Salmon calcitonin distributes into the arcuate nucleus to a subset of NPY neurons in mice

Journal Pre-proof Salmon calcitonin distributes into the arcuate nucleus in mice to a subset of NPY neurons Hannah Louise Zakariassen, Linu Mary John, Jens Lykkesfeldt, Kirsten Raun, Tine Glendorf, Lauge Schaffer, Sofia Lundh, Anna Secher, Thomas Alexander Lutz, Christelle Le Foll PII:

S0028-3908(20)30053-8

DOI:

https://doi.org/10.1016/j.neuropharm.2020.107987

Reference:

NP 107987

To appear in:

Neuropharmacology

Received Date: 8 July 2019 Revised Date:

28 January 2020

Accepted Date: 2 February 2020

Please cite this article as: Zakariassen, H.L., John, L.M., Lykkesfeldt, J., Raun, K., Glendorf, T., Schaffer, L., Lundh, S., Secher, A., Lutz, T.A., Le Foll, C., Salmon calcitonin distributes into the arcuate nucleus in mice to a subset of NPY neurons, Neuropharmacology (2020), doi: https://doi.org/10.1016/ j.neuropharm.2020.107987. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

1

Salmon calcitonin distributes into the arcuate nucleus in mice to a subset of NPY neurons

2 3 4 5

Hannah Louise Zakariassen1,2, Linu Mary John2, Jens Lykkesfeldt1, Kirsten Raun2, Tine

6

Glendorf4, †Lauge Schaffer5, Sofia Lundh3, Anna Secher4, Thomas Alexander Lutz6 and

7

Christelle Le Foll6,*

8 9

1

Section of Experimental Animal Models, Department of Veterinary and Animal Science, Faculty of

10

Health and Medical Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark.

11

2

12

Chemistry, Novo Nordisk A/S, 2760 Måløv, Denmark.

13

University of Zurich, CH-8057 Zurich, Switzerland

Obesity Pharmacology,

3

Pathology and Imaging, and

4

Diabetes Pharmacology 2, 6

5

Research

Institute of Veterinary Physiology,

14 15

*Corresponding authors: [email protected]

16

†

Deceased on 27th August 2019

17 18

Number of figures: 6

19

Number of tables: 1

20

Number of videos (extended data): 3

21

Supplementary figures: 2

1

22

ABSTRACT

23

The amylin receptor (AMY) and calcitonin receptor (CTR) agonists induce acute suppression of food

24

intake in rodents by binding to receptors in the area postrema (AP) and potentially by targeting

25

arcuate (ARC) neurons directly. Salmon calcitonin (sCT) induces more potent, longer lasting

26

anorectic effects compared to amylin. We thus aimed to investigate whether AMY /CTR agonists

27

target key neuronal populations in the ARC, and whether differing brain distribution patterns could

28

mediate the observed differences in efficacy with sCT and amylin treatment. Brains were examined

29

by whole brain 3D imaging and confocal microscopy following subcutaneous administration of

30

fluorescently labelled peptides to male and female mice. We found that sCT, but not amylin,

31

internalizes into a subset of ARC NPY neurons, along with an unknown subset of ARC, AP and

32

dorsal vagal motor nucleus cells. ARC POMC neurons were not targeted. Furthermore, amylin and

33

sCT displayed similar distribution patterns when binding to receptors in the AP, the organum

34

vasculosum of the lamina terminalis (OVLT) and the ARC. Amylin was distributed within the

35

median eminence with only specs of sCT being present in this region, however amylin was only

36

detectable 10 minutes after injection while sCT displayed a residence time of up to 2 hours post

37

injection. We conclude that AMY /CTR agonists bind to receptors in a subset of ARC NPY neurons

38

and in circumventricular organs. Furthermore, the more sustained and greater anorectic efficacy of

39

sCT compared to rat amylin is not attributable to differences in brain distribution patterns but may

40

more likely be explained by greater potency at both CTR and AMY3.

41 42

KEYWORDS: amylin; agonist; whole-brain 3D imaging; area postrema; NPY

2

43 44

HIGHLIGHTS •

45

Whole brain 3D imaging showed that amylin and sCT present similar distribution patterns in the AP, the OVLT and the ARC.

46

•

sCT internalizes into a subset of ARC NPY neurons as well as in AP and DVM nucleus cells.

47

•

Peripherally injected AMY /CTR agonists target specific neuronal populations to induce their

48

metabolic effects.

3

49

1. INTRODUCTION

50 51

The pancreatic hormone amylin is secreted postprandially and induces satiation (Lutz et al., 1995) by

52

binding to amylin receptors (AMY), the constituents of which are distributed throughout the brain

53

(Becskei et al., 2004; Hilton et al., 1995; Nakamoto et al., 2000; Oliver et al., 2001). AMY are

54

composed of two components, the calcitonin receptor (CTR) and one of three receptor activity

55

modifying proteins (RAMP1-3) (Poyner et al., 2002). While amylin binds selectively to AMY,

56

salmon calcitonin binds to both CTR and AMY (Christopoulos et al., 1999; Tilakaratne et al., 2000).

57

Access of circulating factors to the brain are prevented by the blood brain barrier (BBB) except for

58

specific BBB free regions of the brain, namely the circumventricular organs (CVO, e.g., median

59

eminence (ME), subfornical organ (SFO)) that are infiltrated by highly permeable vasculature

60

(Abbott et al., 2010). Additionally, the ARC has been shown to be permeable to circulating factors

61

such as ghrelin by diffusion through the fenestrated capillaries of the ME that branch into the

62

ventromedial region of the arcuate hypothalamus to bind to cognate receptors in NPY/AgRP

63

(neuropeptide Y/ agouti-related protein) and POMC (pro-opiomelanocortin) expressing neurons

64

(Schaeffer et al., 2013). AMY /CTR agonists induce acute anorectic effects by activating neurons in

65

the area postrema (AP), a CVO in the hindbrain. AMY in the AP seem to be necessary for amylin’s

66

satiating effect (Braegger et al., 2014; Lutz et al., 2001; Riediger et al., 2004). From the AP, neuronal

67

projections send signals to other areas in the brain such as the nucleus of solitary tract (NTS), lateral

68

parabrachial nucleus (LPBN) and the lateral hypothalamic area (LHA) (Potes et al., 2010). In the

69

hypothalamus, amylin has been shown to potentiate leptin signaling (Dunn-Meynell et al., 2016;

70

Turek et al., 2010) and regulate several neuropeptide systems involved in energy balance regulation

71

(Barth et al., 2003; Roth et al., 2006).

72

POMC and NPY/AgRP co-expressing neurons in the arcuate nucleus (ARC) exert homeostatic

73

control of feeding behavior by inducing opposing effects of anorexigenic or orexigenic activity, 4

74

respectively (Loh et al., 2015; Yeo and Heisler, 2012). Recently, we found that amylin induces

75

pERK signaling in POMC but not NPY neurons independently of the AP (Lutz et al., 2018), but

76

others have shown that POMC neurons only express low levels of CTRs while NPY/AgRP neurons

77

express high levels (Campbell et al., 2017; Lam et al., 2017; Pan et al., 2018). Hence, these findings

78

indicate that AMY /CTR agonists target neuronal populations in the hypothalamus, but whether

79

NPY/AgRP and/or POMC neurons are targeted directly remains unclear. Treatment with both amylin

80

or calcitonin peptides decreases body weight in rodents (Andreassen et al., 2014; Feigh et al., 2011;

81

Roth et al., 2007; Trevaskis et al., 2010). Both rat amylin and salmon calcitonin have short half-lives

82

of approximately 10 and 30 minutes, respectively (Chaturvedula et al., 2005; Young et al., 1996), but

83

only salmon calcitonin induces a robust acute anorexic response past the reported half-life in plasma

84

(Reidelberger et al., 2001; Reidelberger et al., 2002). This has been suggested to be due to

85

irreversible binding of salmon calcitonin to the CTR (Houssami et al., 1994) and observed

86

differences in potency on cognate receptors. Nevertheless, differences in activation and regulation of

87

CNS signaling pathways have been reported (Braegger et al., 2014; Whiting et al., 2017), although

88

such differences have not been systematically studied.

89

Based on these earlier findings, we aimed to investigate whether fluorescently labelled rat amylin

90

and salmon calcitonin bind directly to POMC and/or NPY/AgRP neurons in the ARC, and if the two

91

peptides have similar distribution patterns within the brain to evaluate whether this could account for

92

part of the differences in acute anorectic effect. We hypothesized that both peptides bind to ARC

93

NPY/AgRP and POMC neurons along with cells in the AP, and that the peptides would show

94

differing distribution patterns. To test this hypothesis, wild type and transgenic mice models NPY-

95

GFP and POMC-cre:ERT2: td-tomato were used.

5

96

2. RESEARCH DESIGN AND METHODS

97 98 99

2.1. Peptides Native peptides

100

Novo Nordisk A/S (Måløv, Denmark) synthesized the native salmon calcitonin in an acetate buffer.

101

Native rat amylin (H9475 Bachem AG, Bubendorf, Switzerland) was diluted in saline at a stock

102

concentration of 0.25 mmol/L and further diluted at 10 nmol/ml in acetate buffer (pH= 4.0; 5 mM

103

acetate; 240 mM propylene glycol, 0.007% tween 20).

104

Synthesis of fluorescently labeled ligands

105

Novo Nordisk A/S (Måløv, Denmark) also synthesized the fluorescently labelled peptides.

