H134R)-mCherry virus
was injected into the NTS of Vgat-ires-Cre mice (Figure 7K),
and the activity of ChR2 within NTS GABAergic neurons was
functionally verified (Figure 7L). Light-evoked IPSCs were then
assessed in randomly selected RPa neurons. Of note, light-
evoked IPSCs were detected in five out of eight RPa neurons
(Figure 7M), with a latency between onset of light and IPSC of
3.1 ± 1.6 ms (mean ± SD, five neurons), and these were
completely blocked by bicuculline (data not shown).
DISCUSSION
RIP-Cre neurons regulate energy balance (Chakravarthy et al.,
2007; Choudhury et al., 2005; Covey et al., 2006; Kubota et al.,
2004; Lin et al., 2004; Mori et al., 2009); however, because
RIP-Cre neurons are located inmany sites, the neurocircuit basis
for this has been unknown. To address this, we undertook
a multistep approach. By deleting VGAT and VGLUT2 from
RIP-Cre neurons, we established that release of GABA, but not
glutamate, from RIP-Cre neurons regulates energy balance.
Remarkably, this regulation was specific for energy expenditure;
food intake was entirely unaffected. Building on this, we hypo-
thesized that GABAergic RIP-Cre neurons located specifically
in the ARC mediate this effect. Using a pharmacogenetic
approach to test this, we selectively stimulated arcuate RIP-
Cre neurons and found that this markedly increased energy
expenditure; again, without any effect on food intake. Notably,
pharmacogenetic stimulation of energy expenditure was entirely
dependent upon release of GABA. These studies establish that
synaptic release of GABA from arcuate RIP-Cre neurons selec-
tively drives energy expenditure.
We then used ChR2-assisted circuit mapping to anatomically
and functionally identify downstream neurons receiving syn-
aptic GABAergic input from arcuate RIP-Cre neurons. We
observed that arcuate RIP-Cre neurons project heavily and
predominately to the PVH. This is notable because it has been
proposed that GABAergic input to the PVH stimulates sympa-
thetic outflow and BAT-mediated energy expenditure (Madden
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. 653
and Morrison, 2009). Importantly, PVH neurons, specifically
those that project to the NTS in the brainstem, a site known to
regulate energy expenditure (Cao et al., 2010), receive mono-
synaptic GABAergic input from arcuate RIP-Cre neurons. In
summary, these studies have uncovered an ARC-initiated neuro-
circuit that selectively drives energy expenditure.
Selective Regulation of Energy ExpenditureThe ability of arcuate RIP-Cre neurons to regulate energy expen-
diture, without affecting food intake, is noteworthy. It is seen
following genetic deletion of Vgat (Figures 2 and 3) and Lepr
(Covey et al., 2006) in RIP-Cre neurons and, importantly, also
following acute pharmacogenetic stimulation of RIP-Cre
neurons (Figure 5). The selectivity of arcuate RIP-Cre neurons
for energy expenditure is remarkable because other arcuate
neurons, for example AgRP and POMC neurons, coordinately
regulate both food intake and energy expenditure. This unique
feature of arcuate RIP-Cre neurons is important because it
provides a scheme for experimentally approaching forebrain
control of energy expenditure. Specifically, neurons receiving
GABAergic output from arcuate RIP-Cre neurons are likely to
play important roles in regulating energy expenditure, but not
food intake. In the present study, as will be discussed below,
we have used this approach to uncover an efferent circuit that
likely drives energy expenditure (arcuate RIP-Cre GABAergic
neurons / PVH neurons / NTS neurons).
Paraventricular Nucleus and Regulationof Energy ExpenditureLocal application of GABAA receptor antagonist to the PVH
decreases sympathetic outflow and BAT-mediated energy
expenditure, suggesting that GABAergic input to the PVH drives
energy expenditure (Madden and Morrison, 2009). The neurons
providing this GABAergic input, however, have been unknown.
The following five points strongly support the view that
arcuate RIP-Cre neurons are a major source of this BAT-, energy
expenditure-stimulating GABAergic input. First, Rip-Cre,
Vgatflox/flox mice have reduced energy expenditure, altered BAT
morphology, reduced BAT temperature, and reduced Ucp1
gene expression—all suggestive of low BAT activity (Figure 2).
Second, Rip-Cre, Vgatflox/flox mice have reduced diet-induced
thermogenesis (Figure 3). Third, Rip-Cre, Vgatflox/flox mice
have an impaired catabolic and BAT thermogenic response
to leptin treatment (Figure 4). Fourth, GABA release by the
arcuate subpopulation of RIP-Cre neurons stimulates BAT
thermogenesis and energy expenditure (Figure 5). Fifth, arcuate
RIP-Cre neurons project primarily and heavily to the PVH
(Figures 6E and 7B).
