The first group of mice was treated with CVN45502 and the second group of mice received only the peanut butter pellet without the CVN45502 compound. Treatment lasted 14 days to mimic DRN and ICV treatments. A lean control group was also evaluated in parallel following the same paradigm. After the 14 treatment days, mice were placed for 7 days on the peanut-butter-only paradigm to assess whether there were irreversible consequences in weight due to the chronic treatment.

**Home-cage indirect calorimetry assessment.** Energy expenditure was assessed through measurement by indirect calorimetry of VO₂, VCO₂, energy expenditure and locomotor activity using an automated home cage phenotyping system (TSE Systems). Mice were singly housed in a climate-controlled chamber (temperature: 22°C; humidity: 55%; 12-hour light–12-hour dark cycle) with ad libitum access to water and chow. After 7 days (TSE Systems) of adaptation to social isolation, the mice were treated with vehicle (4 days) or CNO (6 days) (Fig. 2), or peanut butter (0.1 g) with (6 days) or without (4 days) CVN45502. Data were collected and analyzed as recommended by the manufacturers. Averaged measurements of control periods or treatment periods (CNO/CVN45502) are represented. Locomotor activity was recorded as beam breaks converted into distance/velocity, measuring activity in three dimensions. Beam breaks by mice were analyzed in the metabolic cage using custom software. The respiratory exchange ratio (RER) and energy expenditure were calculated from VO₂ and VCO₂ production data. Statistical assessment of the data was conducted using CalR software and ANCOVA regressions³⁵.

**Statistics and reproducibility**

Statistical parameters reported in the figures and figure legends are displayed as mean ± s.e.m. Significance was defined as *P* < 0.05. Significance annotations are annotated with the actual value. Mice were randomized into control or treatment groups. Control mice were age-matched littermate controls where possible. All statistics and data analysis were performed using GraphPad Prism, CalR Matlab, R or Python. For RNA-seq, transcript abundance and differential expression were performed using cufflinks.

Sample size has been included in all figure legends. Regarding replicates, the biological replicates for imaging studies (Figs. 1 and 8 and Extended Data Figs. 1, 2, 5 and 10) in cells, mice or human donors come from *n* = 3 biologically independent cells, mice or human experiments. For circuit mapping validation studies (Fig. 1 and Extended Data Fig. 1) were performed in two independent experiments assessing feeding, thermogenesis or OFT parameters. Chemogenetics and pharmacology studies in Figs. 2, 4, 5 and 7 and Extended Data Figs. 2–4, 6 and 9 were performed in two independent groups of mice exposed to a HFD (45% calories from fat Research Diets). Feeding and weight loss were measured both manually and automatically using metabolic cages (TSE). Sequencing data analyzed in Fig. 3 and Extended Data Fig. 5 come from three independent pools of three mice injected with an AAV-IV-GFP-L10 (VTRAP) following a GFP-TRAP protocol; details on the data can be found in 'Data availability.' Physiological properties associated with treatments with CVN45502 (Fig. 6) have been conducted in *n* = 3 biologically independent mice. Side effects for calcitonin and SB-334867 treatments presented in Extended Data Fig. 7 were conducted in *n* = 4 biologically independent mice only once owing to limited equipment accessibility. Gene expression analyses associated with CVN45502 in BAT were conducted during review to accommodate reviewers questions, and were performed in one cohort of mice per experiment (lean, 16 weeks exposure to HFD and ob/ob) given the lack of change and the number of animals (Extended Data Figs. 8 and 9). Given that treatments with CVN45502 fail to show effects in thermogenesis or indirect calorimetry, the experiment was not repeated.

**Reporting summary**

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

**Data availability**

Sequencing datasets can be found with the accession number GSE87890 for vTRAP RNA-seq data. All *P* values have been provided in the figures. All other data (iDISCO projection mapping, ISH studies performed with RNAscope and CVN45502 materials) are available from authors on request. Details on the newly synthesized compound have been included in the Supplementary Information. Source data are provided with this paper.

**Code availability**

GENSAT analysis found on Fig. 3 was accomplished following a custom analysis pipeline that can be found as of the date of publication at: https://github.com/RockefellerUniversity/SchneebergerPane_NatureMetabolism2022.

Code for projection mapping quantifications can be achieved using: https://github.com/ChristophKirst/ClearMap2 and https://github.com/AlbertPun/TRAILMAP. Updated code for our specific study is provided along with the Supplementary Information (TubeMapTrail-MAp.py).

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## Acknowledgements

J.M.F. acknowledges support from the JPB Foundation. M.S. acknowledges support from the Mc Cluskey family and the KAVLI Neuroscience Institute. M.S. and J.M.F. acknowledge support from the Robertson Therapeutic Development Fund. This work was supported by the National Institute of Diabetes and Digestive Kidney Diseases (NIDDK) grant K99 DK120869 (M.S.) and 4R00DK1208689. L.P. acknowledges support from The David Rockefeller fellowship program and Boehringer Ingelheim. A.R.N. and P.C. acknowledge support from ADA Pathway to Stop Diabetes. A.R.N. acknowledges support from ADA IBS Core Award and NARSAD Young Investigator Award. N.R. acknowledges support from the program the ERC starting grant ‘NeuroRemod’ (EU Horizon 2020 research program under grant agreement N°758817). N.H. and J.M.F. acknowledge support from the Howard Hughes Medical Institute. We would like to thank P.A. Muller and M. Claret i Carles for critical reading of the article and providing feedback. We would like to thank E.

Stoyanova for comprehensively organizing the GENSAT data. We would like to thank I. Piscitello for all her impeccable assistance.

## Author contributions

M.S. and J.M.F. conceived and designed the study, and developed the research program. K.P., K.J.P., D.F.B., F.M., K.D., R.B., R.N., V.M.M., J.T.S., A.R.N., M.J.O. and L.P. performed experiments. T.S.C., D.B. and N.H. conducted GENSAT based profiling analysis. P.C. conducted energy expenditure study design and helped select the proper panel of BAT gene expression. N.L.B., A.G. and M.C. developed the CVN compound and designed translational experiments. N.R. and T.T. analyzed iDISCO profiling data and generated TRAILMAP analysis. J.M.F. and M.S. wrote the manuscript with input from all the authors.

## Competing interests

N. B., M. C., K. J. P., V. M., K. D., R. B. and D.B. are employed by Cerevance. The other authors declare no competing interests.

## Additional information

Extended data is available for this paper at
https://doi.org/10.1038/s42255-022-00677-8.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42255-022-00677-8.

Correspondence and requests for materials should be addressed to Marc Schneeberger or Jeffrey M. Friedman.

Peer review information *Nature Metabolism* thanks Marc Reitman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement Primary Handling editor: Ashley Castellanos-Jankiewicz, in collaboration with the *Nature Metabolism* team.

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© The Author(s) 2022, corrected publication 2022

<footnote n="1">¹Laboratory of Molecular Genetics, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA. ²Laboratory of Neurovascular Control of Homeostasis, Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA. ³Wu Tsai Institute for Brain and Cognition, Yale School of Medicine, New Haven, CT, USA. ⁴Cerevance, Cambridge, UK. ⁵Laboratory of Molecular Metabolism, The Rockefeller University, New York, NY, USA. ⁶Bioinformatics Resource Center, The Rockefeller University, New York, NY, USA. ⁷Sorbonne Université, Paris Brain Institute, INSERM, CNRS, Hopital de la Pitié Salpetrière, Paris, France. ⁸Takeda Cambridge, Cambridge, UK. ⁹Department of Genetics, Yale School of Medicine, New Haven, CT, USA. ¹⁰College of Physicians and Surgeons, Columbia University, New York, NY, USA. ¹¹Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA. ¹²These authors contributed equally: Nicola L. Brice, Kyle Pellegrino, Luca Parolari, Jordan T. Shaked, Keith J. Page, Francois Marchildon. e-mail: marc.schneebergerpane@yale.edu; friedj@rockefeller.edu</footnote>
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## Acknowledgements

J.M.F. acknowledges support from the JPB Foundation. M.S. acknowledges support from the Mc Cluskey family and the KAVLI Neuroscience Institute. M.S. and J.M.F. acknowledge support from the Robertson Therapeutic Development Fund. This work was supported by the National Institute of Diabetes and Digestive Kidney Diseases (NIDDK) grant K99 DK120869 (M.S.) and 4R00DK1208689. L.P. acknowledges support from The David Rockefeller fellowship program and Boehringer Ingelheim. A.R.N. and P.C. acknowledge support from ADA Pathway to Stop Diabetes. A.R.N. acknowledges support from ADA IBS Core Award and NARSAD Young Investigator Award. N.R. acknowledges support from the program the ERC starting grant ‘NeuroRemod’ (EU Horizon 2020 research program under grant agreement N°758817). N.H. and J.M.F. acknowledge support from the Howard Hughes Medical Institute. We would like to thank P.A. Muller and M. Claret i Carles for critical reading of the article and providing feedback. We would like to thank E.

Stoyanova for comprehensively organizing the GENSAT data. We would like to thank I. Piscitello for all her impeccable assistance.

## Author contributions

M.S. and J.M.F. conceived and designed the study, and developed the research program. K.P., K.J.P., D.F.B., F.M., K.D., R.B., R.N., V.M.M., J.T.S., A.R.N., M.J.O. and L.P. performed experiments. T.S.C., D.B. and N.H. conducted GENSAT based profiling analysis. P.C. conducted energy expenditure study design and helped select the proper panel of BAT gene expression. N.L.B., A.G. and M.C. developed the CVN compound and designed translational experiments. N.R. and T.T. analyzed iDISCO profiling data and generated TRAILMAP analysis. J.M.F. and M.S. wrote the manuscript with input from all the authors.