106

Fluorescently labelled salmon calcitonin and rat amylin was synthesized by conjugating Alexa fluor

107

750-NHS ester (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) to salmon calcitonin

108

(sCT750), Cyanin 7-NHS ester (Amersham by GE Healthcare Life Sciences, Chicago, IL, USA) to rat

109

amylin (rAMYCy7) and Cyanin 5-NHS ester (Amersham by GE Healthcare Life Sciences) to salmon

110

calcitonin (sCTCy5) and rat amylin (rAMYCy5).

111

The fluorescently labelled peptides, described in these experiments, were purified from excess

112

labelling agent by gel filtration on PD10 columns with isocratic elution of 20 mM NH4HCO3. The

113

fractions containing the pure peptide were freeze dried and used in the experiments. The labelled and

114

unlabeled peptide were validated by LC-MS and confirmed the presence of only one fluorophore per

115

peptide. sCTCy5 and sCT750 were confirmed to be single compounds, as the fluorophore sits on the N-

116

terminal amino group while rAMYCy5and rAMYCy7 may be a mixture of regioisomers modified at the

117

N-terminal or the epsilon-amino group of the lysine. All fluorescently labelled peptides were diluted

118

in acetate buffer. Control groups were dosed with saline or acetate buffer.

119

6

120

2.2. Animal husbandry and diet

121

Mice were group housed in temperature controlled (21 ± 2°C) room on a 12:12 h light:dark schedule

122

with ad libitum access to standard chow (no. 3436; Provimi Kliba, Kaiseraugst, Switzerland) and

123

water before and during experiments, unless otherwise stated. Animals were housed in an enriched

124

environment and handled before procedures. The experiments were carried out in accordance with

125

the EU Directive 2010/63/EU of the 22 September 2010 on the protection of animals used for

126

scientific purposes and approved by the Veterinary Office of the Canton Zurich (no. 102/2018) or the

127

Animal Experimentation Inspectorate, Ministry of Environment and Food, Denmark (no. 2012-15-

128

2934-0000069 and no. 2014-15-0201-00388). Studies investigating the efficacy of sCT750 and

129

localization of CTR and POMC in the mouse hypothalamus were performed at Novo Nordisk A/S

130

animal facilities in Måløv, Denmark, while all other studies were performed in the animal facilities at

131

the Institute for Veterinary Physiology, University of Zurich Switzerland.

132 133

2.3. Efficacy of fluorophore conjugated compounds on acute food intake suppression

134

Adult C57BL/6J male mice were purchased from Taconic (Lille Skensved, Denmark), fed a low-fat

135

diet (D12450B, Research Diets Inc., New Brunswick, NJ, USA) and acclimated to BioDAQ cages

136

for 2 weeks before study initiation (single housed with transparent divider with holes in a

137

temperature-controlled room (23 ± 2°C) on a 12-h light/12-h dark light cycle). Before study initiation

138

mice were randomized into three groups (n = 6 – 8), fasted for 4 hours before dark and injected

139

subcutaneously (SC) with vehicle, salmon calcitonin 100 nmol/kg or sCT750 100 nmol/kg 20 minutes

140

before dark initiation. At dark onset, food was returned, and food intake measured continuously by

141

the BioDAQ system (Research Diets Inc.) for 24 hours. After 24 hours, animals were sacrificed by

142

CO2 inhalation.

143

Adult male C57Bl6JRj mice (Elevage Janvier, France) fed a standard chow diet (no. 3436; Provimi

144

Kliba) were acclimated to BioDAQ cages for 1.5 weeks (temperature-controlled room (21 ± 2°C) on 7

145

a 12-h light/12-h dark light cycle). The mice were used in three separate experiments to validate the

146

in vivo efficacy of rAMYCy5, rAMYCy7 and sCTCy5: experiment 1) vehicle, rat amylin 100 nmol/kg or

147

rAMYCy5 100 nmol/kg; experiment 2) vehicle, rat amylin 300 nmol/kg or rAMYCy7 300 nmol/kg;

148

experiment 3) vehicle, salmon calcitonin 100 nmol/kg or sCTCy5 100 nmol/kg. Animals were

149

weighed before each experiment, randomized to new treatment groups (n = 8/group) and fasted for

150

12 hours before SC injection of peptides right before dark onset. At dark onset, food was returned,

151

and food intake measured every 15 min for 24 hours. A wash out period of 4 – 6 days was set

152

between each experiment. Due to unexpected errors in food intake measurements, one mouse was

153

excluded from the rat amylin group in experiment 1, one mouse was excluded from the rAMYCy7

154

group in experiment 2 and two mice were excluded from the vehicle group in experiment 3 before

155

statistical analysis.

156 157

2.4. In vitro validation of the fluorophore conjugated compounds

158

A BHK cell line was stably transfected with the human calcitonin receptor and a cAMP responsive

159

element (CRE) luciferase reporter gene. The cell line was further transfected with receptor modifying

160

protein 3 (RAMP3) thus generating the human amylin 3 receptor. When performing the assays,

161

frozen aliquots of the above mentioned BHK cell lines were thawed, washed in seeding medium

162

(DMEM with phenol red (no. 31966-021; Gibco ThermoFisher Scientific), 10% (v/v) FBS (no.

163

16140-071; Gibco) and 1% (v/v) Penicillin-Streptomycin (no. 15140-122; Gibco) and resuspended in

164

seeding medium. Cells were plated into 384-well plates at 4000 cells/well in a volume of 40 µL and

165

incubated O/N at 37°C and 5% CO2. On the following day, reference and test compounds were

166

prediluted to appropriate concentrations (approximately 10-100 nM) in assay buffer (DMEM w/o

167

phenol red (no. 11880-028; Gibco), 10 mM HEPES (no. 15630-056; Gibco), 1X GlutaMAX™ (no.

168

35020-038; Gibco), 0.1% (w/v) ovalbumin (no. A5503; Sigma Aldrich) and 1% (w/v) HSA (no.

169

A1887; Sigma Aldrich)) and transferred to 96-well plates where 7-fold serial dilutions of each 8

170

compound were performed in duplicates using a Biomek i7 liquid handler. Cells were washed three

171

times in assay medium without HSA and 30 µL of each dilution of reference and test compounds

172

were transferred to the 384-well assay plates. The plates were incubated for 3h at 37°C and 5% CO2

173

and 30 µL of steadyliteplus reagent (no.6066759; PerkinElmer) added. While protected from light,

174

plates were incubated with shaking for 5 minutes at 300 rpm and incubated for an additional 30

175

minutes at room temperature before measuring luminescence on a Synergy 2 (BioTek) plate reader.

176

Data were imported into GraphPad Prism (version 8.0.2 for Windows, GraphPad Software, San

177

Diego, California, USA) and EC50-values determined using non-linear regression (log(agonist) vs.

178

response (four parameters) with a Hill slope of 1.5 and a shared bottom for all curves). rAMYCy7

179

could not be assessed in vitro due to its low purity.

180 181

2.5. Salmon calcitonin and rat amylin binding to arcuate and hindbrain single cells

182

NPY-hrGFP [B6.FVB-Tg(Npy-hrGFP)1Lowl/J, no. 006417; The Jackson Laboratory] and POMC-

183

Cre:ERT2 (Gift from Pr. Joel Elmquist, UT Southwestern, USA) (Berglund et al., 2013) were bred in

184

the animal facilities at the Institute for Veterinary Physiology. NPY-hrGFP allows for identification

185

of NPY neurons without additional immunohistochemistry (IHC) staining due to endogenous GFP

186

fluorescence driven by the NPY promoter. Similarly, the POMC-Cre:ERT2 mice were crossed with

187

tdTomato reporter mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, no. 007914; The

188

Jackson Laboratory) to allow for identification of POMC neurons expressing tdTomato induced by

189

tamoxifen injection. POMC-Cre:ERT2::tdTomato mice were treated daily for 5 days SC with

190

tamoxifen diluted in corn oil 100 mg/kg (T5648, Sigma Aldrich, St. Louis, MO, USA) to induce the

191

Cre recombination in POMC neurons. Mice were randomized into groups (n = 2 – 4/group/strain)

192

before study initiation and injected with either sCTCy5 or rAMYCy5 in the middle of the light period.

193

Both male and female were included in the study (5 males and 2 females from each strain). The

194

selection of dosing regimens was based on pilot studies performed with the fluorescently labelled 9

195

peptides. sCTCy5 100 nmol/kg was injected SC twice with 2 hours in between injections, before

196

termination 2 hours after the last injection. rAMYCy5 100 nmol/kg was injected SC twice with 30

197

minutes in between injections, before termination 10 minutes after the last injection. At termination,

198

mice were perfused with 0.1 mol/L phosphate buffer (PB) followed by 4% paraformaldehyde (PFA)

199

in 0.1 mol/l phosphate buffer (pH 7.2). Brains were post-fixed in 4 % PFA overnight and

200

cryoprotected in 20 % sucrose PB overnight and frozen in hexane before storage at –80°C.

201 202

2.6. Localization of calcitonin receptors on POMC and NPY neurons in the mouse ARC

203

Adult C57BL/6J male mice were purchased from Charles River Laboratories (Lyon, France), housed

204

at temperature-controlled room (23 ± 2°C) on a 12-h light/12-h dark light cycle and fed a low-fat diet

205

(D12450K, Research Diets Inc.). Animals were anaesthetized by isoflurane and were euthanized by

206

cardiac perfusion with a peristaltic pump (NaCl for 2 – 3 minutes and then 10% neutral buffered

207

formalin (NBF) for 10 minutes) (n = 4). The brains were removed and post-fixed in 10% NBF

208

overnight at room temperature.