We used ChR2-assisted circuit mapping to test the aforemen-
tioned assertion that RIP-Cre neurons provide important
synaptic GABAergic input to PVH neurons. Connectivity,
however, was observed for only a small subset (�15%) of
randomly selected PVH neurons. This low rate of connectivity
is related to complexity of the PVH, which contains numerous
cell types each with unique function (Biag et al., 2012; Simmons
and Swanson, 2009). These include (1) neuroendocrine neurons
that secrete oxytocin, vasopressin, or thyrotropin- or cortico-
tropin-releasing hormone; (2) nonneuroendocrine neurons that
654 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
regulate feeding behavior via presently ill-defined pathways
(Gold et al., 1977; Leibowitz, 1978); and finally, (3) nonneuroen-
docrine neurons that regulate either sympathetic or parasympa-
thetic outflow (Geerling et al., 2010; Swanson and Sawchenko,
1983). Because sympathetic outflow is the primary driver of
brown adipose function and energy expenditure (Bachman
et al., 2002; Bartness et al., 2010), the energy expenditure-
promoting action of RIP-Cre neurons is likely mediated by
aminor subset of nonneuroendocrine neurons, specifically those
that control sympathetic tone.
Role of NTS-Projecting PVH NeuronsFor reasons mentioned below, neurons in the NTS, and there-
fore the subset of PVH neurons projecting to the NTS, are strong
candidates for mediating RIP-Cre neuron-driven energy expen-
diture. NTS neurons (1) receive dense input from the PVH; (2)
are known to control sympathetic outflow (Andresen and Kunze,
1994; Guyenet, 2006); (3) are labeled by the transneuronal
retrograde tract tracer, pseudorabies virus, following its injec-
tion into BAT (Bamshad et al., 1999; Cano et al., 2003; Oldfield
et al., 2002); and (4) have recently been shown to regulate
BAT function (Cao et al., 2010). To test if NTS-projecting PVH
neurons are indeed a major target of GABAergic RIP-Cre
neurons, we injected retrogradely transported fluorescent
beads into the NTS (and, due to its close proximity, the nearby
DMV) and performed ChR2-assisted circuit mapping. Of great
interest, most NTS-projecting neurons receive synaptic
GABAergic input from RIP-Cre neurons as assessed by light-
evoked IPSCs in bead-positive PVH neurons (Figure 7C). In
striking contrast, most other PVN neurons, i.e., those not projec-
ting to the NTS, do not receive GABAergic input from RIP-Cre
neurons (Figure 7D). Thus, NTS-projecting PVH neurons are
the major target of energy expenditure-regulating arcuate
GABAergic RIP-Cre neurons.
AgRP neurons, unlike RIP-Cre neurons, are not synaptically
connected to NTS-projecting PVH neurons (Figure 7G). This
distinction is remarkable because, like RIP-Cre neurons, AgRP
neurons are located in the arcuate, are GABAergic, and send
very dense projections to the PVH. However, in sharp contrast
with RIP-Cre neurons, AgRP neurons have qualitatively different
functions—they inhibit, as opposed to stimulate, energy expen-
diture (Krashes et al., 2011; Tong et al., 2008), and in addition,
they also stimulate food intake (Aponte et al., 2011; Krashes
et al., 2011). These different functions must be explained by
differences in efferent circuitry. Consistent with this, the present
study clearly demonstrates that such differences can robustly
be resolved at the level of the PVH; RIP-Cre neurons are synap-
tically connected with NTS-projecting PVH neurons, whereas
AgRP neurons are not. These results suggest a means for de-
convoluting complex circuitry that traverses the PVH. By using
PVH-projecting neurons of defined function as the entry point,
in this case AgRP versus RIP-Cre neurons, and by performing
ChR2-assisted circuit mapping to identify PVH neurons that
are synaptically downstream, in conjunction with retrograde
tracers that subdivide PVH neurons based upon differential
sites of projection, it is possible to ascribe function and to deter-
mine wiring diagrams of circuits that span three synaptically
coupled sites.
Circuitry Linking NTS Neurons with BATThe RPa is a sympathetic preautonomic area that when acti-
vated potently stimulates BAT activity (Morrison and Naka-
mura, 2011). Indeed, the RPa / sympathetic preganglionic /
postganglionic neuron / BAT circuit likely constitutes the final
common pathway by which the brain controls BAT activity.
With this in mind, our observation that most GABAergic input
to RPa neurons comes from the NTS is of interest and sug-
gests the following pathway: arcuate RIP-Cre GABAergic
neurons / PVH neurons / NTS GABAergic neurons /
RPa neurons / / / BAT activity. The inclusion of a second
GABAergic neuron in this circuit, namely the NTS GABAergic
neuron, allows for stimulation (disinhibition) of BAT activity by
RIP-Cre GABAergic neurons. Future studies will be required
to critically test the functionality, with respect to control of
BAT activity, of the aforementioned pathway. In total, the
circuitry uncovered in this study provides a framework for
understanding homeostatic regulation of BAT activity and
energy expenditure.