## Competing interests

N. B., M. C., K. J. P., V. M., K. D., R. B. and D.B. are employed by Cerevance. The other authors declare no competing interests.

## Additional information

Extended data is available for this paper at
https://doi.org/10.1038/s42255-022-00677-8.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42255-022-00677-8.

Correspondence and requests for materials should be addressed to Marc Schneeberger or Jeffrey M. Friedman.

Peer review information *Nature Metabolism* thanks Marc Reitman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement Primary Handling editor: Ashley Castellanos-Jankiewicz, in collaboration with the *Nature Metabolism* team.

Reprints and permissions information is available at
www.nature.com/reprints.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2022, corrected publication 2022

<footnote n="1">¹Laboratory of Molecular Genetics, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA. ²Laboratory of Neurovascular Control of Homeostasis, Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA. ³Wu Tsai Institute for Brain and Cognition, Yale School of Medicine, New Haven, CT, USA. ⁴Cerevance, Cambridge, UK. ⁵Laboratory of Molecular Metabolism, The Rockefeller University, New York, NY, USA. ⁶Bioinformatics Resource Center, The Rockefeller University, New York, NY, USA. ⁷Sorbonne Université, Paris Brain Institute, INSERM, CNRS, Hopital de la Pitié Salpetrière, Paris, France. ⁸Takeda Cambridge, Cambridge, UK. ⁹Department of Genetics, Yale School of Medicine, New Haven, CT, USA. ¹⁰College of Physicians and Surgeons, Columbia University, New York, NY, USA. ¹¹Laboratory of Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA. ¹²These authors contributed equally: Nicola L. Brice, Kyle Pellegrino, Luca Parolari, Jordan T. Shaked, Keith J. Page, Francois Marchildon. e-mail: marc.schneebergerpane@yale.edu; friedj@rockefeller.edu</footnote>
A

<figure><img src="image_55.png" /></figure>

B

<figure><img src="image_56.png" /></figure>

**Extended Data Fig. 1 | Ascending projections from DRN<sup>Vglut3</sup> Neurons**
**Demonstrate Integration to Brain Loci with Previously Reported Roles in**
**Energy Balance Control.** (a) Whole mount axonal projections using clearing/
immunostaining technology IDISCO+ from DRN<sup>Vglut3</sup> neurons to multiple
extrahypothalamic loci with previously reported roles in energy homeostasis
regulation. In particular, we highlight projections to the Lateral Septum and
Amygdala (basolateral and central section) with known feeding regulation
roles and the Parabrachial Nucleus with known implications in both feeding

and thermoregulation. (Left Panels) Allen brain atlas annotations of the
aforementioned regions. (Middle Panels) Coronal reslices of axonal projections
to the aforementioned regions. (Right panels) Whole-brain, 3D reconstruction
images of DRN<sup>Vglut3</sup> projections throughout the brain from multiple viewpoints.
Scale bars 200um. (b) Schema of the most prominent site of projection from
DRN<sup>Vglut3</sup> neurons to the LHA. See also Extended Data Video 1 for animated view
of projection patterns from DRN<sup>Vglut3</sup> neurons and Supplementary Table 1 for
quantification of the projection targets identified in the entire brain.
A

<figure><img src="image_59.png" /></figure>

B

<figure><img src="image_60.png" /></figure>

**Extended Data Fig. 1 | Ascending projections from DRN<sup>Vglut3</sup> Neurons**
**Demonstrate Integration to Brain Loci with Previously Reported Roles in**
**Energy Balance Control.** (a) Whole mount axonal projections using clearing/
immunostaining technology IDISCO+ from DRN<sup>Vglut3</sup> neurons to multiple
extrahypothalamic loci with previously reported roles in energy homeostasis
regulation. In particular, we highlight projections to the Lateral Septum and
Amygdala (basolateral and central section) with known feeding regulation
roles and the Parabrachial Nucleus with known implications in both feeding

and thermoregulation. (Left Panels) Allen brain atlas annotations of the
aforementioned regions. (Middle Panels) Coronal reslices of axonal projections
to the aforementioned regions. (Right panels) Whole-brain, 3D reconstruction
images of DRN<sup>Vglut3</sup> projections throughout the brain from multiple viewpoints.
Scale bars 200um. (b) Schema of the most prominent site of projection from
DRN<sup>Vglut3</sup> neurons to the LHA. See also Extended Data Video 1 for animated view
of projection patterns from DRN<sup>Vglut3</sup> neurons and Supplementary Table 1 for
quantification of the projection targets identified in the entire brain.
A

<figure><img src="image_57.png" /></figure>

Dorsal raphe nucleus, Vglut3-IRES-Cre mouse

B

Dorsal Raphe

<figure><img src="image_58.png" /></figure>

Lateral Hypothalamus

<figure><img src="image_59.png" /></figure>

C

Optogenetic Terminal Stimulation
BLA/PBN/LS

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

D

AAV5-Ef1a-DIO-TVA
AAV5-Ef1a-DIO-GTB (EnvA)SAD-deltaG-GFP

<figure><img src="image_67.png" /></figure>

LHA, Vglut2-IRES-Cre mouse

E

Dorsal Raphe

<figure><img src="image_68.png" /></figure>

Lateral Hypothalamus

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

F

Lateral Hypothalamic Area
Vglut2-IRES-Cre neurons

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>

<figure><img src="image_74.png" /></figure>

G

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

H

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

Extended Data Fig. 2 | See next page for caption.
A

<figure><img src="image_61.png" /></figure>

Dorsal raphe nucleus, Vglut3-IRES-Cre mouse

B

Dorsal Raphe

<figure><img src="image_62.png" /></figure>

Lateral Hypothalamus

<figure><img src="image_63.png" /></figure>

C

Optogenetic Terminal Stimulation
BLA/PBN/LS

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

D

AAV5-Ef1a-DIO-TVA
AAV5-Ef1a-DIO-GTB (EnvA)SAD-deltaG-GFP

<figure><img src="image_71.png" /></figure>

LHA, Vglut2-IRES-Cre mouse

E

Dorsal Raphe

<figure><img src="image_72.png" /></figure>

Lateral Hypothalamus

<figure><img src="image_73.png" /></figure>

<figure><img src="image_74.png" /></figure>

<figure><img src="image_75.png" /></figure>

F

Lateral Hypothalamic Area
Vglut2-IRES-Cre neurons

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

G

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /></figure>

H

<figure><img src="image_81.png" /></figure>

<figure><img src="image_82.png" /></figure>

Extended Data Fig. 2 | See next page for caption.
**Extended Data Fig. 2 | DRN<sup>Vglut3</sup> Neurons Anatomic Analysis Suggests the Blockade of Energy Homeostasis occurs through LHA<sup>Vglut2</sup> neurons.** (a)

Schema for DRN<sup>Vglut3</sup> terminals in the LHA, using and AAV5-DIO-ChR2-eYFP injected into the DRN and an optic fiber placed in the LHA. (b) Representative images of cell bodies of 1 mouse from a samples size of 3 independent biological samples. (Left panel) injected with AAV5-DIO-ChR2-eYFP in the DRN and axonal terminals (Right panel) in the LHA. Dashed line shows optic fiber location in the LHA. (C,Left) Schema representing terminal activation DRN<sup>Vglut3</sup> terminals in the BLA (red), PBN (purple) and LS (green). (c, Right) Stimulation of terminals in the LHA (Laser On phase) significantly suppresses food intake (treatment: p < 0.001) in the BLA and PBN but not in the LS (n = 5 control groups, 5 group BLA, 6 group PBN, 7 group LS). (d) Schema illustrating monosynaptic retrograde tracing from Vglut2 neurons in the LHA to the DRN (n = 3 independent biological samples). (e, Left) Retrogradely labeled (green) neurons were found in the DRN following the monosynaptic tracing experiment (e, Right) Dual labelling for both mCherry

and GFP is observed in the LHA following the monosynaptic tracing experiment, demonstrating both helper virus and rabies virus expression. (F-Left) Schema representing the strategy used to optogentically activate LHA<sup>Vglut2</sup> cell bodies. (F-Right) Stimulation of Vglut2 neurons in the LHA (Laser On phase) significantly suppresses food intake (treatment: p < 0.001) (n = 5 control group, 6 group LHA). (G-H) Activation of LHA<sup>Vglut2</sup> cell bodies does not impair thermoregulation as observed by measurement of BAT temperature using IPTT-300 transponders (g) or core body temperature through anal thermometer measurements. (h) (n = 5 control group, 6 group LHA). Data are presented as mean ± SEM. Two-way repeated measures ANOVA comparing control and treated groups (n = 5-7 mice per group) followed by an ad hoc Sidak's multiple comparison test (C, F, G, H). Blue-shaded region highlights Laser On epoch. Scale bars, 200µm. p < 0.05 is considered significant. LHA (Lateral Hypothalamic Area), DRN (Dorsal Raphe Nucleus), BLA (Basolateral Amygdala), PBN (Parabrachial Nucleus), LS (Lateral Septum) and BAT (Brown adipose tissue).
**Extended Data Fig. 2 | DRN<sup>Vglut3</sup> Neurons Anatomic Analysis Suggests the Blockade of Energy Homeostasis occurs through LHA<sup>Vglut2</sup> neurons.** (a)