209 210

2.7. Salmon calcitonin and rat amylin distribution to the whole brain

211

Adult male and female wild type mice (background 129S2/Sv issued from in-house colony) were

212

randomized into groups (n = 3 – 5/group) before study initiation, and injected with saline, sCT750 or

213

rAMYCy7 in the middle of the light period. Dosing regimen was based on findings from the above-

214

described study. sCT750 100 nmol/kg was injected SC twice with 2 hours between injections, before

215

termination at 30 minutes or 2 hours after the last injection. rAMYCy7 300 nmol/kg was injected SC

216

twice with 5 minutes between injections before termination 10 minutes after the last injection. At

217

termination, mice were perfused as described above, brains post-fixed in 4 % PFA for ~1 week,

218

transferred to PBS and stored at 5 °C.

219 10

220

2.8. Analysis of brains from the ARC and hindbrain cell binding study

221

2.8.1.

Brain sectioning

222

sCTCy5 and rAMYCy5 brains were cryosectioned into 20 µm thick coronal sections onto Superfrost

223

Plus slides (Life Technologies Europe, Zug, Switzerland), isolating the ARC and the AP/NTS/Dorsal

224

motor nucleus of the vagus (DMV) (bregma -1.23 mm to -2.53 mm and bregma -7.31 to -7.91 mm in

225

“Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates” (Paxinos, 2013), respectively).

226

2.8.2. Immunohistochemistry (IHC)

227

DAPI IHC: All sections were stained with the nuclear dye DAPI (diamidino-2-phenylindole,

228

Thermo Fischer Scientific, Waltham, MA, USA). DAPI was diluted in PBS to a concentration of 25

229

mg/ml, and sections stained. Sections where then rinsed in PBS and coverslipped using

230

VECTASHIELD HardSet mounting medium (Vectorlabs, Servion, Switzerland).

231

POMC IHC: Comparison between POMC IHC and td-Tomato endogenous fluorescence was

232

assessed on slide from POMC-Cre:ERT2:td-tomato mice dosed with sCTCy5. Brain sections were

233

washed with 0.02M Potassium PBS (KPBS) for 30 min and blocked for overnight at 4°C (4% NDS,

234

1% bovine serum albumin (BSA), 0.4% Triton X-100 in KPBS). Sections were then incubated for 48

235

h at 4°C in blocking solution containing rabbit anti-POMC antibody (1:1.000; H-029-30, Phoenix

236

Europe, Karlsruhe, Germany). Following rinses, sections were placed in Alexa-Fluor 488 donkey

237

anti-rabbit for 2 hours (1:200; Jackson ImmunoResearch, Luzern, Switzerland), counterstained with

238

DAPI (Life Technologies Europe) and coverslipped using Vectashield Hardset mounting medium

239

(Vectorlabs, Servion, Switzerland) (Lutz et al., 2018).

240

2.8.3. Image analysis and quantitative analysis of fluorescently labelled cells in the ARC

241

Three sections from the ARC and from the AP/NTS/DMV areas of each animal were acquired using

242

Zeiss SP8 confocal system equipped with a 20X/0.75 objective performing sequential scans (DAPI:

243

HyD1 405, laser 7% with 100% gain; hrGFP: HyD3 552, laser 2% with 100% gain; tdTomato: HyD3

244

552, laser 2% with 20% gain; Cyanin 5: HyD3 638, laser 67% with 100% gain; Alexa Fluor 488: 11

245

HyD3 552, laser 5% with 15% gain; all scans: zoom 1, pinhole 1, Z-stack 20 µm, and step of 0.5

246

µm). Images from selected sections were furthermore acquired with an oil-immersed 63X objective

247

performing sequential tile scans (same settings as above).

248

Co-localization of Cyanin 5 with hrGFP/TdTomato: Image analysis was performed using Imaris

249

9.2.1 software (Bitplane AG, Zürich, Switzerland). The average count/ARC section was calculated

250

from three ARC sections for each animal. Quantification and co-localization of hrGFP/tdTomato

251

with Cyanin5 was performed using the spots function and spot co-localization function. hrGFP+,

252

tdTomato+ and Cyanin 5+ cells were defined as signal positive spots co-localizing or lying right

253

adjacent to a DAPI+ spot (spot size hrGFP/tdTomato/Cyanin 5: 12 µm; spot size DAPI: 8 µm; co-

254

localization threshold: 10 µm). Co-localization of hrGFP+/tdTomato+ cells with Cyanin 5+ cells was

255

defined as ≥40 % overlap of spots (co-localization threshold: 5 µm). Results are reported as average

256

count/section (means) and % co-localization (means).

257

Comparison between POMC IHC and td-tomato: Labelled cells were imaged using an L2 Imager

258

upright microscope (Zeiss, Germany). Images of single and double labelled cells were counted using

259

ImageJ (NIH, Bethesda, MD, USA) to allow for quantification. Three consecutive sections as

260

verified by the DAPI counterstain were used for quantification. To ensure similar imaging conditions

261

for all images, the same microscope set-up and acquisition settings were used to acquire all images

262

within the same experiment. Results are reported as average count/section (means) and % co-

263

localization (means). Co-localization of POMC IHC and sCTCy5 could not be performed as no

264

Cyanin 5 signal was evident after IHC processing.

265 266 267

2.9. Analysis of brains for the localization of calcitonin receptors, NPY and POMC in the ARC

268

Brains were paraffin embedded and the ARC (bregma -1.23 mm to -2.53 mm in “Paxinos and

269

Franklin's the Mouse Brain in Stereotaxic Coordinates” (Paxinos, 2013), respectively) cryosectioned 12

270

into 4.5 µm thick coronal sections. Duplex TSA+-based fluorescent in situ hybridization (ISH) for

271

CTR and POMC or NPY was performed on a Bond RX Fully Automated Research Stainer (Leica

272

Biosystems, Wetzlar, Germany). After bake and dewax (30 minutes at 60ºC) and ISH target retrieval

273

(15 minutes of ACD enzyme protease III and 15 minutes of ER2 protease treatment at 95ºC

274

(Advanced Cell Diagnostics, Newark, CA, USA)), the mRNA signal of interest was detected using

275

the RNAscope® LS Multiplex Fluorescent Reagent Kit (Cat No. 322800, Advanced Cell

276

Diagnostics). Specific probes that target mouse CTR, POMC or NPY (RNAscope® 2.5 LS Probe-

277

Mm-Calcr, Cat No. 494078; RNAscope® 2.5 LS Probe-Mm-Pomc-C2, Cat No. 314088-C2;

278

RNAscope® 2.5 LS Probe- Mm-Npy-C2, Cat No. 313328-C2 , Advanced Cell Diagnostics) were

279

used in combination with TSA+ Cy3 or Cy5 amplification systems, according to the manufacturer’s

280

instructions (Advanced Cell Diagnostics; PerkinElmer, Waltham, WA, USA).The slides were

281

counterstained with DAPI (Advanced Cell Diagnostics) and mounted in Fluorescent mounting

282

medium (S3023, Dako, Glostrup, Denmark) before being scanned in an Olympus VS120 slide

283

scanner using a 20x (NA 0.75, 0.33 µm/pixel) objective. 2 – 3 ARC sections were evaluated from

284

each mouse.

285 286

2.10. Analysis of brains from the whole brain distribution study

287

2.10.1. Tissue clearing and whole brain light sheet fluorescence microscopy (LSFM)

288

The sCT750 and rAMYCy7 brains were cleared using a modification of the tetrahydrofuran (THF)

289

clearing protocol (Ertürk et al., 2012). Briefly, brain tissue was dehydrated in THF diluted in dH2O

290

(w/v) 30/50/70/80/96%/2 x 100 % 6 – 12 hours for each step. The brains were subsequently cleared

291

in diBenzylether (DBE) for 6 hours in room temperature. Brains were imaged by LSFM. Image

292

stacks (16-bit tif) were acquired using the UltraMicroscope I or II LSFM system (LaVision Biotec,

293

Bielefeld, Germany) equipped with an Andor Neo 5.5 scmos camera (Andor Technology Ltd.,

294

Belfast, UK) and a SuperK Extreme EXR-15 laser (NKT Photonics, Birkerød, Denmark), with the 13

295

ImSpectorPro software (Lavision Biotec). Step size was set to 10 µm with a 0.8 X magnification.

296

Data acquisition was performed using a 545/25 nm excitation filter and 605/70 nm emission filter for

297

imaging auto-fluorescence and a 710/75 nm excitation filter and a 775/40 nm emission filter for

298

imaging specific signals (sCT750 and rAMYCy7). For quantification of signal intensity, images were

299

captured from all brains by the UltraMicroscope 1, and high-resolution images were captured from

300

selected brains with the UltraMicroscope II.

301

2.10.2. Quantification of brain distribution

302

Image analysis was performed utilizing the Imaris 7.6.5 software (Bitplane AG). Before

303

quantification of brain distribution, spectral unmixing was performed as described earlier (Salinas et

304

al., 2018) to minimize the contribution of tissue auto-fluorescence to the specific sCT750 and

305

rAMYCy7 distribution signals. In brief, the estimated auto-fluorescence contribution in the specific

306

channel was calculated and removed based on ratios of voxel intensities between selected voxels in

307

the unspecific channel, and corresponding voxel in the specific channel. A ratio was computed for 40

308

sets of voxels selected from the histogram of the unspecific channel. To determine regions which

309

displayed consistent specific signal, each brain was mapped to an integrated brain atlas and average

310

3D signals combining individual brain samples constructed for each group. Quantification of specific

311

(sCT750 and rAMYCy7) signal intensity in the ARC/ME and AP was performed by manual

312

segmentation of regions with the contour surfaces function in Imaris 7.6.5 (Bitplane AG). The total

313

signal intensity within each region of interest from the specific unmix channel was utilized for

314

analysis. The results are reported as fold changes (means ± SEM).

