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Nat. Neurosci.; doi:10.1038/nn.3725

Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feedingJae Geun Kim, Shigetomo Suyama, Marco Koch, Sungho Jin, Pilar Argente-Arizon, Jesús Argente, Zhong-Wu Liu, Marcelo R Zimmer, Jin Kwon Jeong, Klara Szigeti-Buck, Yuanqing Gao, Cristina Garcia-Caceres, Chun-Xia Yi, Natalina Salmaso, Flora M Vaccarino, Julie Chowen, Sabrina Diano, Marcelo O Dietrich, Matthias H Tschöp & Tamas L Horvath

In the PDF version of the supplementary information initially published, Supplementary Figures 1a,b and 5a and the corresponding legends were missing. In the HTML version, the legends were present but the figure panels were missing. The errors have been corrected in the HTML and PDF versions of the supplementary information as of 10 June 2014.

Supplementary information Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding Jae   Geun   Kim1,   Shigetomo   Suyama1,   Marco   Koch1,   2,   Sungho   Jin1,   Pilar   Argente-­‐Arizon3,  Jesús  Argente3  Zhong-­‐Wu  Liu1,  Marcelo  R.  Zimmer1,  Jin  Kwon  Jeong1,4,  Klara  Szigeti-­‐Buck1,   Yuanqing   Gao5,   Cristina   Garcia-­‐Caceres5,   Chun-­‐Xia   Yi5,   Natalina  Salmaso6,   Flora   M.   Vaccarino6,7,   Julie   Chowen3,   Sabrina   Diano1,4,7,   Marcelo   O  Dietrich1,  Matthias  H.  Tschöp5  and  Tamas  L.  Horvath1,4,7   1Program in Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, USA. 2Institute of Anatomy, University of Leipzig, 04103 Leipzig, Germany. 3Hospital Infantil Universitario Niño Jesús, Department of Endocrinology, Instituto de Investigación La Princesa and Centro de Investigación Biomédica en Red de la Fistiopatología (CIBER) de Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain. 4Department of Obstetrics, Gynecology, and Reproductive Sciences, New Haven, Connecticut, USA. 5Institute for Diabetes and Obesity, Helmholtz Zentrum München & Technische Universität München, Germany. 6Child Study Center, Yale University School of Medicine, New Haven, Connecticut, USA. 7Department of Neurobiology, Yale University School of Medicine, Connecticut, USA.  

Nature Neuroscience: doi:10.1038/nn.3725

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Supplementary figure 1. Presence of leptin receptors in hypothalamic astrocytes. (a) Double

fluorescence labeling of the astrocyte marker GFAP (red) and leptin receptors (leptin receptor-driven

expression of EGFP, green) shows co-localization of GFAP-immunolabeling with EGFP-tagged

leptin receptor-containing profiles (white arrows) in the arcuate nucleus (Arc). Scale bar = 100 μm.

(b) The truncated leptin receptor (exon 17) allele was confirmed by in situ hybridization combined

with immunohistochemistry. Red fluorescence indicates GFAP-positive astrocyte and green

fluorescence indicates POMC neurons. White dots indicate mRNA signals of the leptin receptor

containing exon 17. White arrows indicate cells expressing mRNA of leptin receptors. White

arrowheads indicate leptin receptor-negative cells. Scale bar = 30 μm. (c) To further confirm

astrocyte-specific expression of leptin receptors, transcript of leptin receptor b (LepRb) was

amplified from RNA bound to the ribosomes selectively in astrocytes isolated from AldH-EGFP-

L10a mice and in AgRP neurons isolated from AgRP-cre:floxed Rpl22HA mice. To check purification

and contamination of the samples, we analyzed enrichment of AgRP (marker for AgRP purification),

NeuN (Marker for neuronal cell contamination) and s100β (marker for astrocyte contamination)

compared to that of input samples. Purple bar graphs represent enrichment of LepRb in the AgRP

neurons or astrocytes of the hypothalamus compared to that of input controls.