EXPERIMENTAL PROCEDURES
Mice
All animal care and experimental procedures were approved by the Beth Israel
Deaconess Medical Center Institutional Animal Care and Use Committee.
Rip-Cre transgenic mice (Postic et al., 1999) were obtained from The Jackson
Laboratory (#003573). lox-Vgat mice were generated previously (Tong et al.,
2008). Chow (Teklad F6 Rodent Diet 8664) or HFDs (Research Diets;
D12331) were used.
Metabolic Studies
Food intake, body weight, fat mass, and locomotor activity was measured as
described by Dhillon et al. (2006). Oxygen consumption was measured with
indirect calorimetry (Columbus Instruments). iBAT temperature was measured
as described by Enriori et al. (2011) with remote biotelemetry (IPTT-300; Bio
Medic Data Systems).
Leptin Treatment Studies
Leptin’s effects on body weight and food intake were measured as reported
by Banno et al. (2010). Leptin’s effects on iBAT temperature were assessed
as reported by Enriori et al. (2011) in animals implanted with biotelemetry
probes (as above). Leptin-induced STAT3 phosphorylation was assessed
in Rip-Cre, lox-GFP mice using methods previously described (Vong et al.,
2011).
Pharmacogenetic Studies
AAV8-Flex-hM3Dq-mCherry virus (Krashes et al., 2011) was stereotaxically
injected into the arcuate of 5- to 6-week-old mice (for oxygen consumption
and iBAT temperature studies) or 3- to 4-week-old Rip-Cre, lox-GFP mice
(for electrophysiological studies). Two to three weeks following viral injection,
electrophysiological responses of mCherry-expressing neurons to 5 mM CNO
or thermogenic responses of animals to 0.3 mg/kg CNO (i.p.) were tested. See
also Extended Experimental Procedures.
ChR2-Assisted Circuitry Mapping
AAV8-Flex-ChR2(H134R)-mCherry virus was stereotaxically injected into sites
of interest of 3- to 4-week-old mice (Atasoy et al., 2008; Zhang et al., 2007).
Two to three weeks after viral injection, light-stimulated firing responses or
IPSCs were tested by whole-cell recordings. For retrograde bead-related
studies, red or green beads (Lumafluor) were stereotaxically injected 5 days
prior to electrophysiological studies. See also Extended Experimental
Procedures.
Statistics
Statistics were performed with GraphPad Prism software.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, seven
figures, and two tables and can be foundwith this article online at http://dx.doi.
org/10.1016/j.cell.2012.09.020
ACKNOWLEDGMENTS
The authors acknowledge J. Lu, J.B. Ding, and members of the B.B.L. lab for
helpful discussion; Z. Yang, J. Yu, B. Choi, X. Hu, S. Ma, and D. Cusher for
technical support; C.B. Saper for insightful suggestions and reading
the manuscript; K. Deisseroth for AAV-Flex-ChR2(H134R)-mCherry; and
B.L. Roth for AAV-FLEX-hM3Dq-mCherry. This work was supported by
Grants R01 DK089044, R01 DK071051, R01 DK075632, R37 DK053477,
and BNORC & BADERC Transgenic Core Grants P30DK046200 and
P30DK057521 (to B.B.L.), P&F from BADERC & BNORC Grants
P30DK057521 and P30DK0460200 (to D.K.), R01 DK092605 and AHA
SDG3280017 (to Q.T.), and NS0736313 (to P.M.F.).
Received: March 30, 2012
Revised: June 26, 2012
Accepted: September 6, 2012
Published: October 25, 2012
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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
AnimalsAll animal care and experimental procedures were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care
and Use Committee. Mice were housed at 22�C–24�C with a 12 hr light/12 hr dark cycle with standard mouse chow (Teklad F6
Rodent Diet 8664; 4.05 kcal/g, 3.3 kcal/gmetabolizable energy, 12.5% kcal from fat; Harlan Teklad, Madison,WI) andwater provided
ad libitum. All diets were provided as pellets. Mice were euthanized by CO2 narcosis.
Generation of Rip-Cre, Vgatflox/flox, and Rip-Cre, Vglut2flox/flox MiceMice with loxp-flanked Vglut2 alleles (Slc17a6) (Vglut2flox/flox) or with loxp-flanked Vgat alleles (Slc32a1) (Vgatflox/flox) have been
described previously (Tong et al., 2007, 2008). Rip-Cre transgenic mice were obtained from the Jackson Laboratory (Strain:
B6.Cg-Tg(Ins2-cre)25Mgn/J; stock number: 003573). Rip-Cre,Vgatflox/+ mice were generated by mating Rip-Cre mice with
Vgatflox/flox mice (129, C57Bl/6 mixed background). Study groups (Vgatflox/flox, Rip-Cre, Vgatflox/+ and Rip-Cre, Vgatflox/flox mice)
were generated by mating Rip-Cre, Vgatflox/+ mice with Vgatflox/flox mice. The same breeding scheme was used to generate study
subjects Rip-Cre, Vglut2flox/flox mice except that Vglut2flox/flox mice were used.