315 316

2.11. Statistical analysis

317

Differences in cumulative food intake over time in the validation of fluorophore conjugated peptides

318

study were tested with repeated measures ANOVA with an autocorrelation matrix, followed by a

319

Tukey’s post hoc test using SAS enterprise Guide 7.1 software (SAS Institute Inc., Cary, NC, USA). 14

320

Differences between groups in specific signal intensity was tested using a Kruskal Wallis analysis

321

followed by a Dunn’s multiple comparisons test or Mann Whitney test using GraphPad Prism 7

322

software (GraphPad Software, La Jolla, CA, USA). A p-value less than 0.05 was considered

323

statistically significant.

15

324

3. RESULTS

325 326

3.1. In vitro potency of fluorescently labelled peptides

327

To ensure that the fluorophore conjugation did not affect the receptor activation properties of rat

328

amylin and salmon calcitonin, in vitro potency assays were undertaken.

329

In cells transfected with the human amylin receptor 3, sCT750 and sCTCy5 displayed EC50-values in a

330

range similar to that of native sCT (Table 1, Suppl. Table 1, Suppl. Fig. 1) whereas rAMYCy5

331

presented a decreased potency compared to pramlintide even though it did not reach significance

332

(Suppl. Table 1). In cells transfected with the human calcitonin receptor, sCT750 and sCTCy5 also

333

displayed EC50-values comparable to that of native sCT, whereas rAMYCy5 and pramlintide

334

expectedly displayed much lower potencies since this cell type did not express RAMP3 (Table 1,

335

Suppl. Table 1).

336 337

3.2. Fluorescently labelled peptides retain anorectic efficacy

338

To ensure that fluorophore conjugation did not eliminate the anorectic properties of rat amylin and

339

salmon calcitonin, acute food intake measurements were compared between fluorescently labelled

340

and native compounds. sCTCy5 and rAMYCy5 had similar effects on suppressing food intake as the

341

native peptides, demonstrating intact in vivo efficacy (Fig 1A and B). Likewise, the efficacy of

342

sCT750 and rAMYCy7 to suppress food intake was on par with that of the native compounds, although

343

the effect of sCT750 was attenuated compared to native salmon calcitonin towards the end of the dark

344

phase (Fig 1C – D). Hence, the acute in vivo efficacy of the compounds was maintained, or slightly

345

attenuated, after fluorescent label conjugation.

346

16

347

3.3. Salmon calcitonin is internalized into a subset of ARC NPY, AP and DMV cells

348

Evaluation of ARC sections from hrGFP-NPY mice treated with sCTCy5 and terminated 2 hours after

349

the last injection revealed that an average of approximately 16 cells/section were sCTCy5 positive in

350

the sCTCy5 group. Furthermore, the sCTCy5 signal was found to be internalized into ARC cells. When

351

evaluated for co-localization with NPY, co-labelling for both sCTCy5 and NPY was observed in 7.5%

352

of all NPY+ neurons and 47 % of all sCTCy5 cells (Fig 2A, B, D and F). Similarly, on average about

353

14 cells/section from the ARC of POMC-Cre:ERT2::tdTomato mice were sCTCy5 positive 2 hours

354

after the last injection, but no co-localization with POMC positive neurons were identified (Fig 2C,

355

G and H). No rAMYCy5 positive cells were identified in either the hrGFP-NPY or POMC-

356

Cre:ERT2::td-tomato mice treated with rAMYCy5 and terminated 10 min post last injection (Fig 2A,

357

B, C and E).

358

Since a POMC-Cre:ERT2::td-tomato mouse model was used, a comparison between POMC td-

359

tomato expression and POMC IHC was performed. The comparison between endogenous POMC in

360

POMC-Cre:ERT2::td-tomato mouse and POMC IHC revealed that the number of POMC neurons was

361

similar between the two stainings, however, the percentage of co-localization of POMC IHC and

362

POMC::td-tomato was around 65-70% suggesting discrepancies between the two POMC labelling

363

techniques similar to what has been shown previously (Padilla et al., 2012; Rau et al., 2018) (Fig 3A

364

– C). No Cy5 fluorescent signal was preserved after POMC IHC staining procedure, whereby co-

365

localization between sCTCy5 and POMC IHC could not be performed. Earlier single cell sequencing

366

investigations suggest that POMC neurons only express low levels of the CTRs in the ARC and ME

367

unlike NPY neurons which display high CTR expression levels (Campbell et al., 2017; Lam et al.,

368

2017; Pan et al., 2018). We therefore investigated whether expression of the CTR co-localized with

369

POMC or NPY expressing neurons in sections from wild type mice. In line with earlier observations,

370

only few 25% of POMC neurons displayed CTR expression, while almost all NPY neurons had

371

abundant CTR expression (Fig 4A – C). 17

372

AP/NTS/DMV sections from hrGFP-NPY and POMC-Cre:ERT2::td-tomato mice treated with sCTCy5

373

or rAMYCy5 were also evaluated for binding to single cells. sCTCy5 positive cells were identified in

374

all AP sections from both mouse strains 2 hours post last injection, with the sCTCy5 being

375

internalized into cells (Fig 5A). No sCTCy5 signal was found in the NTS of any mice, but intra-

376

cellular sCTCy5 was identified in the DMV of most of sections from both mouse strains albeit with

377

lower signal intensity compared to the AP (Fig 5B). No rAMYCy5 positive cells were found in any

378

sections from either mouse strains 10 min after last injection (Fig 5C). This absence of rAMYCy5

379

signal may be explained by the lower in vitro potency compared to pramlintide or sCTCy5 (Table 1).

380 381 382

3.4. Whole brain 3D imaging show that salmon calcitonin and rat amylin distribute into the ARC, AP and OVLT of mouse brain

383

sCT750 was injected into wild type mice twice and brains collected 30 min and 2 hours post injection

384

after which the whole brain was scanned with LSFM. When evaluating the brains, a sCT750 signal

385

was consistently found in the ARC/ME, AP and OVLT both 30 minutes and 2 hours post injection

386

(Fig 6A – B). Supplementary video 6-1 is of a 3D reconstructed whole brain of a mouse

387

summarizing how sCT750 distributes in the brain. No signal was observed in the NTS or the DMV of

388

the hindbrain. sCT750 signal specks were also observed throughout the brain tissue indicative of

389

sCT750 bound to blood vessels. When the total sCT750 signal was quantified in the ARC/ME and AP

390

nuclei, a significantly higher signal was found 30 minutes post injection vs the control group (p<0.05

391

in ARC/ME, p<0.01 in AP) with the signal not significantly increased 2 hours post injection for both

392

regions (Fig 6C – F). sCT750 distribution did not change from 30 minutes to 2 hours post injection.

393

Notably, in the hypothalamus, the majority of the sCT750 signal was found in the medio-basal ARC

394

with only specs present in the ME, but the regions could not be separated in signal intensity analysis

395

(Fig 6D). Supplementary video 6-2 summarizes sCT750 distribution in ARC coronal sections.

18

396

As no signal was observed in the ARC or AP after rAMYCy5 treatment, a tripled dose of rAMYCy7

397

and shortened time interval between injections (5 rather than 30 min) of rAMYCy7 was used for the

398

whole brain distribution study. Ten minutes after the final injection of rAMYCy7 in WT mice, the

399

rAMYCy7 signal was observed in the ARC, ME, AP and OVLT of the brains. Again, rAMYCy7 signal

400

specs were observed in the brain tissue and in the choroid plexus of the brains indicating presence of

401

fluorescently labelled peptide in the vasculature. Quantification of total signal intensity in the

402

ARC/ME and the AP revealed a significantly higher signal in both regions relative to controls 10 min

403

post final injection (p<0.05 in both regions) (Fig 6G – J). Unlike the sCT750, a distinct rAMYCy7

404

signal was observed in the ME along with signal in the medio-basal ARC (Fig 6H). Supplementary

405

video 6-3 summarizes rAMYCy7 distribution in ARC coronal sections. Of note, capture of high-

406

resolution images of rAMYCy7 was not possible, as Cy7 is highly sensitive to light-exposure induced

407

degradation – as such the signal was abolished after the initial scan of the brains.

408

19

409

4. DISCUSSION

410

Our studies demonstrate that salmon calcitonin binds and internalizes into a subset of ARC NPY

411

neurons and unidentified cells in the ARC, AP and DMV of mice. A signal indicating internalization

412

into POMC neurons was not evident in POMC-Cre:ERT2::td-tomato mice. Furthermore, when

413

examining distribution into whole brains, salmon calcitonin distributed into the AP, OVLT and ARC

414

of mice 30 minutes and 2 hours after injection. While rat amylin was not found to bind to single

415

neurons in the ARC or hindbrain at equivalent doses as salmon calcitonin under current study

416

conditions, rat amylin showed a similar distribution pattern to salmon calcitonin in the whole brain

417

visualized by LSFM.