Schema for DRN<sup>Vglut3</sup> terminals in the LHA, using and AAV5-DIO-ChR2-eYFP injected into the DRN and an optic fiber placed in the LHA. (b) Representative images of cell bodies of 1 mouse from a samples size of 3 independent biological samples. (Left panel) injected with AAV5-DIO-ChR2-eYFP in the DRN and axonal terminals (Right panel) in the LHA. Dashed line shows optic fiber location in the LHA. (C,Left) Schema representing terminal activation DRN<sup>Vglut3</sup> terminals in the BLA (red), PBN (purple) and LS (green). (c, Right) Stimulation of terminals in the LHA (Laser On phase) significantly suppresses food intake (treatment: p < 0.001) in the BLA and PBN but not in the LS (n = 5 control groups, 5 group BLA, 6 group PBN, 7 group LS). (d) Schema illustrating monosynaptic retrograde tracing from Vglut2 neurons in the LHA to the DRN (n = 3 independent biological samples). (e, Left) Retrogradely labeled (green) neurons were found in the DRN following the monosynaptic tracing experiment (e, Right) Dual labelling for both mCherry

and GFP is observed in the LHA following the monosynaptic tracing experiment, demonstrating both helper virus and rabies virus expression. (F-Left) Schema representing the strategy used to optogentically activate LHA<sup>Vglut2</sup> cell bodies. (F-Right) Stimulation of Vglut2 neurons in the LHA (Laser On phase) significantly suppresses food intake (treatment: p < 0.001) (n = 5 control group, 6 group LHA). (G-H) Activation of LHA<sup>Vglut2</sup> cell bodies does not impair thermoregulation as observed by measurement of BAT temperature using IPTT-300 transponders (g) or core body temperature through anal thermometer measurements. (h) (n = 5 control group, 6 group LHA). Data are presented as mean ± SEM. Two-way repeated measures ANOVA comparing control and treated groups (n = 5-7 mice per group) followed by an ad hoc Sidak's multiple comparison test (C, F, G, H). Blue-shaded region highlights Laser On epoch. Scale bars, 200µm. p < 0.05 is considered significant. LHA (Lateral Hypothalamic Area), DRN (Dorsal Raphe Nucleus), BLA (Basolateral Amygdala), PBN (Parabrachial Nucleus), LS (Lateral Septum) and BAT (Brown adipose tissue).
A

<figure><img src="image_79.png" /></figure>

B

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_80.png" /></figure>

C

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_81.png" /></figure>

D

<figure><img src="image_82.png" /></figure>

E

<figure><img src="image_83.png" /></figure>

F

<figure><img src="image_84.png" /></figure>

G

Preventive

<figure><img src="image_85.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_86.png" /></figure>

Curative

<figure><img src="image_87.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_88.png" /></figure>

H

Preventive

<figure><img src="image_89.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_90.png" /></figure>

Curative

<figure><img src="image_91.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_92.png" /></figure>

Extended Data Fig. 3 | See next page for caption.
A

<figure><img src="image_83.png" /></figure>

B

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_84.png" /></figure>

C

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_85.png" /></figure>

D

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_86.png" /></figure>

E

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_87.png" /></figure>

F

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

<figure><img src="image_88.png" /></figure>

G

Preventive

● Vglut3Cre Control - Saline

▲ Vglut3Cre hM3Dq - Saline

■ Vglut3Cre Control - CNO

▼ Vglut3Cre hM3Dq - CNO

<figure><img src="image_89.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_90.png" /></figure>

Curative

● Vglut3Cre Control - Saline

▲ Vglut3Cre hM3Dq - Saline

■ Vglut3Cre Control - CNO

▼ Vglut3Cre hM3Dq - CNO

<figure><img src="image_91.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_92.png" /></figure>

H

Preventive

● Vglut3Cre Control - Saline

▲ Vglut3Cre hM3Dq - Saline

■ Vglut3Cre Control - CNO

▼ Vglut3Cre hM3Dq - CNO

<figure><img src="image_93.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_94.png" /></figure>

Curative

● Vglut3Cre Control - Saline

▲ Vglut3Cre hM3Dq - Saline

■ Vglut3Cre Control - CNO

▼ Vglut3Cre hM3Dq - CNO

<figure><img src="image_95.png" /></figure>

▲ Vglut3Cre hM3Dq ● Vglut3Cre Control

▼ Vglut3Cre hM3Dq ■ Vglut3Cre Control

<figure><img src="image_96.png" /></figure>

Extended Data Fig. 3 | See next page for caption.
**Extended Data Fig. 3** | **Chemogenetic Stimulation of DRN<sup>Vglut3</sup> Neurons fed HFD Exhibits a Significant Reduction of Energy Balance in a Preventive Setting.**

(a) Schematic for chronic stimulation of DRN<sup>Vglut3</sup> neurons in HFD fed mice injected with an AAV5-DIO-hm3Dq-mcherry in DRN<sup>Vglut3</sup> neurons using the DREADD agonist clozapine-N-oxide (CNO) twice a day for 11 weeks at the same time of transitioning into a high fat diet. Representative image shows the injection site. (b) Weight curve illustrating weight loss after stimulating DRN<sup>Vglut3</sup> neurons upon DIO. (c) Adiposity assessment using magnetic resonance at the last day of treatment with CNO (n = 7 control group, 6 group HM3Dq). (d) Food intake during the chronic stimulation of DRN<sup>Vglut3</sup> neurons demonstrating hypophagia (n = 7 control group, 6 group HM3Dq). (e) Adaptive thermogenesis at the last day of treatment with CNO (n = 7 control group, 6 group HM3Dq). (f) Core body

temperature assessment at the last day of treatment with CNO. (A-F) Littermate Vglut3-Cre positive DIO mice injected with an AAV2/5-DIO-mCherry were used as controls (n = 7 control group, 6 group HM3Dq). (g) Glucose tolerance test and area under the curve (AUC) of the intraperitoneal glucose (2g/kg) tolerance test during the preventive (CNO and high fat diet given at the same time) (2 left panels) and curative (CNO given after the development of DIO) (2 right panels) setting (n = 7 control group, 6 group HM3Dq). (h) Insulin tolerance test and AUC of the insulin tolerance test during the preventive (2 left panels) and curative (2 right panels) setting (n = 7 control group, 6 group HM3Dq). Data represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons using a Tukey post-hoc approach (B, G-H) or two-tailed unpaired t-test (C-F). Statistical difference established at p < 0.05.
**Extended Data Fig. 3** | **Chemogenetic Stimulation of DRN<sup>Vglut3</sup> Neurons fed HFD Exhibits a Significant Reduction of Energy Balance in a Preventive Setting.**

(a) Schematic for chronic stimulation of DRN<sup>Vglut3</sup> neurons in HFD fed mice injected with an AAV5-DIO-hm3Dq-mcherry in DRN<sup>Vglut3</sup> neurons using the DREADD agonist clozapine-N-oxide (CNO) twice a day for 11 weeks at the same time of transitioning into a high fat diet. Representative image shows the injection site. (b) Weight curve illustrating weight loss after stimulating DRN<sup>Vglut3</sup> neurons upon DIO. (c) Adiposity assessment using magnetic resonance at the last day of treatment with CNO (n = 7 control group, 6 group HM3Dq). (d) Food intake during the chronic stimulation of DRN<sup>Vglut3</sup> neurons demonstrating hypophagia (n = 7 control group, 6 group HM3Dq). (e) Adaptive thermogenesis at the last day of treatment with CNO (n = 7 control group, 6 group HM3Dq). (f) Core body

temperature assessment at the last day of treatment with CNO. (A-F) Littermate Vglut3-Cre positive DIO mice injected with an AAV2/5-DIO-mCherry were used as controls (n = 7 control group, 6 group HM3Dq). (g) Glucose tolerance test and area under the curve (AUC) of the intraperitoneal glucose (2g/kg) tolerance test during the preventive (CNO and high fat diet given at the same time) (2 left panels) and curative (CNO given after the development of DIO) (2 right panels) setting (n = 7 control group, 6 group HM3Dq). (h) Insulin tolerance test and AUC of the insulin tolerance test during the preventive (2 left panels) and curative (2 right panels) setting (n = 7 control group, 6 group HM3Dq). Data represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons using a Tukey post-hoc approach (B, G-H) or two-tailed unpaired t-test (C-F). Statistical difference established at p < 0.05.
A

<figure><img src="image_93.png" /></figure>

AAV5-hSyn-DIO-hM3D(Gq)-mCherry

<figure><img src="image_94.png" /></figure>

<figure><img src="image_95.png" /></figure>

Experimental schema - Curative approach

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

B

<figure><img src="image_98.png" /></figure>

● Vglut3Cre hM3Dq-mCherry_HFD

- Vglut3Cre mCherry_HFD

C

<figure><img src="image_99.png" /></figure>

● Vglut3Cre hM3Dq-mCherry_HFD

- Vglut3Cre mCherry_HFD

D

<figure><img src="image_100.png" /></figure>

● Vglut3Cre hM3Dq-mCherry_HFD

- Vglut3Cre mCherry_HFD

**Extended Data Fig. 4 | DRN<sup>Vglut3</sup> Neurons Drive Reduces Both Food Intake and Body Weight in DIO Female Mice.** (a) Schema illustrating chronic stimulation of DRN<sup>Vglut3</sup> neurons in DIO female mice by injecting an AAV5-DIO-hm3Dq-mcherry in DRN<sup>Vglut3</sup> mice using the DREADD agonist clozapine-N-oxide (CNO) once a day for 14 days after the development of obesity. Representative image shows the injection site. (b) Weight curve illustrating the body weight loss after stimulating DRN<sup>Vglut3</sup> neurons upon DIO in female mice (n = 6 per group). (c) Food intake

during the chronic stimulation of DRN<sup>Vglut3</sup> neurons in females demonstrating hypophagia (n = 6 per group). (d) Adaptive thermogenesis at the last day of treatment with CNO (n = 6 per group). (A-D) Littermate Vglut3-Cre positive DIO mice injected with an AAV2/5-DIO-mCherry were used as controls. Data represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons using a Tukey post-hoc approach (B, C), or two-tailed unpaired t-test (C, D). Statistical difference established at p < 0.05.
A