High-Fat Diet StudiesGroups of male controls (Vgatflox/flox mice and Rip-Cre, Vgatflox/+ mice) and Rip-Cre, Vgatflox/flox littermates were switched to high fat,
high sucrose diet (HFD, Research Diets, D12331, 58% kcal from fat, 26% from sucrose, 5.557kcal/gm) at 6 weeks of age and main-
tained on HFD feeding for additional 20 weeks. Body weight was monitored weekly.
Circadian Rhythm AssaysAdult male littermates at 2 months of age were implanted i.p. with biotelemetry transmitters to record body temperature (Tb) and
locomotor activity as previously described (Fuller et al., 2002, 2008). In brief, all animals were anesthetized, a midline celiotomy
was performed, and a sterilized transmitter (TA11TA-F10; Data Sciences International) was inserted into the peritoneal cavity using
aseptic techniques. All incisions were sutured and treated with topical antibiotics. Following surgery, the animals were individually
housed in standard plastic mouse cages with ad libitum food and water. Each cage was placed on top of a telemetry receiver inter-
faced to a microcomputer data acquisition system. Tb values were recorded at 5 min intervals, and activity data were collected in
5 min bins. The cages were housed inside isolation chambers, which provided ventilation, identical LED-based lighting, an ambient
temperature of 24� ± 1�C, and visual isolation. Placement of the mice cages into the chambers was randomwith respect to genotyp-
ing distribution. Tb and activity data were analyzed and plotted using ClockLab (Coulbourn Instruments).
Energy Expenditure and Food IntakeEnergy expenditure was measured as oxygen consumption rate using indirect calorimetry. Individually housed mice maintained on
chow diet at 7–8 weeks of age were placed in chambers of Comprehensive Lab Animal Monitoring System (CLAMS, Columbus
Instruments, Columbus, OH) with ad libitum food andwater. Mice were acclimatized in the chambers for 48 hr prior to data collection.
Diet-induced thermogenesis was assessed as previously described (Dhillon et al., 2006). In brief, oxygen consumption of chow-fed
animals (2-month old male littermates) was measured for 3 days on chow, followed by 3 days on HFD. The averaged oxygen
consumption over the 3 days on chow and that of the individual days on HFD are presented.
Food intake of individually housed mice was measured for 7 days. For food intake assay on HFD-treated animals, food intake was
measured during the first 2 weeks on HFD.
As for acute studies in mice injected with AAV-Flex-hM3Dq-mCherry virus, mice were studied 3 weeks after viral injection. Each
individually-housed male mouse was i.p. injected with saline on the first day and CNO on the following day at 8:00–8:30 am and
immediately placed back into the metabolic chambers for data collection. CNO was administered at 0.30 mg/kg of body weight.
Saline was delivered at the same volume to maintain consistency in the studies. The mice had free access to food and water during
this study.
iBAT and Subcutaneous Flank TemperatureBAT temperature wasmeasured as previously described (Enriori et al., 2011; Madden, 2012) with remote biotelemetry (IPTT-300, Bio
Medic Data Systems). In brief, temperature probes were implanted beneath the iBAT pad between the scapula under anesthesia.
Animals were allowed 1 week recovery and acclimation was performed with saline injection for up to 5 days. For subcutaneous flank
temperature measurements, probes were implanted subcutaneously on the lower back (a site devoid of iBAT). Recovery and
acclimation was as described above.
Beam-Break AssayLocomotor activity was assessed using an OptoM3 apparatus from Columbus Instruments (Columbus, Ohio). Briefly, individually-
housed male mice were placed in their home cages, and ambulatory counts along the x axis were recorded every min for 1 week
using CI BUS software from Columbus Instruments (Columbus, Ohio).
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. S1
In Situ Hybridization StudiesA VgatmRNA riboprobe corresponding to the exon 2 was used for in situ hybridization. The primers used to amplify Vgat probe were:
Forward, 50 ttggccccgggaaagagg 30, and Reverse, 50 agcagtatcgcagccccaaag 30. The 35S-labeled cRNA probes were prepared and
the in situ hybridizations were performed as previously described (Kong et al., 2010). The in situ hybridization study of the expression
of Vglut2 was described previously (Tong et al., 2007).
Quantitative PCR AssayQuantitative PCRwas performed as previously reported (Kong et al., 2010). In brief, RNAwas extracted frommouse tissues using the
STAT-60 Reagent (Isotex Diagnostics Inc.). cDNA was generated by reverse transcriptase (Clontech Inc.). The assay probes were
purchased from Applied Biosystems, with IDs for Vgat, Ucp1, and Ucp2 as Mm01208836_g1, Mm01244861_m1, and
Mm01274108_g1, respectively. Relative expression of mRNA was determined using standard curves based on the same cDNA
samples, and samples were adjusted for total RNA content by 18S ribosomal RNA quantitative PCR (Applied Biosystems, Foster
City, CA). Quantitative PCR was performed on an Mx3000p instrument (Stratagene).