418

Salmon calcitonin has been found to internalize into cells expressing CTR in vitro (Houssami et al.,

419

1994), but our observation is the first to establish that salmon calcitonin also displays this property in

420

vivo directly targeting ARC NPY/AgRP neurons. Earlier studies have found that salmon calcitonin

421

activates NPY/AgRP neurons (Pan et al., 2018) and that amylin acutely suppresses the activity of

422

this neuronal population in vivo (Su et al., 2017). We have previously shown that pERK signaling in

423

ARC NPY neurons is unaltered after acute amylin treatment (Lutz et al., 2018) which indicates that

424

salmon calcitonin activates and/or suppresses other signaling pathways to elicit effects within this

425

neuronal population. The ARC NPY/AgRP neurons co-expressing leptin receptors and CTRs were

426

recently reported to be important for leptin-mediated metabolic effects since the deletion of leptin

427

receptors from these neurons results in hyperphagia and weight gain (Pan et al., 2018). Thus, the

428

NPY/AgRP neurons targeted by salmon calcitonin in our findings may include a subpopulation of

429

leptin receptor/CTR co-expressing NPY/AgRP neurons that potentially play a role in in leptin/amylin

430

synergism. Nonetheless, an inhibitory effect has not been identified on ARC NPY or AgRP gene

431

expression levels after acute or sub-chronic AMY /CTR agonist treatment in rats (Barth et al., 2003;

432

Le Foll et al., 2015; Roth et al., 2006) suggesting that AMY /CTR mediated satiety may not be a

433

result of direct effects on NPY or AgRP signaling in this neuronal population. However, it has been 20

434

reported that LPBN calcitonin gene related peptide (CGRP) neurons are activated by amylin, and

435

receive tonic GABAergic (γ-aminobutyric acid) inhibitory input from NPY/AgRP neurons in the

436

ARC (Carter et al., 2013; Wu et al., 2009). This suggests that the action of salmon calcitonin on

437

NPY/AgRP neurons with high CTR expression levels may be to regulate GABA levels, a

438

neurotransmitter that is co-expressed with NPY and AgRP in these neurons (Campbell et al., 2017).

439

Thus, AMY /CTR activation by salmon calcitonin on NPY/AgRP neurons may result in direct

440

inhibition of GABAergic input from ARC NPY/AgRP neurons to the LPBN or other neuronal

441

targets. Further studies are needed to ascertain the downstream targets of salmon calcitonin effects

442

on NPY/AgRP neurons.

443

We did not observe any sCTCy5 binding or internalization into ARC POMC neurons of POMC-

444

Cre:ERT2::tdTomato mice, suggesting that they may not be directly targeted by AMY /CTR agonists.

445

In support of this finding, Pan et al. found that salmon calcitonin only activates a low number of

446

ARC POMC neurons (Pan et al., 2018), and in line with earlier studies, we found that few POMC

447

neurons in the ARC displayed CTR expression. This suggests that earlier observed action of amylin

448

inducing intracellular pERK in POMC neurons and increasing ARC POMC expression (Lutz et al.,

449

2018; Roth et al., 2006) may be mediated via action upon other cell populations. AMY /CTR

450

agonists might indirectly activate IL-6 receptor expressing POMC neurons by inducing IL-6

451

production from hypothalamic microglia (Le Foll et al., 2015; Ropelle et al., 2010) or by suppressing

452

inhibitory NPY and GABAergic projections from NPY/AgRP neurons onto POMC neurons (Cowley

453

et al., 2001). However, as only 65-70% of POMC neurons were double labelled for IHC and

454

tdTomato it cannot be rejected that salmon calcitonin might target a subset of POMC neurons we did

455

not identify. Further, due to the labile nature of the sCTCy5 signal, co-localization with POMC-IHC

456

could not be performed. Considering that sCTCy5 was also found to be internalized into a subset of

457

unknown cells in the ARC, we performed Iba1 and GFAP IHC on ARC sections from mice treated

458

with sCTCy5 to test if microglia or astrocytes might be targeted. Unfortunately, the Cyanin 5 21

459

fluorescent signal was not visible after IHC processing and the unknown target cell population was

460

not identified. Furthermore, targeting of neurons producing other neuropeptides cannot be excluded.

461

Despite similar reported half-lives, we only managed to visualize rat amylin within the brain by

462

using a threefold higher dose compared to salmon calcitonin, combined with shorter dosing interval

463

and time to tissue sampling. Salmon calcitonin has been found to bind irreversibly to the CTR (Dal

464

Maso et al., 2018; Gingell et al., 2019; Hilton et al., 2000; Houssami et al., 1994), but this has not

465

been shown for amylin. Hence, rapid dissociation from AMY might diminish internalization

466

potential for amylin, whereby amylin is rapidly broken down and cleared from the extracellular

467

milieu. Rat amylin and salmon calcitonin on the other hand displayed similar distribution patterns

468

within the brain, suggesting that differential distribution does not underlie the observed

469

pharmacodynamic differences on acute food intake suppression. Further, it is likely that amylin

470

targets the same ARC neuronal populations as salmon calcitonin without being internalized. Thus,

471

salmon calcitonin prolonged effect is most likely due to longer residence time at the receptor

472

inducing long-lasting intracellular signaling which has been observed in vitro (Lamp et al., 1981).

473

One caveat of this study was that labelled amylin was not injected in NPY-GFP and POMC-

474

Cre:ERT2::tdTomato mice using the same paradigm as for the whole brain studies. Furthermore, the

475

in vitro potency assay highlighted the fact that rAMYCy5 displayed lower potency than pramlintide

476

and sCTCy5 at the CTR and AMY. These discrepancies in study design and receptor kinetics could

477

underlie the lack of amylin signal in NPY and POMC neurons. Thus, amylin internalization into

478

NPY and POMC neurons cannot be completely rejected based on our findings.

479

Both fluorescently labelled salmon calcitonin and rat amylin distributed into the AP, ARC and

480

OVLT. Alternatively, both rat amylin and salmon calcitonin might gain access to the ARC from the

481

ME via fenestrated capillaries near the ME-ARC border (Rodríguez et al., 2010). Salmon calcitonin

482

was not observed within the ME, while rat amylin was detected at the shorter 10-minute sampling

483

interval. Further, even though we did observe CTR expression in the ME of mice we did not observe 22

484

sCTCy5 internalized into ME cells, and it is unclear why the differential distribution at the ME

485

between sCT and amylin occurs and whether sampling at the shorter interval of 10 minutes would

486

have enabled detection of the salmon calcitonin signal.

487

While our studies confirm that AMY /CTR agonists bind to single cells in the AP, we also found

488

distribution into the OVLT. The OVLT is reported to regulate fluid balance and tonicity (Kaur and

489

Ling, 2017; McKinley et al., 2019), primarily by circulating hormones such as vasopressin and

490

angiotensin II. Several hormones that affect energy balance, have also been reported to influence

491

fluid intake (e.g. GLP-1, PYY CCK and amylin) (Zimmerman et al., 2017). Angiotensin II and

492

amylin has been found to increase water intake by activating the same neuronal population as

493

angiotensin II in the SFO (Riediger et al., 1999). It is thus plausible that AMY /CTRs display a

494

similar action in the OVLT.

495

Salmon calcitonin was also consistently found to be internalized into single DMV cells. However,

496

Sexton et al. reported low to undetectable radioactive amylin binding to the DMV of rat brain

497

sections and no other reports of CTRs in the DMV can be found (Sexton et al., 1994). Further, in

498

vitro studies suggest that calcitonin internalization is CTR mediated (Gingell et al., 2019). In view of

499

this and the lack of sCT750 signal from the DMV in the whole brain study, the significance of this

500

finding is uncertain. High doses of fluorescently labelled salmon calcitonin were utilized in this

501

study, whereby nonspecific binding to the DMV cannot be rejected. However, AMY /CTR agonist

502

have been found to reduce gastric emptying (Andreassen et al., 2014; Reidelberger et al., 2001;

503

Reidelberger et al., 2002), and the mechanism is thought to be centrally mediated being in part

504

dependent on both an intact AP and vagal nerve efferent signaling (Edwards et al., 1998; Wickbom

505

et al., 2008; Young, 2005). Thus, salmon calcitonin might in part modulate gastric motility by

506

binding directly to cells in the DMV, however, functional evidence to support this connection is

507

needed.

23

508

In conclusion, we find that salmon calcitonin distributes to the ARC, targeting and internalizing into

509

NPY/AgRP neurons but not POMC neurons. Further, rat amylin and salmon calcitonin display

510

similar distribution patterns within the mouse brain distributing to the ARC, AP and OVLT,

511

indicating that the underlying difference in their acute anorectic efficacy is due to salmon

512

calcitonin’s ability to be internalized into target cells rather than differential brain distribution.

513 514

5. CONCLUSION

515 516

Analogues of satiety-inducing hormones such as amylin/calcitonin receptor agonists that can act on

517

cognate receptors in the brain to induce beneficial effects on glucose homeostasis and body weight

518

loss are being developed for the treatment of metabolic diseases such as obesity and type 2 diabetes.

519

Earlier studies indicated that AMY /CTR agonists interact with regions of the brain that regulate

520

satiety and homeostatic energy balance, however, it remains unclear to what extent these peptides

521

directly interact with target neuronal populations. Our findings serve to clarify and envision how

522

peripherally injected AMY /CTR agonists target specific neuronal populations to induce their

523

metabolic effects.

524

24

525

ACKNOWLEDGEMENTS:

526

This work was supported by Novo Nordisk A/S, the Swiss National Science Foundation (SNF

527

31003A_175458) to TAL and by the Lifepharm Centre for In Vivo Pharmacology. We would like to

528

thank Wouter Hogendorf his help with the chemistry of the fluorescently labelled peptides along

529

with Lavinia Boccia, Bernd Coester, Salome Gamakharia and Stine Normann Hansen for assistance

530

with experimental work and analytical procedures.

531 532

Conflict of interest statement: KR, LMJ, SL, TG and AS are full-time employees of Novo Nordisk

533

and hold minor share portions as part of their employment. HLZ, JL, TL and CLF declare no

534

competing financial interests.

535

25

536

REFERENCES

537

Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R., Begley, D. J., 2010. Structure and

538

function of the blood–brain barrier. Neurobiol. Dis. 37, 13-25.