<figure><img src="image_97.png" /></figure>

AAV5-hSyn-DIO-hM3D(Gq)-mCherry

<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

Experimental schema - Curative approach

<figure><img src="image_100.png" /></figure>

<figure><img src="image_101.png" /></figure>

B

<figure><img src="image_102.png" /></figure>

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

C

<figure><img src="image_103.png" /></figure>

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

D

<figure><img src="image_104.png" /></figure>

● Vglut3Cre hM3Dq-mCherry_HFD

■ Vglut3Cre mCherry_HFD

**Extended Data Fig. 4 | DRN<sup>Vglut3</sup> Neurons Drive Reduces Both Food Intake and Body Weight in DIO Female Mice.** (a) Schema illustrating chronic stimulation of DRN<sup>Vglut3</sup> neurons in DIO female mice by injecting an AAV5-DIO-hm3Dq-mcherry in DRN<sup>Vglut3</sup> mice using the DREADD agonist clozapine-N-oxide (CNO) once a day for 14 days after the development of obesity. Representative image shows the injection site. (b) Weight curve illustrating the body weight loss after stimulating DRN<sup>Vglut3</sup> neurons upon DIO in female mice (n = 6 per group). (c) Food intake

during the chronic stimulation of DRN<sup>Vglut3</sup> neurons in females demonstrating hypophagia (n = 6 per group). (d) Adaptive thermogenesis at the last day of treatment with CNO (n = 6 per group). (A-D) Littermate Vglut3-Cre positive DIO mice injected with an AAV2/5-DIO-mCherry were used as controls. Data represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons using a Tukey post-hoc approach (B.), or two-tailed unpaired t-test (C, D). Statistical difference established at p < 0.05.
<figure><img src="image_101.png" /></figure>

<figure><img src="image_102.png" /></figure>

<figure><img src="image_103.png" /></figure>

<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /></figure>

<figure><img src="image_107.png" /></figure>

<figure><img src="image_108.png" /></figure>

<figure><img src="image_109.png" /></figure>

**Extended Data Fig. 5** | Translational strategy comparing DRN<sup>Vglut3</sup> molecular profile to the GENSAT Translational Affinity Purification database reveals Druggable Targets for Weight Management. (a) Principal component analysis of the three pairs of samples used for the identification of valuable druggable targets in DRN<sup>Vglut3</sup> neurons using TRAP. (b) Volcano plot for the adjusted p-value logarithmic plots versus the fold change logarithmic plots illustrating the set of markers most enriched in the IP sample versus the input. Red dots highlight differentially enriched markers for drug discovery that will be used to illustrate the enrichment after each filter is applied (CalcR, Hcrt1 and GPR4). (c) Volcano plot for the adjusted p-value logarithmic plots versus the fold change logarithmic plots illustrating the set of markers most enriched in the IP sample the GENSAT database. Red dots highlight differentially enriched markers for drug discovery that will be used to illustrate the enrichment after each filter is applied (CalcR,

Hcrt1 and GPR4). (d) In situ hybridization studies showing colocalization between cell-type specific marker gene Vglut3 and receptors (CalcR or Hcrt1) in the Dorsal Raphe Nucleus. White arrows designate double-labeled cells (n = 3 per group). (e) In situ hybridization studies using RNAscope technology showing expression of the studied receptors in other brain locations such as the Locus Coeruleus, Paraventricular Hypothalamus and Lateral Hypothalamic Area. Scale bars 25um (d) 100um (e). (F-Left) Schema DRN infusion of drug ligands to CalcR and Hcrt1 prior to feeding assessment. (F-middle) Salmon Calcitonin (30ng) infusion into the DRN as a direct ligand for CalcR shows feeding suppressing effects. (F-Right) Agonist (orexin (green)) (200ng) and antagonists (SB334867 (250ng) (cyan) and suvorexant (red)) (250ng) infused locally into the DRN exhibit feeding suppressant effects only at when orexin receptors are antagonized (n = 5 per group). Data are presented as mean ± SEM.
A

<figure><img src="image_105.png" /></figure>

B

<figure><img src="image_106.png" /></figure>

C

<figure><img src="image_107.png" /></figure>

D

<figure><img src="image_108.png" /></figure>

<figure><img src="image_109.png" /></figure>

E

<figure><img src="image_110.png" /></figure>

F

Drug infusion schema

<figure><img src="image_111.png" /></figure>

<figure><img src="image_112.png" /></figure>

<figure><img src="image_113.png" /></figure>

**Extended Data Fig. 5** | Translational strategy comparing DRN<sup>Vglut3</sup> molecular profile to the GENSAT Translational Affinity Purification database reveals Druggable Targets for Weight Management. (a) Principal component analysis of the three pairs of samples used for the identification of valuable druggable targets in DRN<sup>Vglut3</sup> neurons using TRAP. (b) Volcano plot for the adjusted p-value logarithmic plots versus the fold change logarithmic plots illustrating the set of markers most enriched in the IP sample versus the input. Red dots highlight differentially enriched markers for drug discovery that will be used to illustrate the enrichment after each filter is applied (CalcR, Hcrt1 and GPR4). (c) Volcano plot for the adjusted p-value logarithmic plots versus the fold change logarithmic plots illustrating the set of markers most enriched in the IP sample the GENSAT database. Red dots highlight differentially enriched markers for drug discovery that will be used to illustrate the enrichment after each filter is applied (CalcR,

Hcrt1 and GPR4). (d) In situ hybridization studies showing colocalization between cell-type specific marker gene Vglut3 and receptors (CalcR or Hcrt1) in the Dorsal Raphe Nucleus. White arrows designate double-labeled cells (n = 3 per group). (e) In situ hybridization studies using RNAscope technology showing expression of the studied receptors in other brain locations such as the Locus Coeruleus, Paraventricular Hypothalamus and Lateral Hypothalamic Area. Scale bars 25um (d) 100um (e). (F-Left) Schema DRN infusion of drug ligands to CalcR and Hcrt1 prior to feeding assessment. (F-middle) Salmon Calcitonin (30ng) infusion into the DRN as a direct ligand for CalcR shows feeding suppressing effects. (F-Right) Agonist (orexin (green)) (200ng) and antagonists (SB334867 (250ng) (cyan) and suvorexant (red)) (250ng) infused locally into the DRN exhibit feeding suppressant effects only at when orexin receptors are antagonized (n = 5 per group). Data are presented as mean ± SEM.
A

Targeted pharmacology

<figure><img src="image_110.png" /></figure>

dorsal raphe nucleus

drug or vehicle

<figure><img src="image_111.png" /></figure>

Drug infusion schema and treatment

<figure><img src="image_112.png" /></figure>

B

<figure><img src="image_113.png" /></figure>

<figure><img src="image_114.png" /></figure>

C

<figure><img src="image_115.png" /></figure>

D

<figure><img src="image_116.png" /></figure>

E

Targeted pharmacology

<figure><img src="image_117.png" /></figure>

Drug infusion schema and treatment

<figure><img src="image_118.png" /></figure>

F

<figure><img src="image_119.png" /></figure>

G

<figure><img src="image_120.png" /></figure>

H

<figure><img src="image_121.png" /></figure>

I

<figure><img src="image_122.png" /></figure>

J

Extended Data Fig. 6 | See next page for caption.
<figure><img src="image_114.png" /></figure>

<figure><img src="image_115.png" /></figure>

<figure><img src="image_116.png" /></figure>

<figure><img src="image_117.png" /></figure>

<figure><img src="image_118.png" /></figure>

<figure><img src="image_119.png" /></figure>

<figure><img src="image_120.png" /></figure>

<figure><img src="image_121.png" /></figure>

<figure><img src="image_122.png" /></figure>

Extended Data Fig. 6 | See next page for caption.
**Extended Data Fig. 6** | Local and Brain Wide Infusion in the Dorsal Raphe Nucleus of Drug Compounds to CalcR and Hcrtr1 (s-CT and SB-334867) Ameliorates Energy Balance Defects when transitioning from a standard Diet to HFD. (a,Left) Schema and profiling-based drug targeting. (A, Right) Drug infusion schema into the DRN of mice implanted with a local cannula to evaluate food intake when transitioning from chow diet to HFD (preventing obesity). (b) Weight curve illustrating the reduced body weight increase after CalcR agonism or Hcrtr1 antagonism with s-CT (30ng) or SB-334867 (250ng) in the DRN when transitioning from chow diet to HFD. Post-treatment mice are infused with vehicle (n = 5 per group). (c) Weight gain after CalcR agonism or Hcrtr1 antagonism with s-CT (30ng) or SB-334867 (250ng) compared to vehicle treated controls (n = 5 per group). (d) Food intake assessment during the treatment window of s-CT (30ng) or SB-334867 (250ng) into the DRN (n = 5 per group). (e) ICV drug infusion schema of mice to evaluate feeding when transitioning from a