ImmunohistochemistryImmunohistochemistry was performed as described (Kong et al., 2010). In brief, brain sections were pretreated with 0.3% hydro-
gen peroxide in PBS (pH 7.4) for 30 min at room temperature and then were incubated in 3% normal donkey serum (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA) with 0.25% Triton X-100 in PBS (PBT-azide) for 2 hr. Slides were then incu-
bated overnight at room temperature in a rabbit anti-GFP primary antiserum (Molecular Probes, Cat # a-6455; 1:20,000 in PBT-azide).
After washing in PBS, sections were incubated in biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories;
1:1,000) for 2 hr at room temperature, followed by incubation for 1 hr in a solution of avidin-biotin complex (Vectastain Elite ABC
Kit, Vector Laboratories, Burlingame, CA; 1:500) diluted in PBS. The sections were then washed in PBS and incubated in a solution
of 0.04% diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01%hydrogen peroxide in PBS. For fluorescent immunolabeling,
sections treated with anti-GFP antiserum were incubated instead in fluorescein-conjugated donkey anti-rabbit IgG (Jackson
ImmunoResearch Laboratory 1:1000) for 2 hr at room temperature. Sections were mounted onto SuperFrost slides and then visual-
ized with Zeiss Confocal Microscope. For anti-mCherry and anti-tdTomato stainings, primary antibody (anti-DsRed, Invitrogen,
1:2500) and secondary antibody (Alex594-anti-rabbit IgG, Invitrogen, 1:200) were used instead.
For leptin-induced pSTAT3 staining, 7- to 8-week-old Rip-Cre, lox-GFPmalemice were fasted overnight, injected intraperitoneally
with 4 mg/kg recombinant mouse leptin (A.F. Parlow, National Hormone and Peptide Program). 45 min later, the animals were
perfused and the brains were fixed for sectioning at 25 mm. pSTAT3 IHC was performed as described (Munzberg et al., 2004;
Vong et al., 2011) with anti-pSTAT3 antibody. Results were imaged by confocal microscopy (Zeiss LSM 510). Cell counts were per-
formed on one of the five brain series. Sections were first organized systematically in a rostral-to-caudal manner. All brain sections in
the series that contained positive cells in each nucleus were analyzed. Cell counts were obtained from both sides of the brain in each
section, and all GFP- and pSTAT3-positive cells in each hypothalamic region were counted irrespective of staining intensity.
c-fos AssayAnimals were handled for 12 consecutive days before the assay to reduce stress response, and then injected with either saline of
CNO (0.3 mg/kg; i.p.). 150 min later, the animals were sacrificed with 7% chloral hydrate diluted in saline (350 mg/kg) for histological
assay.
Leptin Effects on Body Weight, Food Intake, and iBAT TemperatureA protocol similar to that previously described was used for this study (Banno et al., 2010). Briefly, male Vgatflox/flox and Rip-Cre,
Vgatflox/flox littermates (6–7 weeks old) were individually housed and acclimated by handling for a week. These mice were then i.p.
administered with saline (0.9% NaCl) or leptin (A.F. Parlow, NHPP, 2 mg/kg/injection, 8:00 am and 8:00 pm) every 12 hr for
3 days. Body weight and food intake was measured at baseline, and on each day with saline injections or leptin injections. Body
weight and food intake were averaged during the 3 days prior to injections to obtain the baseline values used for calculating percent
changes. Leptin’s effects on iBAT temperature was measured as previously described (Enriori et al., 2011) and 6 mg/kg leptin or
saline was i.p. injected to the animals implanted with remote biotelemetry (IPTT-300, BMDS).
Stereotaxic InjectionsStereotaxic injections were performed as described (Krashes et al., 2011). Mice were anesthetized with 7% chloral hydrate diluted in
saline (350mg/kg) and placed into a stereotaxic apparatus (KOPFModel 963). After exposing the skull via small incision, a small hole
was drilled for injection. A pulled-glass pipette with 20–40 mm tip diameter was inserted into the brain and virus was injected by an air
pressure system. A micromanipulator (Grass Technologies, Model S48 Stimulator) was used to control injection speed at 25 nl/min
and the pipette was withdrawn 5 min after injection. AAV-Flex-hM3Dq-mCherry, serotype 8 (titer 1.23 1013 genomes copies per ml)
or AAV-Flex-ChR2(H134R)-mCherry, serotype 8 (titer 6 3 1012 genomes copies per ml) was injected bilaterally into the arcuate
nucleus of 5–6 weeks (for DREADD-related energy expenditure and iBAT temperature studies) or 3–4 weeks (for ChR2/DREADD-
related electrophysiological studies) old mice (100 nl, coordinates, bregma: AP:�1.40 mm, DV:�5.80 mm, L: ±0.25 mm). For
S2 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
postoperative care, mice were injected intraperitoneally with meloxicam (0.5 mg/kg). For the PVH-related injections, we used coor-
dinate as bregma: AP: �0.8 mm, DV:�5.0 mm, L: ±0.25 mm. All stereotaxic injection sites were verified under electrophysiological
microscopy (for electrophysiology-related studies) or by immunohistochemistry (for anatomy and in vivo studies).