539

Andreassen, K. V., Feigh, M., Hjuler, S. T., Gydesen, S., Henriksen, J. E., Beck-Nielsen, H.,

540

Christiansen, C., Karsdal, M. A., Henriksen, K., 2014. A novel oral dual amylin and calcitonin

541

receptor agonist (KBP-042) exerts antiobesity and antidiabetic effects in rats. Am. J. Physiol.

542

Endocrinol. Metab. 307, E24-E33.

543

Barth, S. W., Riediger, T., Lutz, T. A., Rechkemmer, G., 2003. Differential effects of amylin and

544

salmon calcitonin on neuropeptide gene expression in the lateral hypothalamic area and the

545

arcuate nucleus of the rat. Neurosci. Lett. 341, 131-134.

546 547

Becskei, C., Riediger, T., Zünd, D., Wookey, P., Lutz, T. A., 2004. Immunohistochemical mapping of calcitonin receptors in the adult rat brain. Brain Res. 1030, 221-233.

548

Berglund, E. D., Liu, C., Sohn, J.-W., Liu, T., Kim, M. H., Lee, C. E., Vianna, C. R., Williams, K.

549

W., Xu, Y., Elmquist, J. K., 2013. Serotonin 2C receptors in pro-opiomelanocortin neurons

550

regulate energy and glucose homeostasis. J. Clin. Invest. 123, 5061-5070.

551

Braegger, F. E., Asarian, L., Dahl, K., Lutz, T. A., Boyle, C. N., 2014. The role of the area postrema

552

in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping.

553

Eur. J. Neurosci. 40, 3055-3066.

554

Campbell, J. N., Macosko, E. Z., Fenselau, H., Pers, T. H., Lyubetskaya, A., Tenen, D., Goldman,

555

M., Verstegen, A. M., Resch, J. M., McCarroll, S. A., 2017. A molecular census of arcuate

556

hypothalamus and median eminence cell types. Nat. Neurosci. 20, 484.

557 558

Carter, M. E., Soden, M. E., Zweifel, L. S., Palmiter, R. D., 2013. Genetic identification of a neural circuit that suppresses appetite. Nature 503, 111-+.

26

559

Chaturvedula, A., Joshi, D. P., Anderson, C., Morris, R. L., Sembrowich, W. L., Banga, A. K., 2005.

560

In vivo iontophoretic delivery and pharmacokinetics of salmon calcitonin. Int. J. Pharm. 297,

561

190-196.

562

Christopoulos, G., Perry, K. J., Morfis, M., Tilakaratne, N., Gao, Y. Y., Fraser, N. J., Main, M. J.,

563

Foord, S. M., Sexton, P. M., 1999. Multiple Amylin Receptors Arise from Receptor Activity-

564

Modifying Protein Interaction with the Calcitonin Receptor Gene Product. Mol. Pharmacol. 56,

565

235-242.

566

Cowley, M. A., Smart, J. L., Rubinstein, M., Cerdán, M. G., Diano, S., Horvath, T. L., Cone, R. D.,

567

Low, M. J., 2001. Leptin activates anorexigenic POMC neurons through a neural network in

568

the arcuate nucleus. Nature 411, 480-484.

569

Dal Maso, E., Just, R., Hick, C., Christopoulos, A., Sexton, P. M., Wootten, D., Furness, S. G., 2018.

570

Characterization of signalling and regulation of common calcitonin receptor splice variants and

571

polymorphisms. Biochem. Pharmacol. 148, 111-129.

572

Dunn-Meynell, A. A., Le Foll, C., Johnson, M. D., Lutz, T. A., Hayes, M. R., Levin, B. E., 2016.

573

Endogenous VMH amylin signaling is required for full leptin signaling and protection from

574

diet-induced obesity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 310, R355-R365.

575

Edwards, G., Gedulin, B., Jodka, C., Dilts, R., Miller, C., Young, A., 1998. Area postrema (AP)-

576

lesions block the regulation of gastric emptying by amylin. Gastroenterology 114, A748.

577

Ertürk, A., Becker, K., Jährling, N., Mauch, C. P., Hojer, C. D., Egen, J. G., Hellal, F., Bradke, F.,

578

Sheng, M., Dodt, H.-U., 2012. Three-dimensional imaging of solvent-cleared organs using

579

3DISCO. Nat. Protoc. 7, 1983.

580

Feigh, M., Henriksen, K., Andreassen, K. V., Hansen, C., Henriksen, J. E., Beck-Nielsen, H.,

581

Christiansen, C., Karsdal, M. A., 2011. A novel oral form of salmon calcitonin improves

582

glucose homeostasis and reduces body weight in diet-induced obese rats. Diabetes Obes.

583

Metab. 13, 911-920. 27

584 585

Gingell, J. J., Hendrikse, E. R., Hay, D. L., 2019. New Insights into the Regulation of CGRP-Family Receptors. Trends Pharmacol. Sci. 40, 71-83.

586

Hilton, J. M., Chai, S. Y., Sexton, P. M., 1995. IN VITRO AUTORADIOGRAPHIC

587

LOCALIZATION OF THE CALCITONIN RECEPTOR ISOFORMS, C1a AND C1b, IN

588

RAT BRAIN. Neuroscience 69, 1223-1237.

589

Hilton, J. M., Dowton, M., Houssami, S., Sexton, P. M., 2000. Identification of key components in

590

the irreversibility of salmon calcitonin binding to calcitonin receptors. J. Endocrinol. 166, 213-

591

226.

592

Houssami, S., Findlay, D. M., Brady, C. L., Myers, D. E., Martin, T. J., Sexton, P. M., 1994.

593

Isoforms of the Rat Calcitonin Receptor: Consequences for Ligand Binding and Signal

594

Transduction. Endocrinology 135, 183-190.

595

Kaur, C., Ling, E.-A., 2017. The circumventricular organs. Histol. Histopathol. 32, 879-892.

596

Lam, B. Y., Cimino, I., Polex-Wolf, J., Kohnke, S. N., Rimmington, D., Iyemere, V., Heeley, N.,

597

Cossetti, C., Schulte, R., Saraiva, L. R., 2017. Heterogeneity of hypothalamic pro-

598

opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing. Mol. Metab. 6,

599

383-392.

600

Lamp, S. J., Findlay, D. M., Moseley, J. M., Martin, T. J., 1981. Calcitonin Induction of a Persistent

601

Activated State of Adenylate Cyclase in Human Breast Cancer Cells (T 47D). J. Biol. Chem.

602

256, 2269-2274.

603

Le Foll, C., Johnson, M. D., Dunn-Meynell, A. A., Boyle, C. N., Lutz, T. A., Levin, B. E., 2015.

604

Amylin-Induced Central IL-6 Production Enhances Ventromedial Hypothalamic Leptin

605

Signaling. Diabetes 64, 1621-1631.

606 607

Loh, K., Herzog, H., Shi, Y. C., 2015. Regulation of energy homeostasis by the NPY system. Trends Endocrinol. Metab. 26, 125-135.

28

608

Lutz, T., Mollet, A., Rushing, P., Riediger, T., Scharrer, E., 2001. The anorectic effect of a chronic

609

peripheral infusion of amylin is abolished in area postrema/nucleus of the solitary tract

610

(AP/NTS) lesioned rats. Int. J. Obes. 25, 1005.

611

Lutz, T. A., Coester, B., Whiting, L., Dunn-Meynell, A. A., Boyle, C. N., Bouret, S. G., Levin, B. E.,

612

Le Foll, C., 2018. Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of

613

the Hypothalamus. Diabetes 67, 805-817.

614 615

Lutz, T. A., Geary, N., Szabady, M. M., Delprete, E., Scharrer, E., 1995. Amylin Decreases Meal Size in Rats. Physiol. Behav. 58, 1197-1202.

616

McKinley, M. J., Denton, D. A., Ryan, P. J., Yao, S. T., Stefanidis, A., Oldfield, B. J., 2019. From

617

sensory circumventricular organs to cerebral cortex: Neural pathways controlling thirst and

618

hunger. J. Neuroendocrinol., e12689.

619

Nakamoto, H., Soeda, Y., Takami, S., Minami, M., Satoh, M., 2000. Localization of calcitonin

620

receptor mRNA in the mouse brain: coexistence with serotonin transporter mRNA. Brain Res.

621

Mol. Brain Res. 76, 93-102.

622

Oliver, K. R., Kane, S. A., Salvatore, C. A., Mallee, J. J., Kinsey, A. M., Koblan, K. S., Keyvan-

623

Fouladi, N., Heavens, R. P., Wainwright, A., Jacobson, M., Dickerson, I. M., Hill, R. G., 2001.

624

Cloning, characterization and distribution of receptor activity central nervous system

625

modifying proteins in the rat. Eur. J. Neurosci. 14, 618-628.

626

Padilla, S. L., Reef, D., Zeltser, L. M., 2012. Defining POMC neurons using transgenic reagents:

627

impact of transient Pomc expression in diverse immature neuronal populations. Endocrinology

628

153, 1219-1231.

629

Pan, W., Adams, J. M., Allison, M. B., Patterson, C., Flak, J. N., Jones, J., Strohbehn, G., Trevaskis,

630

J., Rhodes, C. J., Olson, D. P., 2018. Essential Role for Hypothalamic Calcitonin Receptor‒

631

Expressing Neurons in the Control of Food Intake by Leptin. Endocrinology 159, 1860-1872.

29

632 633 634 635

Paxinos, G., 2013. Paxinos and Franklin's the mouse brain in stereotaxic coordinates. In: Franklin, K. B. J., (Ed). Elsevier/Academic Press, Amsterdam. Potes, C. S., Lutz, T. A., Riediger, T., 2010. Identification of central projections from amylinactivated neurons to the lateral hypothalamus. Brain Res. 1334, 31-44.