chow diet to a HFD (preventing obesity). (f) Dipsogenic Angiotensin II infusion (10 ng) schematic for correct cannula placement evaluating drinking response (n = 13 per group). (g) Weight curve after ICV CalcR agonism or Hcrtr1 antagonism with s-CT (300ng) or SB-334867 (2.5 ug) respectively when transitioning from a chow diet to a HFD. Post-treatment mice are infused with vehicle (n = 5 per group). (h) Weight gain after CalcR agonism or Hcrtr1 antagonism with s-CT (300ng) or SB-334867 (2.5ug) compared to vehicle treated controls (n = 5 per group). (i) Weight gain during the treatment window (n = 5 per group). (j) Food intake assessment during the treatment window (14 days) of ICV s-CT (300ng) or SB-334867 (2.5 ug) infusion (n = 5 per group). Data represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons (Tukey post-hoc) (B-C, G-H), one-way ANOVA with Bonferroni post-hoc multiple comparison analysis (D, I-J) or two-tailed unpaired t-test (F). Statistical difference established at p < 0.05.
**Extended Data Fig. 6** | Local and Brain Wide Infusion in the Dorsal Raphe Nucleus of Drug Compounds to CalcR and Hcrtr1 (s-CT and SB-334867) Ameliorates Energy Balance Defects when transitioning from a standard Diet to HFD. (a,Left) Schema and profiling-based drug targeting. (A, Right) Drug infusion schema into the DRN of mice implanted with a local cannula to evaluate food intake when transitioning from chow diet to HFD (preventing obesity). (b) Weight curve illustrating the reduced body weight increase after CalcR agonism or Hcrtr1 antagonism with s-CT (30ng) or SB-334867 (250ng) in the DRN when transitioning from chow diet to HFD. Post-treatment mice are infused with vehicle (n = 5 per group). (c) Weight gain after CalcR agonism or Hcrtr1 antagonism with s-CT (30ng) or SB-334867 (250ng) compared to vehicle treated controls (n = 5 per group). (d) Food intake assessment during the treatment window of s-CT (30ng) or SB-334867 (250 ng) into the DRN (n = 5 per group). (e) ICV drug infusion schema of mice to evaluate feeding when transitioning from a

chow diet to a HFD (preventing obesity). (f) Dipsogenic Angiotensin II infusion (10 ng) schematic for correct cannula placement evaluating drinking response (n = 13 per group). (g) Weight curve after ICV CalcR agonism or Hcrtr1 antagonism with s-CT (300ng) or SB-334867 (2.5 ug) respectively when transitioning from a chow diet to a HFD. Post-treatment mice are infused with vehicle (n = 5 per group). (h) Weight gain after CalcR agonism or Hcrtr1 antagonism with s-CT (300ng) or SB-334867 (2.5ug) compared to vehicle treated controls (n = 5 per group). (i) Weight gain during the treatment window (n = 5 per group). (j) Food intake assessment during the treatment window (14 days) of ICV s-CT (300ng) or SB-334867 (2.5 ug) infusion (n = 5 per group). Data represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons (Tukey post-hoc) (B-C, G-H), one-way ANOVA with Bonferroni post-hoc multiple comparison analysis (D, I-J) or two-tailed unpaired t-test (F). Statistical difference established at p < 0.05.
A

Targeted pharmacology

<figure><img src="image_123.png" /></figure>

Drug infusion schema and treatment

<figure><img src="image_124.png" /></figure>

B

<figure><img src="image_125.png" /></figure>

C

<figure><img src="image_126.png" /></figure>

D

<figure><img src="image_127.png" /></figure>

E

<figure><img src="image_128.png" /></figure>

F

<figure><img src="image_129.png" /></figure>

G

<figure><img src="image_130.png" /></figure>

H

<figure><img src="image_131.png" /></figure>

I

<figure><img src="image_132.png" /></figure>

J

<figure><img src="image_133.png" /></figure>

K

<figure><img src="image_134.png" /></figure>

**Extended Data Fig. 7** | Infusion to the Dorsal Raphe Nucleus of s-CT and SB-334867 Does Not Induce Major Side Effects in Mice. (a) Drug infusion schema into DRN of mice implanted with a cannula to evaluate food intake effects chronically after induction of DIO. (B-I) 20-hour automated cage observation of mice behavior in a clear glass cage to evaluate whether s-CT or SB-334867 lead to debilitating side effects in the DRN (n = 4 per group). Behavioral paradigms evaluated are time spent (b) Ingesting food and (c) Sleeping. In addition, automated system evaluates the locomotion activity of mice as time spent (d) Walking Left, (e) Walking Right, (f) Walking Slowly or ambulating. As a function of that parameter, it provides the (g) Total Distance Travelled. Recordings provide

information regarding time spent (h) Grooming or (i) Twitching in the behavioral cage which would be associated to stress behaviors. (j-k) Information about eating and sleeping is also calculated as amount of eating and sleeping bouts. Total eating and sleeping bouts of mice treated with ACSF, s-CT and SB-334867. In line with our manually acquired data feeding suppression is observed in both drug treatments (j) and in line with previous literature there is an enhancement of sleeping time in both treatments. Data are represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons (Tukey post-hoc approach) (B-I), one-way ANOVA with Bonferroni post-hoc multiple comparison analysis (J-K). Statistical difference established at p < 0.05.
A

Targeted pharmacology

<figure><img src="image_123.png" /></figure>

Drug infusion schema and treatment

<figure><img src="image_124.png" /></figure>

B

<figure><img src="image_125.png" /></figure>

C

<figure><img src="image_126.png" /></figure>

D

<figure><img src="image_127.png" /></figure>

E

<figure><img src="image_128.png" /></figure>

F

<figure><img src="image_129.png" /></figure>

G

<figure><img src="image_130.png" /></figure>

H

<figure><img src="image_131.png" /></figure>

I

<figure><img src="image_132.png" /></figure>

J

<figure><img src="image_133.png" /></figure>

K

<figure><img src="image_134.png" /></figure>

**Extended Data Fig. 7** | Infusion to the Dorsal Raphe Nucleus of s-CT and SB-334867 Does Not Induce Major Side Effects in Mice. (a) Drug infusion schema into DRN of mice implanted with a cannula to evaluate food intake effects chronically after induction of DIO. (B-I) 20-hour automated cage observation of mice behavior in a clear glass cage to evaluate whether s-CT or SB-334867 lead to debilitating side effects in the DRN (n = 4 per group). Behavioral paradigms evaluated are time spent (b) Ingesting food and (c) Sleeping. In addition, automated system evaluates the locomotion activity of mice as time spent (d) Walking Left, (e) Walking Right, (f) Walking Slowly or ambulating. As a function of that parameter, it provides the (g) Total Distance Travelled. Recordings provide

information regarding time spent (h) Grooming or (i) Twitching in the behavioral cage which would be associated to stress behaviors. (j-k) Information about eating and sleeping is also calculated as amount of eating and sleeping bouts. Total eating and sleeping bouts of mice treated with ACSF, s-CT and SB-334867. In line with our manually acquired data feeding suppression is observed in both drug treatments (j) and in line with previous literature there is an enhancement of sleeping time in both treatments. Data are represented as Mean ± SEM. P-value calculated as a 2-way ANOVA with multiple comparisons (Tukey post-hoc approach) (B-I), one-way ANOVA with Bonferroni post-hoc multiple comparison analysis (J-K). Statistical difference established at p < 0.05.
<figure><img src="image_135.png" /></figure>

**Extended Data Fig. 8** | Treatment with CVN45502 (30 mg/kg) does not lead to major changes in gene expression of brown adipose tissue thermogenesis markers. (a) Normalized gene expression of thermogenic, brown adipose and mitochondrial genes of brown adipose samples collected from mice treated with CVN45502 (30 mg/kg) during 7 days and fed a standard chow diet. Genes selected have been extensively validated as key markers for thermogenic processes in the BAT. PPARgc1a is the only marker exhibiting a reduction in gene expression suggesting no major changes exist in BAT functionality (n = 3 control group, n = 4 CVN45502 group). (b) Normalized gene expression of thermogenic, brown

adipose and mitochondrial genes of brown adipose samples collected from mice treated with CVN45502 (30 mg/kg) during 7 days and fed a high fat diet during 16 weeks to develop diet induced obesity. Genes selected have been extensively validated as key markers for thermogenic processes in the BAT. None of the gene expression markers exhibit a significant difference in gene expression suggesting no major changes exist in BAT functionality (n = 3 control group, n = 4 CVN45502 group). Data represented as Mean ± SEM. P-value calculated as one-way ANOVA with Tukey post hoc assessment. Statistical difference established at p < 0.05.
<figure><img src="image_135.png" /></figure>

**Extended Data Fig. 8** | Treatment with CVN45502 (30 mg/kg) does not lead to major changes in gene expression of brown adipose tissue thermogenesis markers. (a) Normalized gene expression of thermogenic, brown adipose and mitochondrial genes of brown adipose samples collected from mice treated with CVN45502 (30 mg/kg) during 7 days and fed a standard chow diet. Genes selected have been extensively validated as key markers for thermogenic processes in the BAT. PPARgc1a is the only marker exhibiting a reduction in gene expression suggesting no major changes exist in BAT functionality (n = 3 control group, n = 4 CVN45502 group). (b) Normalized gene expression of thermogenic, brown

adipose and mitochondrial genes of brown adipose samples collected from mice treated with CVN45502 (30 mg/kg) during 7 days and fed a high fat diet during 16 weeks to develop diet induced obesity. Genes selected have been extensively validated as key markers for thermogenic processes in the BAT. None of the gene expression markers exhibit a significant difference in gene expression suggesting no major changes exist in BAT functionality (n = 3 control group, n = 4 CVN45502 group). Data represented as Mean ± SEM. P-value calculated as one-way ANOVA with Tukey post hoc assessment. Statistical difference established at p < 0.05.
<figure><img src="image_136.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_137.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_138.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_139.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_140.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_141.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_142.png" /></figure>