For retrograde beads-related tracing studies, red or green beads (Lumafluor Inc.) were stereotaxically injected to the PVH (30 nl),
the NTS (30 nl), and the RPa (25 nl) 5 days prior to assays. Fluoro-Gold (i.p. 15 mg/kg) was injected to label neuroendocrine cells as
previously described (Luther et al., 2002; Merchenthaler, 1991).
Electrophysiology—Effects of DREADD Activation and LeptinThe protocols of slice preparation and whole cell recording have previously been described (Dhillon et al., 2006). In brief, 5–7 weeks
old mice were anesthetized with isoflurane before decapitation and removal of the entire brain. Brains were immediately submerged
in ice-cold, carbogen-saturated (95% O2, 5% CO2) high sucrose solution (238 mM sucrose, 26 mM NaHCO3, 2.5 mM KCl, 1.0 mM
NaH2PO4, 5.0 mM MgCl2, 10.0 mM CaCl2, 11 mM glucose). Then, 200 mM thick coronal sections were cut with a Leica VT1000S
Vibratome and incubated in oxygenated recording aCSF (126 mM NaCl, 21.4 mM NaHCO3, 2.5 mM KCl, 1.2 mM NaH2PO4,
1.2 mM MgCl2, 2.4 mM CaCl2, 10 mM glucose) at room temperature (21–24�C) for at least 1 hr before recording. The hypothalamic
slice was put in a recording chamber and bathed in oxygenated aCSF at a flow rate of approximately 2 ml/min.
Whole-cell recordings were then made on identified neurons under IR-DIC optics by using a MultiClamp 700B Amplifier (Axon
Instrument) with the software pClamp 10.2 (Axon Instrument). Recording electrodes had resistances of 3.5–5 MU when filled with
the K-gluconate internal solution, which contained (in mM) K-gluconate 128, HEPES 10, EGTA 1, KCl 10, MgCl2 1, CaCl2 0.3, Mg-
ATP 3, and Na-GTP 0.3 (pH 7.35 with KOH).
When the effects of CNO were assessed on RIP-Cre neurons, 5- to 7-week-old Rip-Cre, lox-GFP mice injected with AAV-Flex-
hM3Dq-mCherry into the arcuate nucleus 2–3 weeks before were used. Clozapine-N-oxide (CNO) was applied to bath solution
through perfusion as previously reported (Krashes et al., 2011). After acquisition of stable whole-cell recordings for 2–5 min,
aCSF solution containing 5 mM CNO was perfused into the brain slice preparation.
When the effects of leptin on electrophysiological responses of ARC RIP-Cre neurons were tested, 5- to 7-week-old Rip-Cre, lox-
GFP mice were used. Effects of leptin were assessed as previously described (Dhillon et al., 2006). Since it is difficult to wash out
leptin, brain slices were changed and the system was flushed between each leptin addition electrophysiology study. For assays
in Figure S5, synaptic blockers (1mMKynurenate and 100 mMPTX) were included in the aCSF to synaptically isolate RIP-Cre neurons
and minimize indirect effects of leptin.
ChR2 and CRACMViral Channelrhodopsin-2 expression and activation were similarly performed as previously described (Atasoy et al., 2008; Hauben-
sak et al., 2010; Higley et al., 2011; Zhang et al., 2007). Two to three weeks after viral injection, mice were euthanized for slice prep-
aration. ChR2-expressing neurons were identified by mCherry fluorescence emission using a custom filter set (excitation, 560AF55;
emission, 645AF75; dichroic, 595DP longpass; Omega), and then visually targeted with infrared differential interference optics. For
whole-cell current-clamp recordings, glass electrodes (3.5–5 MU) were filled with internal solution containing (in mM): 128 K gluco-
nate, 10 KCl, 10 HEPES, 1 EGTA, 1MgCl2, 0.3 CaCl2, 5 Na2ATP, 0.3 NaGTP, adjusted to pH 7.3 with KOH. In some RIP-Cre cells that
were detected with frequent spontaneous action potentials, we applied a current (�5 to �30 pA) to hyperpolarize the cell for obser-
vation of light-stimulated action potentials.