636

Poyner, D. R., Sexton, P. M., Marshall, I., Smith, D. M., Quirion, R., Born, W., Muff, R., Fischer, J.

637

A., Foord, S. M., 2002. International Union of Pharmacology. XXXII. The mammalian

638

calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol.

639

Rev. 54, 233-246.

640

Rau, A. R., Hughes, A. R., Hentges, S. T., 2018. Various transgenic mouse lines to study

641

proopiomelanocortin cells in the brain stem label disparate populations of GABAergic and

642

glutamatergic neurons. Am. J. Physiol. Regul. Integr. Comp. Physiol. 315, R144-R152.

643

Reidelberger, R. D., Arnelo, U., Granqvist, L., Permert, J., 2001. Comparative effects of amylin and

644

cholecystokinin on food intake and gastric emptying in rats. Am. J. Physiol. Regul. Integr.

645

Comp. Physiol. 280, R605-R611.

646

Reidelberger, R. D., Kelsey, L., Heimann, D., 2002. Effects of amylin-related peptides on food

647

intake, meal patterns, and gastric emptying in rats. Am. J. Physiol. Regul. Integr. Comp.

648

Physiol. 282, R1395-R1404.

649 650

Riediger, T., Rauch, M., Schmid, H. A., 1999. Actions of amylin on subfornical organ neurons and on drinking behavior in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 276, R514-R521.

651

Riediger, T., Zuend, D., Becskei, C., Lutz, T. A., 2004. The anorectic hormone amylin contributes to

652

feeding-related changes of neuronal activity in key structures of the gut-brain axis. Am. J.

653

Physiol. Regul. Integr. Comp. Physiol. 286, R114-R122.

654

Rodríguez, E. M., Blázquez, J. L., Guerra, M., 2010. The design of barriers in the hypothalamus

655

allows the median eminence and the arcuate nucleus to enjoy private milieus: the former opens

656

to the portal blood and the latter to the cerebrospinal fluid. Peptides 31, 757-776. 30

657

Ropelle, E. R., Flores, M. B., Cintra, D. E., Rocha, G. Z., Pauli, J. R., Morari, J., de Souza, C. T.,

658

Moraes, J. C., Prada, P. O., Guadagnini, D., 2010. IL-6 and IL-10 anti-inflammatory activity

659

links exercise to hypothalamic insulin and leptin sensitivity through IKKβ and ER stress

660

inhibition. PLoS Biol. 8, e1000465.

661

Roth, J. D., Coffey, T., Jodka, C. M., Maier, H., Athanacio, J. R., Mack, C. M., Weyer, C., Parkes,

662

D. G., 2007. Combination therapy with amylin and peptide YY 3-36 in obese rodents:

663

Anorexigenic synergy and weight loss additivity. Endocrinology 148, 6054-6061.

664

Roth, J. D., Hughes, H., Kendall, E., Baron, A. D., Anderson, C. M., 2006. Antiobesity effects of the

665

beta-cell hormone amylin in diet-induced obese rats: Effects on food intake, body weight,

666

composition, energy expenditure, and gene expression. Endocrinology 147, 5855-5864.

667

Salinas, C. B. G., Lu, T. T.-H., Gabery, S., Marstal, K., Alanentalo, T., Mercer, A. J., Cornea, A.,

668

Conradsen, K., Hecksher-Sørensen, J., Dahl, A. B., 2018. Integrated brain atlas for unbiased

669

mapping of nervous system effects following liraglutide treatment. Sci. Rep. 8, 10310.

670

Schaeffer, M., Langlet, F., Lafont, C., Molino, F., Hodson, D. J., Roux, T., Lamarque, L., Verdié, P.,

671

Bourrier, E., Dehouck, B., 2013. Rapid sensing of circulating ghrelin by hypothalamic

672

appetite-modifying neurons. Proc. Natl. Acad. Sci. U. S. A. 110, 1512-1517.

673 674 675 676 677

Sexton, P., Paxinos, G., Kenney, M., Wookey, P., Beaumont, K., 1994. In vitro autoradiographic localization of amylin binding sites in rat brain. Neuroscience 62, 553-567. Su, Z., Alhadeff, A. L., Betley, J. N., 2017. Nutritive, post-ingestive signals are the primary regulators of AgRP neuron activity. Cell Rep. 21, 2724-2736. Tilakaratne, N., Christopoulos, G., Zumpe, E. T., Foord, S. M., Sexton, P. M., 2000. Amylin receptor

678

phenotypes

derived

from

human

calcitonin

receptor/RAMP

coexpression

exhibit

679

pharmacological differences dependent on receptor isoform and host cell environment. J.

680

Pharmacol. Exp. Ther. 294, 61-72.

31

681

Trevaskis, J. L., Turek, V. F., Griffin, P. S., Wittmer, C., Parkes, D. G., Roth, J. D., 2010. Multi-

682

hormonal weight loss combinations in diet-induced obese rats: Therapeutic potential of

683

cholecystokinin? Physiol. Behav. 100, 187-195.

684

Turek, V. F., Trevaskis, J. L., Levin, B. E., Dunn-Meynell, A. A., Irani, B., Gu, G. B., Wittmer, C.,

685

Griffin, P. S., Vu, C., Parkes, D. G., Roth, J. D., 2010. Mechanisms of Amylin/Leptin Synergy

686

in Rodent Models. Endocrinology 151, 143-152.

687

Whiting, L., McCutcheon, J. E., Boyle, C. N., Roitman, M. F., Lutz, T. A., 2017. The area postrema

688

(AP) and the parabrachial nucleus (PBN) are important sites for salmon calcitonin (sCT) to

689

decrease evoked phasic dopamine release in the nucleus accumbens (NAc). Physiol. Behav.

690

176, 9-16.

691

Wickbom, J., Herrington, M. K., Permert, J., Jansson, A., Arnelo, U., 2008. Gastric emptying in

692

response to IAPP and CCK in rats with subdiaphragmatic afferent vagotomy. Regul. Pept. 148,

693

21-25.

694 695 696 697

Wu, Q., Boyle, M. P., Palmiter, R. D., 2009. Loss of GABAergic signaling by AgRP neurons to the parabrachial nucleus leads to starvation. Cell 137, 1225-1234. Yeo, G. S. H., Heisler, L. K., 2012. Unraveling the brain regulation of appetite: lessons from genetics. Nat. Neurosci. 15, 1343-1349.

698

Young, A., 2005. Inhibition of gastric emptying. Adv. Pharmacol. 52, 99-121.

699

Young, A. A., Vine, W., Gedulin, B. R., Pittner, R., Janes, S., Gaeta, L. S. L., Percy, A., Moore, C.

700

X., Koda, J. E., Rink, T. J., Beaumont, K., 1996. Preclinical pharmacology of Pramlintide in

701

the Rat: Comparisons With Human and Rat Amylin. Drug. Dev. Res. 37, 231-248.

702 703

Zimmerman, C. A., Leib, D. E., Knight, Z. A., 2017. Neural circuits underlying thirst and fluid homeostasis. Nat. Rev. Neurosci. 18, 459.

704 32

705

FIGURE LEGENDS

706 707

Figure 1. In vivo efficacy of fluorescently labelled peptides in mice. Cumulative food intake

708

(mean ± SEM) (g) after a single subcutaneous injection in C57Bl6 mice of A) vehicle, rAMY (100

709

nmol/kg) or rAMYCy5 (100 nmol/kg); B) vehicle, sCT (100 nmol/kg) or sCTCy5 (100 nmol/kg); C)

710

vehicle, rAMY (300 nmol/kg) or rAMYCy7 (300 nmol/kg); D) vehicle, sCT (100 nmol/kg) or sCT750

711

(100 nmol/kg). Cumulative food intake plotted every 1 hour 0 – 24 hours after injection. Shading =

712

dark period. N=6-8 per group. *p< 0.05: native peptide vs. vehicle; #p< 0.05: fluorescently labelled

713

peptide vs. vehicle. sCT = salmon calcitonin. rAMY = rat amylin.

714 715

Figure 2. Salmon calcitonin internalizes in a subset of NPY neurons but not POMC neurons in

716

reporter mice. hrGFP-NPY and POMC-Cre:ERT2::tdTomato mice were used to investigate whether

717

fluorescently labelled salmon calcitonin (sCTCy5) and rat amylin (rAMYCy5) distribute into the ARC

718

and binds to NPY and/or POMC neurons. A-D) Results from hrGFP-NPY mice injected with sCTCy5

719

or rAMYCy5 (n = 2 – 3/group) depicting average count/ARC section (mean) of NPY+, Cy5+ and

720

double-labelled cells (A), and the percentage of NPY+ and Cy5+ cells (mean) which were double

721

labelled in ARC sections (C). An orthogonal projection of a representative 20X image of the ARC

722

from hrGFP-NPY mice injected with rAMYCy5 showing NPY neurons (green), but no Cy5 cells (red)

723

(B). An orthogonal projection of a representative 20X image of Cy5 (red), NPY (green) and double

724

labelled (yellow, white arrows) cells in the ARC after sCTCy5 injection, where Cy5 internalization

725

into NPY neurons is visualized in the X, Y and Z planes (lines cross) (left). High resolution 63X

726

images of the marked section are depicted with (bottom) or without (top) the NPY channel showing

727

co-localization of Cy5 and NPY in the same neurons (right) (D). E-F) Results from POMC-

728

Cre:ERT2::tdTomato mice injected with sCTCy5 or rAMYCy5 (n = 3 – 4/group) depicting average

729

count/ARC section (mean) of POMC+, Cy5+ and double-labelled cells (E). An orthogonal projection 33

730

of a representative 20X picture of Cy5 (red, a selection marked with arrowheads) and POMC (green)

731

cells in the ARC after sCTCy5 injection, showing that Cy5 does not internalize into POMC neurons

732

(left). High resolution 63X image of the marked section where accumulation of Cy5 is not evident in

733

POMC expressing neurons (right) (F). 3V = 3rd ventricle. ARC = Arcuate nucleus. ME = Median

734

eminence.