Nature Neuroscience: doi:10.1038/nn.3725

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Supplementary figure 2. Strategy of transgenic mice with selective impairment of leptin receptor

signaling in astrocyte. (a) Schematic drawing of floxed leptin receptor construct, which was used to

generate a mouse line allowing cell and time-specific knockout of leptin receptors in astrocytes. (b) RT-

PCR shows a deletion of exon 17 of the leptin receptor in primary astrocytes from GFAP-LepR KO mice.

Representative images were selected from at least 3 time repeated experiments.

Nature Neuroscience: doi:10.1038/nn.3725

Tamoxifen

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Supplementary figure 3. Verification of astrocyte-specific Cre-recombination. (a) Schematic

drawing of human GFAP-driven Cre promoter construct under control of estrogen receptors. (b) GFAP-

CreERT2 mice were crossed with tdTomato-loxP reporter mice to confirm successful Cre-mediated

recombination in GFAP-positive cells. (c) Ai14 tdTomato mice express red fluorescence in GFAP-

positive cells following cre-mediated recombination. tdTomato fluorescences were not co-localized with

signals of Iba-1, a biomarker for microglia or NeuN, a biomarker for neuron. White arrows indicate

double-labeled cells. Representative images were selected from at least 3 time repeated experiments.

Scale bar = 50 μm.

Nature Neuroscience: doi:10.1038/nn.3725

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Supplementary figure 4. Effect of astrocyte-specific deletion of leptin receptors on morphology of

astrocytes in the hippocampal CA3. (a) To confirm expression of leptin receptor in hippocampal

astrocytes, we performed ribosome profiling to isolated RNA bound to the ribosomes selectively from

hippocampal astrocytes and analyzed enrichment of LepRb. Purple bar graph represents enrichment of

LepRb in astrocytes of the hippocampus compared to that of input control. (b) Representative image of

GFAP-immunolabeling in the hippocampal CA3 of GFAP-LepR+/+ (+/+) or GFAP-LepR–/– (–/–) mice. (c

The number of GFAP-positive cells (n=5 slices for GFAP-LepR+/+ ; n=4 slices for GFAP-LepR–/–,

p=0.7150, t(7)=0.3803) and (d) length (n=8 cells for GFAP-LepR+/+; n=8 cells for GFAP-LepR–/–,

p>0.9999, t(14)=0.0) and (e) number (n=8 cells for GFAP-LepR+/+; n=8 cells for GFAP-LepR–/–, p=0.456

t(14)=0.7667) of astrocyte primary projections did not differ between GFAP-LepR+/+ and GFAP-LepR–/–

mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed

Student’s t-test.

Nature Neuroscience: doi:10.1038/nn.3725

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Supplementary figure 5. Leptin receptor signaling in astrocytes affects contact formation

between astrocytes and melanocortin cells. (a) Representative electron micrograph showing

astrocyte coverage (green pseudo-color and blue arrows) onto POMC-labeled cells. POMC cells of

GFAP-LepR–/– mice had less coverage of their perikaryal membranes by astrocytic processes

compared to controls. Scale bar = 1 μm. Coronal sections labeled with GFP in AgRP or POMC cells

were incubated with GFAP antibody. (b, d) Representative pictures of double-labeled AgRP or

POMC-GFP and GFAP-positive cells in the Arc of GFAP-LepR+/+ or GFAP-LepR–/– mice.

Percentage of (c) POMC (n=7 slices for GFAP-LepR+/+; n=6 slices for GFAP-LepR–/–, p<0.0001,

t(11)=8.226) or (e) AgRP cells (n=8 slices for GFAP-LepR+/+; n=8 slices for GFAP-LepR–/–,

p=0.0004, t(14)=4.672) contacted with GFAP-fiber signals was reduced in GFAP-LepR–/– mice.

White arrows indicate cells interacted with astrocyte fibers. ***, p<0.001 versus GFAP-LepR+/+

mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed

Student’s t-test. Scale bar = 100 μm.