Photostimulation-evoked IPSCs in the PVH and RPa neurons were recorded in the whole cell voltage clampmode, with membrane
potential clamped at �60 mV. The intracellular solution contained the following (in mM): 140 CsCl, 1 BAPTA, 10 HEPES, 5 MgCl2,
5 Mg-ATP, 0.3 Na2GTP, and 10 lidocaine N-ethyl bromide (QX-314), pH 7.35 and 290 mOsm. Photostimulation was performed
with 1 mM kynurenic acid to block excitatory postsynaptic currents. All recordings were made using an Axopatch 200B amplifier,
and data were filtered at 2 kHz and digitized at 10 kHz. To photostimulate Channelrhodopsin2-positive fibers, a laser or LED light
source (473 nm; Opto Engine LLC; Thorlabs) was used. The blue light was focused on to the back aperture of the microscope objec-
tive, producing a wide-field exposure around the recorded cell of 10–20 mW/mm2. The light power at the specimen was measured
using an optical power meter PM100D (ThorLabs). The light output is controlled by a programmable pulse stimulator, Master-8 (AMPI
Co. ISRAEL) and the pClamp 10.2 software (AXON Instruments); 20 mM bicuculline was applied to the bath when light-responsive
neurons were recorded. Evoked IPSCs with short latency (%6 ms) upon light stimulation were considered as light-driven IPSCs.
As discussed by others, such currents are most likely monosynaptic (Petreanu et al., 2007).
Double-injection experiments were performed similarly as described (Haubensak et al., 2010). 30 nl retrograde green beads (Green
Retrobeads, Lumafluro Inc.) was stereotaxically injected into the NTS 5 days prior to the recordings. Beads-positive and adjacent
beads-negative neurons were patched for photostimulation recordings.
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Figure S1. Further Assessment of Energy Balance in Mice Lacking VGAT in RIP-Cre Neurons, Related to Figure 2
(A) Subcutaneous flank temperature measured with subcutaneously implanted temperature probes (mean ± SEM, n = 8).
(B) Immunohistochemistry for GFP (DAB, brown stain) in the hypothalamus (Bregma �0.48 mm) of Rip-Cre, lox-GFP transgenic mice.
(C) In situ hybridization for VgatmRNA (35S-labbeld cRNA probe, silver grains) in control (Vgatflox/flox) andRip-Cre, Vgatflox/flox littermates. Arrows indicate the SCN
with notable reduction of Vgat mRNA signal in Rip-Cre, Vgatflox/flox mice.
(D and E) Circadian rhythms of body temperature (Tb) over 30 days recorded in 2-month old control (Vgatflox/flox, n = 3) and Rip-Cre, Vgatflox/flox (n = 5) male
littermates using i.p. biotelemetry implants. The animals were housed under 12:12 light-dark cycles (LD) for 15 days, followed by exposure to constant darkness
(DD); Two representative actograms from each genotype are shown. Under LD conditions, the diurnal rhythm of Tb in Rip-Cre, Vgatflox/flox mice exhibited stable
entrainment with a phase angle that was comparable to that of the wild-types. Similarly, under DD conditions, Rip-Cre, Vgatflox/flox mice showed a persisting
consolidated ‘‘free-running’’ circadian Tb rhythm with a period close to or slightly shorter than 24 hr, indicating that deletion of GABA release from SCN RIP-Cre
neurons does not disturb circadian rhythms.
(F) Serum T4 level and (G) serum corticosterone level (mean ± SEM, n = 8).
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. S5
Figure S2. Energy Balance in Mice Lacking VGLUT2 in RIP-Cre Neurons, Related to Figure 2
(A and B) In situ hybridization for Vglut2 mRNA (35S-labbeld cRNA probe, silver grains) in the brain of (A) control (Vglut2flox/flox) and (B) Rip-Cre, Vglut2flox/flox
littermates.
(C) Body weight, (D) oxygen consumption, and (E) daily food intake of ad libitum chow-fed male mice at 3 months of age (mean ± SEM; n = 8–10).
S6 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
Figure S3. Leptin-Induced Energy Expenditure and Leptin-Responsive RIP-Cre Neurons in the Hypothalamus, Related to Figure 4
(A and B) Subcutaneous flank temperature of control (Vgatflox/flox) and Rip-Cre, Vgatflox/flox mice, upon (A) saline or (B) leptin (6 mg/Kg, i.p.) stimulation (mean ±
SEM, n = 8–10).
(C–N) Double immunohistochemistry for GFP (green), leptin-induced phosphorylated STAT3 (Tyr105, pSTAT3) (purple) in the ARC (C–E), VMH (F–H), DMH (I–K),
and SCN (L–N) ofRip-Cre, lox-GFPmice. Neuronswith co-expressedGFP and pSTAT3 (indicated by arrows) weremainly observed in the ARC and VMH,with few
in the DMH and none in the SCN. See also Figure 4I for quantification.
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. S7
Figure S4. Pharmacogenetic Activation of Arcuate RIP-Cre Neurons, Related to Figure 5
(A) A representative voltage tracing recorded in an ARCRIP-Cre neuron that was negative for mCherry fluorescence. The brain slice was prepared from aRip-Cre,
lox-GFP mouse that was injected with AAV-Flex-hM3Dq-mCherry virus into the ARC. 5 mM CNO was applied to the bath as indicated.