735 736

Figure 3. Comparison between POMC positive cells in POMC-Cre:ERT2::tdTomato mice and

737

POMC IHC. ARC sections from POMC-Cre:ERT2::tdTomato mice expressing tdTomato in POMC

738

neurons were stained for POMC with IHC, and POMC expression compared (n = 4). The

739

identification number of the mouse is indicated next to the symbol to allow for direct comparison. A)

740

Average counts / ARC section (mean) of POMC+ after IHC staining or with endogenous tdTomato

741

expression. B) The percentage of POMC neurons which were double labelled for IHC+ and

742

tdTomato+ expression. C) Representative 20X image of DAPI, POMC-IHC and POMC tdTomato

743

expression in the ARC. 3V = 3rd ventricle. ARC = arcuate nucleus. IHC = immunohistochemistry.

744 745

Figure 4. CTR is more abundantly expressed in ARC NPY neurons than in POMC neurons.

746

ARC sections from wild type mice investigated for CTR expression in NPY and POMC expressing

747

neurons with ISH (n = 4). A) Depicted is a representative 20X image of an ARC/ME section with a

748

close-up of the marked section showing that few POMC expressing neurons (red) also express the

749

CTR (yellow) (white arrows). B) Depicted is a representative 20X image of an ARC/ME section

750

with a close-up of the marked section showing that almost all NPY expressing neurons (red) express

751

abundant levels of the CTR (yellow). C) Quantification of CTR-NPY and CTR-POMC neurons in

752

the ARC. 3V = 3rd ventricle. ARC = Arcuate nucleus. CTR = calcitonin receptor.

753

Immunohistochemistry. ISH = In situ hybridization.

IHC =

754 34

755

Figure 5. Salmon calcitonin internalizes into AP and DMV cells of mice. hrGFP-NPY and

756

POMC-Cre:ERT2::tdTomato mice were used to investigate if fluorescently labelled salmon calcitonin

757

(sCTCy5) and rat amylin (rAMYCy5) binds to cells in hindbrain nuclei (n = 5 – 7/group, both mouse

758

strains combined). A-B) An orthogonal projection of a representative 20X image from mice treated

759

with sCTCy5 of the AP (A) and DVM (B) showing that Cy5 (red) is internalized into cells in the X, Y

760

and Z planes (lines cross and marked with arrowheads) in both regions. High resolution 63x image of

761

marked section (insets) shows Cy5 accumulation in the cytoplasm of cells. C) An orthogonal

762

projection of a representative 20X image of the AP from mice treated with rAMYCy5, showing no

763

Cy5 signal (red) in the AP. AP = area postrema; DVM = Dorsal motor nucleus of the vagus; NTS =

764

Nucleus of solitary tract.

765 766

Figure 6. Distribution of fluorescently labelled salmon calcitonin and rat amylin in the mouse

767

brain. Wild type mice were injected with fluorescently labelled salmon calcitonin (sCT750) or rat

768

amylin (rAMYCy7) to investigate how the peptides distributes into the brain of mice (auto-

769

fluorescence from tissue = green, specific signal = white). A – B) Representative images of the

770

reconstructed 3D whole brain 30 min after last injection of sCT750 in the horizontal (A) and sagittal

771

(B) plane. C – F) Quantification (mean fold change ± SEM) of specific sCT750 signal in the

772

ARC/ME and AP of the brain 30 minutes and 2 hours post last injection of sCT750 in mice (C), and

773

representative images of the ARC/ME (D), AP (E) and OVLT (F) from mice terminated 30 min after

774

injection of sCT750. G – J) Quantification (mean fold change ± SEM) of specific rAMYCy7 signal in

775

the ARC/ME and AP of the brain 10 minutes post last injection of rAMYCy7 in mice (G), and

776

representative images of the ARC/ME (H), AP (I) and OVLT (J) from mice injected with rAMYCy7.

777

A-B) Scale bars = 1000 µm. D-F, H-J) scale bars = 100 µm. N=5 per group. *p< 0.05 and **p< 0.01

778

vs. control group. ARC = arcuate nucleus; AP = area postrema; ME = median eminence; OVLT =

779

vascular organ of the lamina terminalis. 35

780 781

Supplementary video 6-1.

782

A representative reconstructed 3D whole brain summarizing sCT750 distribution in the entire brain 2

783

hours post injection (autofluorescence from tissue = green, specific signal = white).

784 785

Supplementary video 6-2.

786

A representative reconstruction of ARC coronal sections summarizing sCT750 distribution in the

787

ARC 30 min post injection (autofluorescence from tissue = green, specific signal = white).

788 789

Supplementary video 6-3.

790

A representative reconstruction of ARC coronal sections summarizing rAMYCy7 distribution in the

791

ARC 10 min post injection (autofluorescence from tissue = green, specific signal = white).

36

792

Table 1: In vitro potency data (EC50-values) for pramlintide, sCT, sCT750, rAMYCy5 and sCTCy5

793

measured in BHK cell lines stably expressing the human calcitonin receptor (CTR) or amylin

794

receptor 3 (AMY3) and the CRE-luciferase reporter gene. Data are shown as means with 95%

795

confidence intervals (CI) based on three independent experiments. See statistical analysis in

796

supplementary table 1. Compound

AMY3

CTR

Pramlintide

EC50 (95% CI) [pM] 2.4 (1.3 to 4.3) 45.9 (21.2 to 99.4)

sCT sCT750 sCTCy5 rAMYCy5

0.9 (0.4 to 1.8) 1.9 (0.6 to 5.6) 0.8 (0.5 to 1.4) 9.8 (7.5 to 12.8)

1.7 (0.4 to 6.3) 2.6 (1.1 to 6.3) 1.6 (0.5 to 4.9) 167.7 (134.6 to 208.9)

797

37

B) Cumulative food intake (g)

Cumulative food intake (g)

A) 5 4 3

#

*, #

2

#

Vehicle rAMY 100 nmol/kg rAMYCy5 100 nmol/kg

#

1 0

*, #

4 3 2

Vehicle sCT 100 nmol/kg sCTCy5 100 nmol/kg

1 0

5

10 15 Time (h)

20

C)

0

5

10 15 Time (h)

20

D) *, #

5 4 #

3 2 Vehicle rAMY 300 nmol/kg rAMYCy7 300 nmol/kg

1 0

Cumulative food intake (g)

0

Cumulative food intake (g)

5

Vehicle sCT 100 nmol/kg sCT750 100 nmol/kg

5 4

*, # 3 2 1 0

0

5

10 15 Time (h)

20

0

5

10 15 Time (h)

20

120

rAMYCy5 sCTCy5

100 80 60

3V

40 20 0

NPY+

Cy5+

Cy5+ + NPY+

ME DAPI NPY rAMYCy5

ARC Cy5+ NPY+ co-loc. (%)

80 60 40

3V

20 0

Cy5+ NPY+ of NPY+

Cy5+ NPY+ of Cy5+

ME DAPI NPY sCTCy5 ARC

Average count / section

Average count / section

ARC

60 50 40 30 20

3V

10 0

POMC+

Cy5+

Cy5+ +POMC+

DAPI POMCsCTCy5

ME

B) 80

100

4 1

60

14 3

2

40

3

2

20 0

POMC IHC+ tdTomato+ co-loc. (%)

Average count / section

A)

IHC+ tdTomato+

C)

80 60

1 3 4

1 4 2 3

2

40 20 0 IHC+ tdTomato+ IHC+ tdTomato+ of IHC+ of tdTomato+

"5'

#%&'8 *+,-./*-

!"#$

#%&'($)'

"5' :;

6789.

#%&'(*+,-./*-

%0123/4

ARC

A)

ARC

DAPI POMC CTR

3V 3V

ME

B) DAPI NPY CTR

ARC

3V

Average count / section

ME

150

100

50

0

POMC+

NPY+

CTR+ CTR+ %CTR+ %CTR+ +POMC+ +NPY+ +POMC+ +NPY+

A)

B)

C)

AP AP

NTS DMV

DAPI sCTCy5

DAPI sCTCy5

DAPI rAMYCy5

A)

B)

OVLT

AP

ARC

AP OVLT

Fold change (sCT750 / Control)

C)

D)

sCT750 30 min sCT750 2 hours

35 30

E)

F)

**

25 20 15 10

ARC

*

5 ARC/ME 80 60

AP

ME

AP

G) Fold change (rAMYCy7 / Control)

ARC

H)

I)

OVLT

J)

rAMYCy7 10 min

*

*

40

ARC

20

AP 0 ARC/ME

AP

ME

OVLT

AUTHOR CONTRIBUTIONS:

Conceptualization, H.L.Z., L.M.J., T.A.L. and C.L.F.; Methodology, H.L.Z. and C.L.F.; Investigation, H.L.Z., T.G., S.L. and C.L.F.; Writing – Original Draft, H.L.Z and C.L.F. ; Writing – Review & Editing, H.L.Z., L.M.J., J.L., T.G., A.S., T.A.L. and C.L.F. Resources, L.M.J., K.R. and T.A.L.; Funding Acquisition, K.R., J.L. and T.A.L. C.L.F. is the guarantor of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.