+/+ _/_

Nature Neuroscience: doi:10.1038/nn.3725

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Supplementary figure 6. The probability distribution and average peak amplitude of miniature

postsynaptic currents on AgRP or POMC neurons. (a, b) Cummulative probability distribution and

average peak amplitude (inserts) of mIPSC or mEPSC onto AgRP neurons (Fig. 6a: n=9 cells for GFAP-

LepR+/+; n=9 cells for GFAP-LepR–/–, p=0.7715, t(798)=0.2906; Fig. 6b: n=9 cells for GFAP-LepR+/+;

n=9 cells for GFAP-LepR–/–, p=0.2788, t(798)=1.084). No-differences were observed between control

and GFAP-LepR–/– mice. (c) GFAP-LepR–/– mice showed a significantly increase in probability

distribution and average peak amplitude (inserts) of mIPSC onto POMC neurons (n=9 cells for GFAP-

LepR+/+; n=9 cells for GFAP-LepR–/–, p<0.0001, t(896)=7.604). (d) GFAP-LepR–/– mice revealed an

increase in probability distribution of mEPSC amplitude onto POMC neurons (n=23 cells for GFAP-

LepR+/+; n=25 cells for GFAP-LepR–/–, p<0.0001, t(3998)=5.255). **, p<0.01; ***, p<0.001 versus

GFAP-LepR+/+. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-

tailed Student’s t-test.

Nature Neuroscience: doi:10.1038/nn.3725

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Supplementary figure 7. Metabolic phenotypes of GFAP-LepR+/+ and GFAP-LepR–/– mice. Five-

week-old male mice were administrated tamoxifen (Tx) or vehicle (Veh) and then analyzed metabolic

phenotype at three-month-old age. GFAP-LepR–/– mice did not show significant changes of (a) body

weight (n=6 mice for GFAP-LepR+/+-veh; n=8 mice for GFAP-LepR+/+-tx; n=9 mice for GFAP-LepR–/–-

tx, p=0.3720, t(12)=0.9274 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.08, t(15)=1.874 for

GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx), (b) fat mass (n=6 mice for GFAP-LepR+/+-veh; n=6 mice

for GFAP-LepR+/+-tx; n=5 mice for GFAP-LepR–/–-tx, p=0.588, t(10)=0.5630 for GFAP-LepR+/+-veh

versus GFAP-LepR+/+-tx; p=0.1974, t(9)=1.392 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (c) lean

mass (n=6 mice for GFAP-LepR+/+-veh; n=6 mice for GFAP-LepR+/+-tx; n=5 mice for GFAP-LepR–/–-tx,

p=0.405, t(10)=0.8686 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.1410, t(9)=1.614 for

GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (d) food intake (n=5 mice for GFAP-LepR+/+-veh; n=7 mice

for GFAP-LepR+/+-tx; n=7 mice for GFAP-LepR–/–-tx, p=0.8471, t(10)=0.1979 for GFAP-LepR+/+-veh

versus GFAP-LepR+/+-tx; p=0.6162, t(12)=0.05146 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (e, f)

energy expenditure (n=8 mice for GFAP-LepR+/+; n=8 mice for GFAP-LepR–/–, p=0.8378, t(14)=0.2086

for light period; p=0.8641, t(14)=0.1743 for dark period; p=0.9966, t(14)=0.004295 for total) and (g, h)

physical activity (n=8 mice for GFAP-LepR+/+; n=8 mice for GFAP-LepR–/–, p=0.1184, t(14)=1.663 for

light period; p=0.2703, t(14)=1.148 for dark period; p=0.1692, t(14)=1.149 for total) when compared to

littermate control mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed

by two-tailed Student’s t-test. Nature Neuroscience: doi:10.1038/nn.3725

a:leptin+/+

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Supplementary fig. 8. Representative images of Fos-positive POMC or AgRP neurons in the arcuate of GFAP-LepR+/+ and GFAP-LepR- /- mice. (a) Representative

images show double labeled POMC-GFP and Fos cells in the Arc of GFAP-LepR+/+ and

GFAP-LepR- /- mice. Scale bar = 100 μm. (b) Representative images show double

labeled AgRP-GFP and Fos cells in the Arc of GFAP-LepR+/+ and GFAP-LepR- /- mice.

Scale bar = 100 μm.

Nature Neuroscience: doi:10.1038/nn.3725