Extended Data Fig. 9 | See next page for caption.
<figure><img src="image_136.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_137.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_138.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_139.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_140.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_141.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_142.png" /></figure>

Extended Data Fig. 9 | See next page for caption.
**Extended Data Fig. 9** | Treatment with CVN45502 (30 mg/kg) leads to a reduction in food intake with no major changes in energy expenditure in leptin deficient genetic obese ob/ob mice. (a-f) Energy balance phenotyping using a home-cage metabolic cage system. Parameters measured in a day/ night cycle were (A) feeding, (B) CO2 production, (C) oxygen consumption, (D) locomotor activity and, (E) energy expenditure (n = 7 control group, n = 8 CVN45502 group). Significant changes supportive of weight loss only existed in food intake, similar to CVN45502 treatment in DIO mice (Fig. 7). (f) body weight gain of ob/ob mice treated with CVN45502 (n = 6 control group, n = 5 CVN45502 group). (g) Normalized gene expression of thermogenic, brown adipose and

mitochondrial genes of brown adipose samples collected from mice treated with CVN45502 (30 mg/kg) during 7 days and fed a standard chow diet. Genes selected have been extensively validated as key markers for thermogenic processes in the BAT. None of the gene expression markers exhibit a significant difference in gene expression suggesting no major changes exist in BAT functionality (n = 7 control group, n = 6 CVN45502 group). Data represented as Mean ± SEM. P-value calculated using CalR software and ANCOVA regression analysis taking body weight into account (A-E) or two-way ANOVA with repeated measures and Tukey post hoc assessment. p < 0.05 is considered significant.
**Extended Data Fig. 9** | Treatment with CVN45502 (30 mg/kg) leads to a reduction in food intake with no major changes in energy expenditure in leptin deficient genetic obese ob/ob mice. (a-f) Energy balance phenotyping using a home-cage metabolic cage system. Parameters measured in a day/ night cycle were (A) feeding, (B) CO2 production, (C) oxygen consumption, (D) locomotor activity and, (E) energy expenditure (n = 7 control group, n = 8 CVN45502 group). Significant changes supportive of weight loss only existed in food intake, similar to CVN45502 treatment in DIO mice (Fig. 7). (f) body weight gain of ob/ob mice treated with CVN45502 (n = 6 control group, n = 5 CVN45502 group). (g) Normalized gene expression of thermogenic, brown adipose and

mitochondrial genes of brown adipose samples collected from mice treated with CVN45502 (30 mg/kg) during 7 days and fed a standard chow diet. Genes selected have been extensively validated as key markers for thermogenic processes in the BAT. None of the gene expression markers exhibit a significant difference in gene expression suggesting no major changes exist in BAT functionality (n = 7 control group, n = 6 CVN45502 group). Data represented as Mean ± SEM. P-value calculated using CalR software and ANCOVA regression analysis taking body weight into account (A-E) or two-way ANOVA with repeated measures and Tukey post hoc assessment. p < 0.05 is considered significant.
<figure><img src="image_143.png" /><figcaption>Transient FFPE</figcaption></figure>

<figure><img src="image_144.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_145.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_146.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_147.png" /></figure>

<figure><img src="image_148.png" /></figure>

Extended Data Fig. 10 | See next page for caption.
<figure><img src="image_143.png" /><figcaption>Transient FFPE</figcaption></figure>

<figure><img src="image_144.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_145.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_146.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_147.png" /><figcaption>Human brain donor dorsal raphe (IHC)</figcaption></figure>

<figure><img src="image_148.png" /></figure>

Extended Data Fig. 10 | See next page for caption.
**Extended Data Fig. 10** | *Hcrtr1 is expressed in the DRN and colocalizes with Vglut3 in humans.*

( a ) In vitro studies in chemical competent cells (control) as well as chemical competent cells transfected with Hcrtr1 to validate the specificity of the probe used (Staining in blue). ( b ) In vitro immunohistochemistry (IHC) study (top) and immunofluorescence (IF) study (bottom) in Formalin Fixed Paraffin Embedded HEK 293 cells expressing Hcrtr1 demonstrating antibody specificity to move forward with human anatomy studies with the selected antibody. The cells used in the bottom image are Hcrtr1-FLAG-tagged HEKs. Cells were observed with a 20-fold objective lens. ( c ) Immunohistochemistry of Hcrtr1 in brain tissue of human donor at the Locus Coeruleus region. Extensive evidence shows Hcrtr1 expression in this region

of the brain, hence this data validates in vivo the selected antibody. (d) In situ
hybridization studies using an RNAscope probe for human Hcrtr1 in the locus
coeruleus of human donors. This data validates the selected probe for studies
in the DRN. (e) In situ hybridization studies staining for Hcrtr1 in DRN sections
of human donors confirming labelling of Hcrtr1 in this brainstem region and
recapitulating our initial starting point of this study, the DRN, in humans (Left).
Immunohistochemistry of Hcrtr1 in brain tissue of human donor at the DRN
region further demonstrating expression of Hcrtr1, using a validated antibody for
Hcrtr1 (Right). All imaging studies were performed in 3 biological independent
experiments in all cell culture or mice work. Scale bars = 50–100 um.
**Extended Data Fig. 10** | *Hcrtr1 is expressed in the DRN and colocalizes with Vglut3 in humans.*

( a ) In vitro studies in chemical competent cells (control) as well as chemical competent cells transfected with Hcrtr1 to validate the specificity of the probe used (Staining in blue). ( b ) In vitro immunohistochemistry (IHC) study (top) and immunofluorescence (IF) study (bottom) in Formalin Fixed Paraffin Embedded HEK 293 cells expressing Hcrtr1 demonstrating antibody specificity to move forward with human anatomy studies with the selected antibody. The cells used in the bottom image are Hcrtr1-FLAG-tagged HEKs. Cells were observed with a 20-fold objective lens. ( c ) Immunohistochemistry of Hcrtr1 in brain tissue of human donor at the Locus Coeruleus region. Extensive evidence shows Hcrtr1 expression in this region

of the brain, hence this data validates in vivo the selected antibody. (d) In situ
hybridization studies using an RNAscope probe for human Hcrtr1 in the locus
coeruleus of human donors. This data validates the selected probe for studies
in the DRN. (e) In situ hybridization studies staining for Hcrtr1 in DRN sections
of human donors confirming labelling of Hcrtr1 in this brainstem region and
recapitulating our initial starting point of this study, the DRN, in humans (Left).
Immunohistochemistry of Hcrtr1 in brain tissue of human donor at the DRN
region further demonstrating expression of Hcrtr1, using a validated antibody for
Hcrtr1 (Right). All imaging studies were performed in 3 biological independent
experiments in all cell culture or mice work. Scale bars = 50–100 um.
<figure><img src="image_149.png" /></figure>

# Reporting Summary

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## Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed

<figure><img src="image_150.png" /></figure>

<figure><img src="image_151.png" /></figure>

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement

<figure><img src="image_152.png" /></figure>

<figure><img src="image_153.png" /></figure>

A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

<figure><img src="image_154.png" /></figure>

<figure><img src="image_155.png" /></figure>

The statistical test(s) used AND whether they are one- or two-sided
*Only common tests should be described solely by name; describe more complex techniques in the Methods section.*

<figure><img src="image_156.png" /></figure>

<figure><img src="image_157.png" /></figure>

A description of all covariates tested

<figure><img src="image_158.png" /></figure>

<figure><img src="image_159.png" /></figure>

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons

<figure><img src="image_160.png" /></figure>

<figure><img src="image_161.png" /></figure>

A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

<figure><img src="image_162.png" /></figure>

<figure><img src="image_163.png" /></figure>

For null hypothesis testing, the test statistic (e.g. *F*, *t*, *r*) with confidence intervals, effect sizes, degrees of freedom and *P* value noted
*Give *P* values as exact values whenever suitable.*

<figure><img src="image_164.png" /></figure>

<figure><img src="image_165.png" /></figure>

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings

<figure><img src="image_166.png" /></figure>

<figure><img src="image_167.png" /></figure>

For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes

<figure><img src="image_168.png" /></figure>

<figure><img src="image_169.png" /></figure>

Estimates of effect sizes (e.g. Cohen's *d*, Pearson's *r*), indicating how they were calculated

*Our web collection on statistics for biologists contains articles on many of the points above.*

## Software and code

Policy information about [availability of computer code](#)

<table><tr><td>Data collection</td><td>Zen 2.31 was used for confocal imaging, Inspector Pro (version 4.0.207) for light sheet imaging, Ethovision XT for open field measurements and TSE phenomaster suite for indirect calorimetry. R packages were used for molecular profiling GENSAT comparisons.</td></tr><tr><td>Data analysis</td><td>GraphPAD prism 8.0.2 (statistics/graphics) was used for all statistic measurements besides indirect calorimetry which used CalR and Molecular Profiling that used R.<br/><br/>Image J/FIJI v2.0.0.0-rc-69/1.52p was used for ISH and IHC cell counting and image reconstruction.<br/><br/>Imaris 9.1 was used for light sheet imaging representation and video generation.<br/><br/>Trailmap and Clearmap 2.0 was used for light sheet projection mapping quantifications. Code for Clearmap 2.0 can be found at https://github.com/CristophKirst/ClearMap2 and for TrailMap at https://github.com/AlbertPun/TRAILMAP. Code to account for imaging corrections specific to our data set are located in supplementary information and can be run in ClearMap2.0 as TubeMapTrailMAP.py<br/><br/>R packages DEseq2 v1.20.0, Salmon v0.8.2, Tximport version 1.8.0, Rsubread subjunct version 1.30.6, Rtracklayer version 1.40.6, GSVA version 1.34.0, Pheatmap, Metap and complex Heatmap Bioconductor packages were used for molecular profiling characterization and comparison to GENSAT database. Code availability for the specific runs can be found at https://github.com/RockefellerUniversity/SchneebergerPane_NatureMetabolism2022.</td></tr></table>

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio [guidelines for submitting code & software](#) for further information.
<figure><img src="image_149.png" /></figure>

# Reporting Summary

Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our [Editorial Policies](#) and the [Editorial Policy Checklist](#).

## Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed

<figure><img src="image_150.png" /></figure>

<figure><img src="image_151.png" /></figure>

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement

<figure><img src="image_152.png" /></figure>

<figure><img src="image_153.png" /></figure>

A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

<figure><img src="image_154.png" /></figure>

<figure><img src="image_155.png" /></figure>

The statistical test(s) used AND whether they are one- or two-sided
*Only common tests should be described solely by name; describe more complex techniques in the Methods section.*

<figure><img src="image_156.png" /></figure>

<figure><img src="image_157.png" /></figure>

A description of all covariates tested

<figure><img src="image_158.png" /></figure>

<figure><img src="image_159.png" /></figure>

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons

<figure><img src="image_160.png" /></figure>

<figure><img src="image_161.png" /></figure>

A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

<figure><img src="image_162.png" /></figure>

<figure><img src="image_163.png" /></figure>

For null hypothesis testing, the test statistic (e.g. *F*, *t*, *r*) with confidence intervals, effect sizes, degrees of freedom and *P* value noted
*Give *P* values as exact values whenever suitable.*

<figure><img src="image_164.png" /></figure>

<figure><img src="image_165.png" /></figure>

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings

<figure><img src="image_166.png" /></figure>

<figure><img src="image_167.png" /></figure>

For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes

<figure><img src="image_168.png" /></figure>

<figure><img src="image_169.png" /></figure>

Estimates of effect sizes (e.g. Cohen's *d*, Pearson's *r*), indicating how they were calculated

*Our web collection on statistics for biologists contains articles on many of the points above.*

## Software and code

Policy information about [availability of computer code](#)

<table><tr><td>Data collection</td><td>Zen 2.31 was used for confocal imaging, Inspector Pro (version 4.0.207) for light sheet imaging, Ethovision XT for open field measurements and TSE phenomaster suite for indirect calorimetry. R packages were used for molecular profiling GENSAT comparisons.</td></tr><tr><td>Data analysis</td><td>GraphPAD prism 8.0.2 (statistics/graphics) was used for all statistic measurements besides indirect calorimetry which used CalR and Molecular Profiling that used R.<br/><br/>Image J/FIJI v2.0.0.0-rc-69/1.52p was used for ISH and IHC cell counting and image reconstruction.<br/><br/>Imaris 9.1 was used for light sheet imaging representation and video generation.<br/><br/>Trailmap and Clearmap 2.0 was used for light sheet projection mapping quantifications. Code for Clearmap 2.0 can be found at https://github.com/CristophKirst/ClearMap2 and for TrailMap at https://github.com/AlbertPun/TRAILMAP. Code to account for imaging corrections specific to our data set are located in supplementary information and can be run in ClearMap2.0 as TubeMapTrailMAp.py<br/><br/>R packages DEseq2 v1.20.0, Salmon v0.8.2, Tximport version 1.8.0, Rsubread subjunct version 1.30.6, Rtracklayer version 1.40.6, GSVA version 1.34.0, Pheatmap, Metap and complex Heatmap Bioconductor packages were used for molecular profiling characterization and comparison to GENSAT database. Code availability for the specific runs can be found at https://github.com/RockefellerUniversity/SchneebergerPane_NatureMetabolism2022.</td></tr></table>

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio [guidelines for submitting code & software](#) for further information.
# Data

Policy information about availability of data

All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

- Accession codes, unique identifiers, or web links for publicly available datasets

- A description of any restrictions on data availability

- For clinical datasets or third party data, please ensure that the statement adheres to our policy

RNAseq data from the vTRAP experiment in DRNvglut3 neurons is publicly available following the GSE87890 published in Nectow AR et al. 2017 Cell.

Allen Brain Atlas ISH Database is used for validation of ISH studies conducted with RNAscope technology and is publicly available.

# Human research participants

Policy information about studies involving human research participants and Sex and Gender in Research.

Reporting on sex and gender

For ISH studies in human tissue 3 samples from frozen sections of non-diseased donors were used. In particular we used samples from a 59 year old male, from a 58 year old female and from a 78 year old female.

Population characteristics

Population selected was from healthy donors since the sole purpose of the experiments was to confirm the expression of the same target (Hcrtr1) in the same neurons Slc17A8 in humans.

Recruitment

Cerevance acquired the samples with full consent from the Tissues for Research biobank located in the UK and from the General Section of the Douglas-Bell Canada Brain Biobank.

Ethics oversight

Cerevance acquired samples already for more than 11000 donors at multiple Biobanks, all with appropriate ethical consents.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

# Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

<figure><img src="image_170.png" /></figure>

Life sciences

<figure><img src="image_171.png" /></figure>

Behavioural & social sciences

<figure><img src="image_172.png" /></figure>

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see [nature.com/documents/nr-reporting-summary-flat.pdf](https://nature.com/documents/nr-reporting-summary-flat.pdf)

# Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size

No statistical methods were used to calculate the sample size ahead of each study.

Sample size was determined based on prior studies and literature in the field as well as animal availability in transgenic breedings to meet age matchings. PMID: 2409368, PMID: 21209617, PMID: 22801496, PMID: 27616062

Data exclusions

No data were excluded

Replication

All data were successfully replicated and data from multiple experiments were pooled in acute experiments. These information is expanded in Statistics and Reproducibility section in the manuscript.

Chronic experiments in HFD were performed in large sample sizes and were only performed once given the amount of available drugs and for timing consideration, multiple experiments coming to similar biological conclusions or using different paradigms strengthen the conclusions.

Randomization

For all experiments mice were randomly assigned assuring that mice were age, sex and weight matched prior to weight loss assessments.

Blinding

Data were collected bind, and posthoc registered to the treatment conditions and analyzed to prevent bias.

# Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
# Data

Policy information about availability of data

All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

- Accession codes, unique identifiers, or web links for publicly available datasets

- A description of any restrictions on data availability

- For clinical datasets or third party data, please ensure that the statement adheres to our policy

RNAseq data from the vTRAP experiment in DRNVglut3 neurons is publicly available following the GSE87890 published in Nectow AR et al. 2017 Cell.

Allen Brain Atlas ISH Database is used for validation of ISH studies conducted with RNAscope technology and is publicly available.

# Human research participants

Policy information about studies involving human research participants and Sex and Gender in Research.

Reporting on sex and gender

For ISH studies in human tissue 3 samples from frozen sections of non-diseased donors were used. In particular we used samples from a 59 year old male, from a 58 year old female and from a 78 year old female.

Population characteristics

Population selected was from healthy donors since the sole purpose of the experiments was to confirm the expression of the same target (Hcrtr1) in the same neurons Slc17A8 in humans.

Recruitment

Cerevance acquired the samples with full consent from the Tissues for Research biobank located in the UK and from the General Section of the Douglas-Bell Canada Brain Biobank.

Ethics oversight

Cerevance acquired samples already for more than 11000 donors at multiple Biobanks, all with appropriate ethical consents.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

# Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

<figure><img src="image_170.png" /></figure>

Life sciences

<figure><img src="image_171.png" /></figure>

Behavioural & social sciences

<figure><img src="image_172.png" /></figure>

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see [nature.com/documents/nr-reporting-summary-flat.pdf](https://nature.com/documents/nr-reporting-summary-flat.pdf)

# Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size

No statistical methods were used to calculate the sample size ahead of each study.

Sample size was determined based on prior studies and literature in the field as well as animal availability in transgenic breedings to meet age matchings. PMID: 2409368, PMID: 21209617, PMID: 22801496, PMID: 27616062

Data exclusions

No data were excluded

Replication

All data were successfully replicated and data from multiple experiments were pooled in acute experiments. These information is expanded in Statistics and Reproducibility section in the manuscript.

Chronic experiments in HFD were performed in large sample sizes and were only performed once given the amount of available drugs and for timing consideration, multiple experiments coming to similar biological conclusions or using different paradigms strengthen the conclusions.

Randomization

For all experiments mice were randomly assigned assuring that mice were age, sex and weight matched prior to weight loss assessments.

Blinding

Data were collected bind, and posthoc registered to the treatment conditions and analyzed to prevent bias.

# Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td><input type="checkbox" checked/></td><td>Antibodies</td><td><input type="checkbox" checked/></td><td>ChIP-seq</td></tr><tr><td><input type="checkbox" checked/></td><td>Eukaryotic cell lines</td><td><input type="checkbox" checked/></td><td>Flow cytometry</td></tr><tr><td><input type="checkbox" checked/></td><td>Palaeontology and archaeology</td><td><input type="checkbox" checked/></td><td>MRI-based neuroimaging</td></tr><tr><td><input type="checkbox" checked/></td><td>Animals and other organisms</td><td></td><td></td></tr><tr><td><input type="checkbox" checked/></td><td>Clinical data</td><td></td><td></td></tr><tr><td><input type="checkbox" checked/></td><td>Dual use research of concern</td><td></td><td></td></tr></tbody></table>

Methods

Antibodies

Antibodies used

The following primary antibodies were used, and unless otherwise indicated concentrations apply to all staining techniques. These reagents and others can be found now as Supplementary Table 5. We have used antibodies across multiple lots, and found the results to be comparable across animals and tissues:

Anti-Hcrtr1-(human)- PA5-33838 (1:100) Thermofisher Catalog # PA5-33838; RRID: AB_2551207

Alexa Fluor 488 AffiPure Donkey Anti-Mouse IgG (H+L) (1:1000) Thermofisher Catalog # A-21202; RRID: AB_141607

Alexa Fluor 647 AffiPure Donkey Anti-Rabbitt IgG (H+L) (1:1000) Thermofisher Catalog # A-31573; RRID: AB_2536183

Alexa Fluor 488 AffiPure Donkey Anti-Rabbitt IgG (H+L) (1:1000) Thermofisher Catalog # A-21206; RRID: AB_2535792

Chicken-anti-GFP (1:2000) AVES-LABS Catalog # GFP-1020; RRID: AB_2307313

Anti-GFAP rabbit polyclonal antibody (1:5000) Abcam Catalog # AB7260

Anti-RFP (RABBIT) Antibody Min X Hu Ms and Rt Serum Proteins (1:2000) ROCKLAND Catalog # 600-401-379

RNA-scope technologies (ACD-BIO) in situ probes:

Slc32a1-C3 mouse Acdbio Catalog # 319191

Slc17a6-C3 mouse Acdbio Catalog # 319171

Slc17a8-C2-human Acdbio Catalog # 487431

Hcrtr1-C1-human Acdbio Catalog # 312588

Slc17a8-C3 mouse Acdbio Catalog # 431261

CalcR-C2 mouse Acdbio Catalog # 317518

Hcrtr1-C2 mouse Acdbio Catalog # 466638

Gpcr4-C2 mouse Acdbio Catalog # 427948

Validation

Antibodies used are commercially available and have been thoroughly validated in the literature and by the manufacturer as it can be seen in their webpage.

Human antibody against Hcrtr1 was additionally validated in HEK Cells expressing Hcrtr1 or not (see Extended Data Fig 10).

RFP (PMIDs: 31257028, 33854415)

GFP (PMIDs 31372393, 31257028)

GFAP (PMIDs: 33034847, 33094475)

Alexa 488 donkey anti-Rabbit (PMID: 36130959, 36134661)

Alexa 647 donkey anti-Rabbit (PMID: 36163184, 36151203)

ALexa 488 donkey anti-Mouse (PMID: 35259845, 35240875)

RNAscope probes are all validated by Acdbio before being send and are computationally matched guaranteeing a 100% success..
Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td><input type="checkbox" checked/></td><td>Antibodies</td><td><input type="checkbox" checked/></td><td>ChIP-seq</td></tr><tr><td><input type="checkbox" checked/></td><td>Eukaryotic cell lines</td><td><input type="checkbox" checked/></td><td>Flow cytometry</td></tr><tr><td><input type="checkbox" checked/></td><td>Palaeontology and archaeology</td><td><input type="checkbox" checked/></td><td>MRI-based neuroimaging</td></tr><tr><td><input type="checkbox" checked/></td><td>Animals and other organisms</td><td></td><td></td></tr><tr><td><input type="checkbox" checked/></td><td>Clinical data</td><td></td><td></td></tr><tr><td><input type="checkbox" checked/></td><td>Dual use research of concern</td><td></td><td></td></tr></tbody></table>

Methods

Antibodies

Antibodies used

The following primary antibodies were used, and unless otherwise indicated concentrations apply to all staining techniques. These reagents and others can be found now as Supplementary Table 5. We have used antibodies across multiple lots, and found the results to be comparable across animals and tissues:

Anti-Hcrtr1-(human)- PA5-33838 (1:100) Thermofisher Catalog # PA5-33838; RRID: AB_2551207

Alexa Fluor 488 AffiPure Donkey Anti-Mouse IgG (H+L) (1:1000) Thermofisher Catalog # A-21202; RRID: AB_141607

Alexa Fluor 647 AffiPure Donkey Anti-Rabbitt IgG (H+L) (1:1000) Thermofisher Catalog # A-31573; RRID: AB_2536183

Alexa Fluor 488 AffiPure Donkey Anti-Rabbitt IgG (H+L) (1:1000) Thermofisher Catalog # A-21206; RRID: AB_2535792

Chicken-anti-GFP (1:2000) AVES-LABS Catalog # GFP-1020; RRID: AB_2307313

Anti-GFAP rabbit polyclonal antibody (1:5000) Abcam Catalog # AB7260

Anti-RFP (RABBIT) Antibody Min X Hu Ms and Rt Serum Proteins (1:2000) ROCKLAND Catalog # 600-401-379

RNA-scope technologies (ACD-BIO) in situ probes:

Slc32a1-C3 mouse Acdbio Catalog # 319191

Slc17a6-C3 mouse Acdbio Catalog # 319171

Slc17a8-C2-human Acdbio Catalog # 487431

Hcrtr1-C1-human Acdbio Catalog # 312588

Slc17a8-C3 mouse Acdbio Catalog # 431261

CalcR-C2 mouse Acdbio Catalog # 317518

Hcrtr1-C2 mouse Acdbio Catalog # 466638

Gpcr4-C2 mouse Acdbio Catalog # 427948

Validation

Antibodies used are commercially available and have been thoroughly validated in the literature and by the manufacturer as it can be seen in their webpage.

Human antibody against Hcrtr1 was additionally validated in HEK Cells expressing Hcrtr1 or not (see Extended Data Fig 10).

RFP (PMIDs: 31257028, 33854415)

GFP (PMIDs 31372393, 31257028)

GFAP (PMIDs: 33034847, 33094475)

Alexa 488 donkey anti-Rabbit (PMID: 36130959, 36134661)

Alexa 647 donkey anti-Rabbit (PMID: 36163184, 36151203)

ALexa 488 donkey anti-Mouse (PMID: 35259845, 35240875)

RNAscope probes are all validated by Acdbio before being send and are computationally matched guaranteeing a 100% success..
# Eukaryotic cell lines

Policy information about **cell lines and Sex and Gender in Research**

**Cell line source(s)**
HEK293FT (Invitrogen R70007), Formalin-Fixed Paraffin-Embedded cell line was obtained from AMSBIO. Data using these lines is presented in Extended Data Figure 10

**Authentication**
HEK 293FT was purchased by Cerevance from Invitrogen. FFPE line was purchased by Cerevance from AMSBIO. Cells were not authenticated by us.

**Mycoplasma contamination**
They were all negative from mycoplasma contamination.

**Commonly misidentified lines**
(See ICLAC register)
No commonly misidentified lines.

# Animals and other research organisms

Policy information about **studies involving animals; ARRIVE guidelines** recommended for reporting animal research, and **Sex and Gender in Research**

**Laboratory animals**
Adult male and female mice from strains: C57Bl6 (wild type #000664, The Jackson Laboratory), ob/ob (#000632, The Jackson Laboratory) Vglut3-IRES-Cre (Lou et al. 2013)-gift, Vglut2-IRES-Cre (#016963, The Jackson Laboratory). All animals were obtained at 5 weeks of age and HFD started at week 6 until week 22 of age to ensure DIO. Experiments not dependent on DIO were conducted between 12-14 weeks of age.

**Wild animals**
The study did not include wild animals

**Reporting on sex**
Most studies were conducted in male mice after basic responses on vglut3 neurons in body weight loss were confirmed for both sexes to minimize animal use.

**Field-collected samples**
this study did not involve field study samples

**Ethics oversight**
Rockefeller University IACUC

Note that full information on the approval of the study protocol must also be provided in the manuscript.
# Eukaryotic cell lines

Policy information about **cell lines and Sex and Gender in Research**

**Cell line source(s)**
HEK293FT (Invitrogen R70007), Formalin-Fixed Paraffin-Embedded cell line was obtained from AMSBIO. Data using these lines is presented in Extended Data Figure 10

**Authentication**
HEK 293FT was purchased by Cerevance from Invitrogen. FFPE line was purchased by Cerevance from AMSBIO. Cells were not authenticated by us.

**Mycoplasma contamination**
They were all negative from mycoplasma contamination.

**Commonly misidentified lines**
(See ICLAC register)
No commonly misidentified lines.

# Animals and other research organisms

Policy information about **studies involving animals; ARRIVE guidelines** recommended for reporting animal research, and **Sex and Gender in Research**

**Laboratory animals**
Adult male and female mice from strains: C57Bl6 (wild type #000664, The Jackson Laboratory), ob/ob (#000632, The Jackson Laboratory) Vglut3-IRES-Cre (Lou et al. 2013)-gift, Vglut2-IRES-Cre (#016963, The Jackson Laboratory). All animals were obtained at 5 weeks of age and HFD started at week 6 until week 22 of age to ensure DIO. Experiments not dependent on DIO were conducted between 12-14 weeks of age.

**Wild animals**
The study did not include wild animals

**Reporting on sex**
Most studies were conducted in male mice after basic responses on vglut3 neurons in body weight loss were confirmed for both sexes to minimize animal use.

**Field-collected samples**
this study did not involve field study samples

**Ethics oversight**
Rockefeller University IACUC

Note that full information on the approval of the study protocol must also be provided in the manuscript.