(B) Food intake over the first 4 hr following saline or CNO injection in AAV-Flex-hM3Dq-mCherry virus-injected Vgatflox/flox,(left), Rip-Cre (middle), and Rip-Cre,
Vgatflox/flox (right) mice (mean ± SEM, n = 8).
(C) Subcutaneous flank temperature following saline or CNO injection in AAV-Flex-hM3Dq-mCherry virus-injected Rip-Cre mice (mean ± SEM, n = 6).
S8 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
Figure S5. Leptin Action on ARC RIP-Cre Neurons, Related to Figure 4
(A) Diagram illustrating experimental design in Rip-Cre, lox-GFP mice.
(B) Fluorescent image indicating the injection site of retrograde red beads.
(C) Immunohistochemistry against GFP (green) and native fluorescence of retrograde red beads (red) in the ARC of beads-injected Rip-Cre, lox-GFP animals.
Arrows indicate RIP-Cre neurons that were labeled with both GFP and red beads (i.e., PVH-projecting RIP-Cre neurons). An adjacent RIP-Cre neuron that was
labeled with GFP only but not with red beads is also shown (i.e., a RIP-Cre neuron that does not project to the PVH). There were also many ARC neurons labeled
with red beads only but not with GFP (i.e., PVH-projecting, non-RIP-Cre neurons) (data not shown).
(D and E) Whole-cell current-clamp recordings were performed in ARC neurons labeled with (D) both GFP and red beads or with (E) GFP only. Synaptic blockers
(1 mM Kynurenate and 100 mM PTX) were included in aCSF to minimize leptin’s indirect effects. (D) Representative tracing of PVH-projecting ARC RIP-Cre
neurons: (D i) that were activated by leptin (membrane potential: from�60.7 ± 2.1 mV to�54.3 ± 1.7mV (n = 9, p < 0.001), firing rate: from 0.12 ± 0.09 Hz to 0.54 ±
0.23Hz (n = 9, p < 0.05); (D ii) that did not respond to leptin (membrane potential: from �59.4 ± 2.8 mV to �59.9 ± 2.8 mV [n = 6], firing rate: from 0.40 ± 0.28 Hz
to 0.42 ± 0.30 Hz [n = 6]). (E) Representative tracing of non-PVH-projecting ARC RIP-Cre neurons: (E i) that did not respond to leptin (membrane potential:
from �59.5 ± 2.0 mV to �60.1 ± 1.8 mV (n = 9), firing rate: from 0.9 ± 0.3 Hz to 1.0 ± 0.4 Hz (n = 9); (E ii) that were inhibited by leptin (membrane potential:
from �60.2 ± 1.1 mV to �66.7 ± 0.9 mV (n = 3, p < 0.001), firing rate: from of 0.72 ± 0.58Hz to 0.06 ± 0.03 Hz [n = 3]). (mean ± SEM, unpaired t test).
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. S9
Figure S6. Optogenetic Stimulation of ARC RIP-Cre Neurons, Related to Figure 6
(A–C) Voltage traces showing light-driven spikes in a current-clamped ARC RIP-Cre neuron with mCherry expression in acute brain slice. AAV-Flex-
ChR2(H134R)-mCherry virus was stereotaxically injected into the ARCofRip-Cre transgenicmice at 3–4weeks of age and patch-clamp analyseswere performed
3 weeks later. Light pulses (473 nm) were applied at the frequencies indicated above the traces. Light-evoked spikes at 4Hz (B) and at 10Hz (C) are shown in
a time-expanded view. These results demonstrate that ARC RIP-Cre neurons spike faithfully following ChR2-based photostimulation at a wide range of
frequencies. 0.5 Hz photostimulation was employed in Figures 6 and 7.
S10 Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc.
Figure S7. Retrograde Tracing Studies in the NTS and RPa, Related to Figure 7
(A) Diagram and fluorescent image indicating the injection site of retrograde green beads into the NTS/DMV and (B) retrogradely labeled beads-positive neurons.
(C) PVH neurons labeled with i.p. injected Fluoro-Gold and with NTS-injected retrograde green beads. No colocalization of Fluoro-Gold and green beads was
observed, indicating that the PVH neurons that project to the NTS are not neuroendocrine cells.
(D) Representative voltage tracing showing light-driven spikes in a mCherry+ AgRP neuron from AAV-Flex-ChR2(H134R)-mCherry virus-injected Agrp-ires-Cre
mice.
(E) Diagram and fluorescent image indicating the injection site of retrograde green beads into the RPa and (F and G) retrogradely labeled bead-positive neurons.
(H) Immunoreactivity of tdTomato (red) and native fluorescence of green beads (green) of Vgat-ires-Cre, lox-tdTomato mice with green beads injected into the
RPa. In summary, 1/51 bead-positive neurons in the MPO, 0/74 in the DMH, 3/46 in the PAG, and 0/44 in the DR, are tdTomato immunoreactive.
Cell 151, 645–657, October 26, 2012 ª2012 Elsevier Inc. S11
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